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Loss of Heterozygosity Drives Adaptation in Hybrid Yeast Caiti S. Smukowski Heil, 1 Christopher G. DeSevo, ‡,2 Dave A. Pai, §,2 , Cheryl M. Tucker, ¶,2 Margaret L. Hoang, k,3,4 and Maitreya J. Dunham* ,1 1 Department of Genome Sciences, University of Washington, Seattle, WA 2 Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 3 Department of Embryology, Howard Hughes Medical Institute, Carnegie Institution, Baltimore, MD 4 Department of Biology, Johns Hopkins University, Baltimore, MD ‡ Present address: Exelixis, Dallas, TX § Present address: BioNano Genomics, San Diego, CA Present address: Wall High School, Wall Township, NJ k Present address: Nanostring, Seattle, WA *Corresponding author: E-mail: [email protected] Associate editor: Jianzhi Zhang Abstract Hybridization is often considered maladaptive, but sometimes hybrids can invade new ecological niches and adapt to novel or stressful environments better than their parents. The genomic changes that occur following hybridization that facilitate genome resolution and/or adaptation are not well understood. Here, we examine hybrid genome evolution using experimental evolution of de novo interspecific hybrid yeast Saccharomyces cerevisiae Saccharomyces uvarum and their parentals. We evolved these strains in nutrient-limited conditions for hundreds of generations and sequenced the resulting cultures identifying numerous point mutations, copy number changes, and loss of heterozygosity (LOH) events, including species-biased amplification of nutrient transporters. We focused on a particularly interesting example, in which we saw repeated LOH at the high-affinity phosphate transporter gene PHO84 in both intra- and interspecific hybrids. Using allele replacement methods, we tested the fitness of different alleles in hybrid and S. cerevisiae strain backgrounds and found that the LOH is indeed the result of selection on one allele over the other in both S. cerevisiae and the hybrids. This is an example where hybrid genome resolution is driven by positive selection on existing heterozygosity and demonstrates that even infrequent outcrossing may have lasting impacts on adaptation. Key words: hybrid, adaptation, loss of heterozygosity, experimental evolution, Saccharomyces uvarum, Saccharomyces cerevisiae. Introduction Hybridization is now recognized as a common phenomenon across the tree of life. Historically however, the detection of hybrids has been difficult, and its incidence may be under- reported for both plants and animals, and almost certainly for certain eukaryotes like insects and fungi (Bullini 1994; Albertin and Marullo 2012). Its importance as an evolutionary force has thus been maligned, as hybrids appeared both rare and typically at a reduced fitness. In addition to potential postre- productive barriers, the hybrid is theorized to be ill-adapted to its environment and will also suffer minority cytotype dis- advantage, because other hybrids are uncommon and back- crosses to parental species may be unfit (Mallet 2007). However, hybrids can have a variety of advantages over their parents, including heterozygote advantage, extreme pheno- typic traits, and reproductive isolation (usually resulting from polyploidy), and can thus facilitate adaptation to novel or stressful conditions, invade unoccupied ecological niches, and even increase biodiversity. Some hybridization events lead to new hybrid species (Rieseberg 1997; Nolte et al. 2005; Mavarez et al. 2006; Meyer et al. 2006; Soltis and Soltis 2009; Schumer et al. 2014), whereas most result in introgression from hybrid back- crosses to the more abundant parental species (Dowling et al. 1989; Taylor and Hebert 1993; Wayne 1993; Grant et al. 2005; Dasmahapatra et al. 2012). Hybridization introduces genetic variation into a population at orders of magnitude greater than what mutation alone can achieve, in a sense operating as a “multi-locus macro-mutation” (Grant and Grant 1994; Barton 2001; Mallet 2007; Abbott et al. 2013). Therefore, hy- bridization via introgression, polyploidy, or homoploid hybrid speciation may offer a rapid strategy for adaptation to chang- ing environmental conditions. For example, in Darwin’s finches, adaptive introgression supplied the morphological variation that allowed the species to survive following an El Ni~ no event (Grant and Grant 2010, 2002), and in ancient humans, introgression allowed adaptation to high altitudes (Huerta-Sanchez and Casey 2015), among other traits (Racimo et al. 2015). The most iconic example comes from the hybrid sunflower species Helianthus anomalus, Helianthus deserticola, and Helianthus paradoxus, from the parents Helianthus annuus and Helianthus petiolaris. These three hy- brid species are locally adapted to extreme desert, salt marsh, Article ß The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons. org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Open Access Mol. Biol. Evol. 0(0):1–17 doi:10.1093/molbev/msx098 Advance Access publication March 29, 2017 1

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Page 1: Loss of Heterozygosity Drives Adaptation in Hybrid Yeastdunham.gs.washington.edu/SmukowskiHeil.pdf · 2017-05-20 · Loss of Heterozygosity Drives Adaptation in Hybrid Yeast Caiti

Loss of Heterozygosity Drives Adaptation in Hybrid Yeast

Caiti S Smukowski Heil1 Christopher G DeSevoDagger2 Dave A Paisect2 Cheryl M Tuckerpara2 Margaret LHoangk34 and Maitreya J Dunham1

1Department of Genome Sciences University of Washington Seattle WA2Lewis-Sigler Institute for Integrative Genomics Princeton University Princeton NJ3Department of Embryology Howard Hughes Medical Institute Carnegie Institution Baltimore MD4Department of Biology Johns Hopkins University Baltimore MDDaggerPresent address Exelixis Dallas TXsectPresent address BioNano Genomics San Diego CAparaPresent address Wall High School Wall Township NJkPresent address Nanostring Seattle WA

Corresponding author E-mail maitreyauwedu

Associate editor Jianzhi Zhang

Abstract

Hybridization is often considered maladaptive but sometimes hybrids can invade new ecological niches and adapt tonovel or stressful environments better than their parents The genomic changes that occur following hybridization thatfacilitate genome resolution andor adaptation are not well understood Here we examine hybrid genome evolutionusing experimental evolution of de novo interspecific hybrid yeast Saccharomyces cerevisiae Saccharomyces uvarumand their parentals We evolved these strains in nutrient-limited conditions for hundreds of generations and sequencedthe resulting cultures identifying numerous point mutations copy number changes and loss of heterozygosity (LOH)events including species-biased amplification of nutrient transporters We focused on a particularly interesting examplein which we saw repeated LOH at the high-affinity phosphate transporter gene PHO84 in both intra- and interspecifichybrids Using allele replacement methods we tested the fitness of different alleles in hybrid and S cerevisiae strainbackgrounds and found that the LOH is indeed the result of selection on one allele over the other in both S cerevisiae andthe hybrids This is an example where hybrid genome resolution is driven by positive selection on existing heterozygosityand demonstrates that even infrequent outcrossing may have lasting impacts on adaptation

Key words hybrid adaptation loss of heterozygosity experimental evolution Saccharomyces uvarum Saccharomycescerevisiae

Introduction

Hybridization is now recognized as a common phenomenonacross the tree of life Historically however the detection ofhybrids has been difficult and its incidence may be under-reported for both plants and animals and almost certainly forcertain eukaryotes like insects and fungi (Bullini 1994 Albertinand Marullo 2012) Its importance as an evolutionary forcehas thus been maligned as hybrids appeared both rare andtypically at a reduced fitness In addition to potential postre-productive barriers the hybrid is theorized to be ill-adaptedto its environment and will also suffer minority cytotype dis-advantage because other hybrids are uncommon and back-crosses to parental species may be unfit (Mallet 2007)However hybrids can have a variety of advantages over theirparents including heterozygote advantage extreme pheno-typic traits and reproductive isolation (usually resulting frompolyploidy) and can thus facilitate adaptation to novel orstressful conditions invade unoccupied ecological nichesand even increase biodiversity

Some hybridization events lead to new hybrid species(Rieseberg 1997 Nolte et al 2005 Mavarez et al 2006

Meyer et al 2006 Soltis and Soltis 2009 Schumer et al2014) whereas most result in introgression from hybrid back-crosses to the more abundant parental species (Dowling et al1989 Taylor and Hebert 1993 Wayne 1993 Grant et al 2005Dasmahapatra et al 2012) Hybridization introduces geneticvariation into a population at orders of magnitude greaterthan what mutation alone can achieve in a sense operating asa ldquomulti-locus macro-mutationrdquo (Grant and Grant 1994Barton 2001 Mallet 2007 Abbott et al 2013) Therefore hy-bridization via introgression polyploidy or homoploid hybridspeciation may offer a rapid strategy for adaptation to chang-ing environmental conditions For example in Darwinrsquosfinches adaptive introgression supplied the morphologicalvariation that allowed the species to survive following an ElNi~no event (Grant and Grant 2010 2002) and in ancienthumans introgression allowed adaptation to high altitudes(Huerta-Sanchez and Casey 2015) among other traits(Racimo et al 2015) The most iconic example comes fromthe hybrid sunflower species Helianthus anomalus Helianthusdeserticola and Helianthus paradoxus from the parentsHelianthus annuus and Helianthus petiolaris These three hy-brid species are locally adapted to extreme desert salt marsh

Article

The Author 2017 Published by Oxford University Press on behalf of the Society for Molecular Biology and EvolutionThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (httpcreativecommonsorglicensesby40) which permits unrestricted reuse distribution and reproduction in any medium provided the original work isproperly cited Open AccessMol Biol Evol 0(0)1ndash17 doi101093molbevmsx098 Advance Access publication March 29 2017 1

and dune habitats respectively and show traits such as in-creased drought or salt tolerance relative to their parents(Heiser 1954 Rieseberg 1991 Schwarzbach et al 2001Rosenthal et al 2002)

Agriculture and industry use both intra- and interspecifichybrids as a tool to increase yield or robustness introduceresistance to pests and create novel phenotype or flavorprofiles For example plant breeders have crossed domesti-cated species to wild species to introduce resistance to avariety of pathogens in wheat potato and canola (Masonand Batley 2015) and almost all maize grown in the UnitedStates is grown from intraspecific hybrid seeds which hasincreased yield and provided improved resistance to bioticand abiotic factors (Crow 1998) Vintners and brewers havecreated interspecific hybrids to select for traits such as loweracetic acid concentration (Bellon et al 2015) and many inci-dental fungal hybrids have been discovered in brewing andindustry including Pichia sorbitophila (Louis et al 2012) andvarious hybrids across the Saccharomyces clade (Gonzalezet al 2006 2008 Muller and McCusker 2009 Hittinger2013 Bellon et al 2015) most notably the lager-brewing yeastSaccharomyces pastorianus (Tamai et al 1998 Dunn andSherlock 2008 Walther et al 2014 Baker et al 2015 Gibsonand Liti 2015 Peris et al 2016) It is presumed that the severeselection pressures exerted during industrial processes haveselected for interspecific hybrid genomes that may be moreable to cope with the extreme environments

At the genomic level hybridization induces chromosomelossaneuploidy chromosomal rearrangements gene losschanges in gene expression changes in epigenetic modificationstransposable element mobilization and large-scale loss of het-erozygosity (LOH) in which the allele of one species is lost andthe allele of the other species is retained and may even beduplicated via gene conversion or break-induced replication(Masly et al 2006 Landry et al 2007 Doyle et al 2008Michalak 2009 Ainouche and Jenczewski 2010 Albertin andMarullo 2012 Abbott et al 2013 Soltis 2013 Borneman et al2014 Soltis et al 2014) These extensive changes can result in achimeric stabilized hybrid although the period of time for ge-nome stabilization to occur can range dramatically (Soltis et al2014) It is unknown whether there are structural and functionalbiases in the ways in which genesalleles are lost or modifiedBoth drift and selection influence the resolution of the hybridgenome but their contributions are difficult to untangle

Researchers have long been exploring the genetics of hy-brid traits in the lab particularly in agricultural crops al-though this is often slowed by infertility and reducedviability in many interspecific hybrids (Perez-Prat and vanLookeren Campagne 2002 Hajjar and Hodgkin 2007Ouyang et al 2010) The Saccharomyces genus which in-cludes the budding yeast Saccharomyces cerevisiae lends itselfparticularly well to experimental study Many hybrids of thisgenus have been discovered in brewing industrial and natu-ral environments indeed the genus itself is speculated tohave been founded by the product of an ancient hybridiza-tion event (Hittinger 2013 Marcet-Houben and Gabaldon2015 Barbosa et al 2016 Leducq et al 2016) Viable interspe-cific hybrids can be created de novo in the lab (Marinoni et al

1999 Greig et al 2002) and their ability to grow mitoticallymeans that the catastrophic postzygotic barriers to speciationthat generally doom other obligate sexually reproducing hy-brids can be avoided This experimental system allows us toobserve evolution in real time in the laboratory environmentand the genetic and genomic tools available in this modelgenus facilitate characterization of the connection betweengenotype and phenotype including fitness

Previous work in our lab group has utilized experimentalevolution to investigate adaptive events in haploid andhomozygous diploid S cerevisiae (Gresham et al 2008Payen et al 2014 Sunshine et al 2015) To investigate genomeevolution post hybridization we utilize an interspecific hybridS cerevisiae Saccharomyces uvarum and its parentals ahomozygous diploid S uvarum and an intraspecific hybridS cerevisiae GRF167 S cerevisiae S288C This allows us tounderstand the impact of varying levels of heterozygosity onadaptation and genome evolution ranging from none(S uvarum and previous S cerevisiae experiments) to intra-specific heterozygosity (S cerevisiae GRF167 S cerevisiaeS288C) to the most extreme case of interspecific hybridsSaccharomyces uvarum is one of the most distantly relatedspecies of S cerevisiae in the Saccharomyces clade separatedby 20 My and 20 sequence divergence at coding sites (Kelliset al 2003 Cliften et al 2006) Despite this extensive diver-gence S cerevisiae and S uvarum are largely syntenic andcreate hybrids though less than 1 of spores are viable(Greig 2009) The two species differ in their stress tolerancesfor example S cerevisiae being more thermotolerant S uva-rum being cryotolerant (Almeida et al 2014) Previous evolu-tion experiments using lab-derived hybrids have revealednovel andor transgressive phenotypes for ammonium limi-tation ethanol tolerance and growth on xylose (Belloch et al2008 Wenger et al 2010 Piotrowski et al 2012 Dunn et al2013) Notably Dunn et al (2013) revealed several LOHevents and a repeatable nonreciprocal translocation that pro-duces a gene fusion at the high-affinity ammonium permeaseMEP2 after selection in ammonium limitation offering insightinto potential mutational events in the adaptation andorstabilization of S cerevisiae S uvarum hybrids

Here we evolved these hybrids and diploids in replicate inthree nutrient-limited conditions for hundreds of genera-tions Using whole genome sequencing we found wholechromosome aneuploidy genome rearrangements copynumber variants de novo point mutations and LOH Wesought to determine how initial heterozygosity affects adap-tation to novel conditions and explore whether neutral orselective forces are influencing the resolution of the hybridgenome over time In particular we investigated a reoccur-ring LOH event observed in both intra- and interspecifichybrids and found support for the hypothesis that LOH atthis locus is due to selection

Results

Experimental Evolution of Hybrid and Parental SpeciesAn interspecific hybrid was created by crossing S cerevisiaeand S uvarum (strains in supplementary table S1

Smukowski Heil et al doi101093molbevmsx098 MBE

2

Supplementary Material online) and evolved in continu-ous culture in the chemostat (Monod 1949 Novick andSzilard 1950a 1950b) In parallel homozygous diploidS uvarum and heterozygous diploid S cerevisiae(GRF167 S288C) were also evolved Each strain wasgrown in two or more replicate independent culturesunder three different nutrient limitationsmdashglucose phos-phate and sulfatemdashfor 85ndash557 generations (median 158)at 30 C except for S uvarum which was unable toachieve steady state in all conditions at 30 C and sowas evolved at 25 C The population sizes were largelysimilar across strains species and conditions

Evolved clones were isolated from each population andsubsequently competed individually against the appropriategreen fluorescent protein (GFP)-tagged ancestor to gaugerelative fitness As expected evolved hybrid and parentalclones generally exhibit higher fitness than their unevolvedancestor with typical relative fitness gains between 20 and30 (tables 1 and 2) To explore whether these fitness gainsare general or condition specific we additionally competedeach hybrid clone in the two nutrient-limited conditions inwhich the clone was not evolved Results are variable withsome clones having negative or neutral fitness in the alternateconditions suggesting condition-specific adaptation andsome clones experiencing fitness gains in multiple conditionssuggesting more general growth benefits (table 1) Only oneclone exhibited fitness gains in all three nutrient environ-ments and no clones have a greater fitness gain in an alter-nate condition than the condition it was evolved in signifyingthat clones are largely specifically adapted to the particularcondition in which they were evolved

Mutations Are Recovered in Both Novel andPreviously Observed Gene Targets in InterspecificHybridsTo identify mutations in the evolved hybrids we generatedwhole genome sequencing data for 16 clones from the endpoints of the evolution experiments (table 1) We thus cap-tured data from a range of nutrient limitations (phosphate 6glucose 3 sulfate 7) and generations (100ndash285 median 154)Each clone had an average of 24 point mutations a numberof which have been previously identified in prior S cerevisiaeevolution experiments For example a nonsynonymous mu-tation in the S cerevisiae allele of the glucose-sensing geneSNF3 has been identified in glucose-limited experiments inS cerevisiae (Kvitek and Sherlock 2013 Selmecki et al 2015)To our knowledge 2027 coding point mutations are uniqueto these experiments (Payen et al 2016)

In evolved parentals we again sequenced one clone fromthe end point of each population In total we sequenced 16clones 6 from each of the 3 nutrients (2 S uvarum diploidsand 4 S cerevisiae diploids) except in glucose limitation inwhich only 2 S cerevisiae populations were sampled Thegenerations ranged from 234 to 557 (median 477) in S uva-rum with an average of 283 mutations per clone and from127 to 190 (median 1665) in S cerevisiae with an average of09 point mutations per clone (table 2) This discrepancy in

point mutations between S cerevisiae and S uvarum may beexplained by differences in generation number

With the limited number of samples we have from hybridand parental clones it is difficult to draw any conclusionsregarding unique point mutations in hybrids Furthermorewe have not tested the fitness of these mutations to provethey are adaptive However one class of mutations that maybe of particular interest in hybrids are genomic mutationsthat may interact with the mitochondria as previous workhas shown that nuclearndashmitochondria interactions can un-derlie hybrid incompatibility (Lee et al 2008 Chou and Leu2010 Meiklejohn et al 2013) Other studies have found thatonly the S cerevisiae mitochondria are retained in S cerevisiae S uvarum hybrids (Antunovics et al 2005) and we reca-pitulate these findings potentially setting the stage for con-flicting interactions between the S uvarum nuclear genomeand the foreign mitochondria We observe several mitochon-dria-related mutations in hybrids in both S cerevisiae andS uvarum alleles For example one point mutation a nonsyn-onymous mutation in the S cerevisiae allele of the mitochon-drial ribosomal protein gene MHR1 was seen in two separateclones independently evolved in phosphate limitation Thisgene may be of particular interest as it was discovered in aprevious screen as being haploproficient (increased fitness of19) in hybrids in which the S cerevisiae allele is missing andthe S uvarum allele is retained (Lancaster S Dunham MJunpublished data) suggesting that this mutation may alteror disable the S cerevisiae protein in some way Another ex-ample involves the gene IRC3 a helicase responsible for themaintenance of the mitochondrial genome which has a non-synonymous mutation in the S uvarum allele in clone Gh3and is deleted in clone Gh2 potentially suggesting that theS uvarum allele is deleterious in the hybrid backgroundAlthough our sample size is small 427 point mutations inhybrids are related to mitochondria function compared with026 in parentals and may represent interesting targets forfurther exploration

Copy Number Variants Frequently Involve theAmplification of Nutrient TransportersYeasts in both natural and artificial environments are knownto frequently experience changes in copy number rangingfrom single genes to whole chromosomes (Dunham et al2002 Gresham et al 2008 Dunn et al 2012 Kvitek andSherlock 2013 Payen et al 2014 Selmecki et al 2015Sunshine et al 2015 Zhu et al 2016) This holds true in ourevolution experiments We observe copy number changesacross all genetic backgrounds (fig 1 supplementary figsS1ndashS3 Supplementary Material online) Clones were com-pared with array comparative genomic hybridization of pop-ulations to confirm that clones are representative ofpopulations (see Materials and Methods) The evolved hybridclones displayed an average of 15 copy number variants(CNVs) per clone (fig 1 table 1 Supplementary fig S3Supplementary Material online) as defined by the numberof segmental or whole chromosome amplificationsdeletions(though it is likely that some of these CNVs were created inthe same mutational event) The evolved S cerevisiae clones

Adaptation in Hybrids doi101093molbevmsx098 MBE

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Smukowski Heil et al doi101093molbevmsx098 MBE

4

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17T

RM

3Sy

no

nym

ou

sS2

01S

cer

chrV

342

563

Inte

rgen

icu

vach

rX7

6976

8SP

O77

No

nsy

no

nym

ou

sD

418G

uva

chrX

990

873

LEU

35rsquo

-up

stre

amu

vach

rXII

192

491

Inte

rgen

icu

vach

rXIV

251

38EG

T2

Syn

on

ymo

us

T16

8Tu

vach

rII

7703

118

1318

422

gen

esi

ncl

ud

ing

SUL1

CN

V(a

mp

lifica

tio

n)

cer

chrV

III

321

gen

esC

NV

(am

plifi

cati

on

)u

vaSh

5ch

rIV

310

881

RX

T3

No

nsy

no

nym

ou

sP

87T

uva

263

465

26

494

(S)

679

61

35(G

)3

726

723

(P)

chrV

III

1691

1In

terg

enic

uva

chrI

I78

6040

813

184

11ge

nes

incl

ud

ing

SUL1

CN

V(a

mp

lifica

tio

n)

cer

Sh6

chrV

269

392

Inte

rgen

icce

r27

347

52

63

69(S

)2

606

125

(G)

447

66

04(P

)ch

rXIV

746

688

Inte

rgen

icce

rch

rIV

413

046

Inte

rgen

icu

vach

rII7

7894

28

131

8414

gen

esin

clu

din

gSU

L1C

NV

(am

plifi

cati

on

)ce

rSh

7ch

rII

2388

75In

terg

enic

cer

129

314

46

049

(S)

187

61

99(G

)8

746

922

(P)

chrX

VI

4906

31SV

L3N

on

syn

on

ymo

us

A24

5Vce

rch

rXV

I86

106

YP

L245

WN

on

syn

on

ymo

us

A17

4Dce

rch

rII

2732

96In

terg

enic

uva

chrI

I737

875

813

184

42ge

nes

in

clu

din

gSU

L1C

NV

(am

plifi

cati

on

)ce

r

Poin

tm

uta

tio

ns

cop

yn

um

ber

vari

ants

(CN

Vs)

an

dlo

sso

fhet

ero

zygo

sity

even

ts(L

OH

)ar

ere

cord

edfo

rea

chev

olv

edh

ybri

dcl

on

eC

lon

esar

eid

enti

fied

by

nu

trie

nt

(Gg

luco

selim

itat

ion

Pp

ho

sph

ate

limit

atio

na

nd

Ssu

lfate

limit

atio

n)

anldquoh

rdquod

eno

tes

hyb

rid

an

dth

en

um

ber

ind

icat

esit

sd

eriv

atio

nfr

om

ind

epen

den

tp

op

ula

tio

ns

Gen

esin

un

der

line

hav

eb

een

fou

nd

toh

ave

po

int

mu

tati

on

sin

pri

or

exp

erim

ents

No

teth

atm

uta

tio

ns

inth

eS

uvar

umge

no

me

use

Suv

arum

chro

mo

som

esan

dco

ord

inat

esA

llb

reak

po

ints

wer

eca

lled

by

visu

alin

spec

tio

no

fseq

uen

cin

gre

ads

and

are

thu

sap

pro

xim

ate

Rel

ativ

efi

tnes

sis

rep

ort

edw

ith

stan

dar

der

ror(

SE)a

nd

the

con

dit

ion

the

clo

ne

was

evo

lved

inlis

ted

firs

tfo

llow

edb

yth

etw

oal

tern

ativ

eco

nd

itio

ns

seve

ralc

lon

esar

ere

po

rted

wit

ho

ut

SEd

ue

tote

chn

ical

diffi

cult

ies

wit

hre

plic

ates

Adaptation in Hybrids doi101093molbevmsx098 MBE

5

Tab

le2

Mu

tati

on

san

dFi

tnes

so

fEv

olv

edPa

ren

talC

lon

es

Clo

ne

Loca

tio

nG

ene(

s)M

uta

tio

nSp

ecie

sG

ener

atio

ns

Rel

ativ

eFi

tnes

s6

SE

Gc1

chrX

IV0

561

000

6322

507

8433

329

8ge

nes

79

gen

esC

NV

(am

plifi

cati

on

of

chr

14L

favo

rin

gG

RF1

67

del

etio

no

fch

r14R

)ce

r16

316

42

63

42

chrV

160

000

576

874

220

gen

esLO

H(f

avo

rsG

RF1

67)

Gc2

chrV

431

750

576

874

71ge

nes

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

167

103

66

058

chrX

V7

1000

01

0912

9119

6ge

nes

LOH

CN

V(m

on

oso

my

favo

rin

gS2

88C

)G

u1

chrX

V59

7ge

nes

CN

V(w

ho

lech

rom

oso

me

amp

lifica

tio

n)

uva

468

180

36

212

chrI

I911

925

917

281

HX

T6

7C

NV

(am

plifi

cati

on

)ch

rXV

385

930

NEL

1N

on

syn

on

ymo

us

N12

9Ich

rII9

1190

9In

terg

enic

par

to

fth

eH

XT

67

amp

lifica

tio

nG

u2

chrX

V59

7ge

nes

CN

V(w

ho

lech

rom

oso

me

amp

lifica

tio

n)

uva

486

131

2ch

rII9

1192

59

1728

1H

XT

67

CN

V(a

mp

lifica

tio

n)

chrI

V1

0029

3R

GT

2N

on

syn

on

ymo

us

G10

7Vch

rV4

2093

FRD

1N

on

syn

on

ymo

us

G12

8Ach

rII9

1719

1H

XT

7Sy

no

nym

ou

sH

53H

chrX

I155

787

Inte

rgen

icP

c1ch

rXII

I03

9000

(LO

H)

01

9662

8(C

NV

3co

pie

s)

1966

283

7300

0(C

NV

2co

pie

s)LO

H1

5ge

nes

incl

ud

ing

PH

O84

C

NV

201

gen

esLO

HC

NV

(am

plifi

cati

on

fav

ori

ng

GR

F167

)ce

r15

221

22

60

81

Pc2

chrX

III0

411

00(L

OH

)0

196

628

(CN

V3

cop

ies)

19

6628

373

000

(CN

V2

cop

ies)

LOH

16

gen

esin

clu

din

gP

HO

84

CN

V2

01ge

nes

LOH

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

149

181

36

103

chrV

III5

2034

9In

terg

enic

Pc3

chrX

III0

390

00(L

OH

)0

196

628

(CN

V3

cop

ies)

19

6628

373

000

(CN

V2

cop

ies)

LOH

15

gen

esin

clu

din

gP

HO

84

CN

V2

01ge

nes

LOH

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

127

194

9

Pc4

chrX

III0

855

00(L

OH

)0

196

628

(CN

V3

cop

ies)

19

6628

373

000

(CN

V2

cop

ies)

LOH

40

gen

esin

clu

din

gP

HO

84

CN

V2

01ge

nes

LOH

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

132

209

66

141

chrX

II2

6400

01

0781

7743

7ge

nes

LOH

(fav

ori

ng

S288

C)

chrX

V1

0231

97P

IP2

No

nsy

no

nym

ou

sE6

QP

u1

uva

240

1

686

110

Pu

2ch

rIX

144

80Y

PS6

5rsquo-u

pst

ream

uva

234

213

06

073

chrI

X2

2531

4SE

C6

No

nsy

no

nym

ou

sI1

84L

chrX

III

1295

67T

CB

3N

on

syn

on

ymo

us

E625

GSc

1ch

rXIV

01

0200

0(C

NV

3co

pie

s)6

3200

07

8433

3(C

NV

1co

py)

LO

H1

0000

07

8433

348

gen

es7

9ge

nes

367

gen

esLO

HC

NV

(am

plifi

cati

on

of

chr

14L

del

etio

no

fch

r14R

LO

Hfa

vori

ng

S288

C)

cer

182

380

66

175

chrV

III2

0796

7SM

F2N

on

syn

on

ymo

us

W10

5Sch

rXII

I190

000

196

500

RR

N11

CA

T2

VP

S71

LOH

CN

V(d

elet

ion

fav

ori

ng

GR

F167

)ch

rII7

8718

07

9735

0V

BA

1SU

L1P

CA

1C

NV

(am

plifi

cati

on

)Sc

2ch

rXII

578

gen

esC

NV

(wh

ole

chro

mo

som

eam

plifi

cati

on

fa

vori

ng

GR

F167

)ce

r17

640

21

61

33

chrX

II6

9200

01

0781

7719

3ge

nes

LOH

(fav

ori

ng

GR

F167

)ch

rII7

7322

08

1318

418

gen

esin

clu

din

gSU

L1C

NV

(am

plifi

cati

on

)Sc

3ch

rVI9

4104

FRS2

No

nsy

no

nym

ou

sV

303I

cer

201

413

46

677

chrV

III3

0890

3T

RA

1N

on

syn

on

ymo

us

V20

48A

chrX

IV2

3226

6P

OP

1N

on

syn

on

ymo

us

S477

ch

rXV

291

219

TLG

2N

on

syn

on

ymo

us

D28

6Ych

rXV

309

86H

PF1

Syn

on

ymo

us

T20

7Tch

rII7

8180

07

9223

05

gen

esin

clu

din

gB

SD2

and

SUL1

CN

V(a

mp

lifica

tio

n)

(co

nti

nu

ed)

Smukowski Heil et al doi101093molbevmsx098 MBE

6

Tab

le2

Co

nti

nu

ed

Clo

ne

Loca

tio

nG

ene(

s)M

uta

tio

nSp

ecie

sG

ener

atio

ns

Rel

ativ

eFi

tnes

s6

SE

Sc4

chrI

I275

000

813

184

289

gen

esLO

H(f

avo

rin

gG

RF1

67)

cer

190

312

56

613

chrI

I788

608

795

833

SUL1

PC

A1

CN

V(a

mp

lifica

tio

n)

chrX

I517

650

666

816

68ge

nes

CN

V(a

mp

lifica

tio

n)

chrX

III1

9000

01

9650

0R

RN

11C

AT

2V

PS7

1LO

HC

NV

(del

etio

nf

avo

rin

gG

RF1

67)

chrX

IV6

3200

07

8433

379

gen

esLO

HC

NV

(del

etio

n)

chrX

V3

3670

03

4200

034

2000

109

1291

2ge

nes

384

gen

esLO

H(f

avo

rin

gG

RF1

67f

avo

rin

gS2

88C

)ch

rIX

233

67C

SS1

No

nsy

no

nym

ou

sD

914N

Su1

chrX

177

350

345

680

96ge

nes

incl

ud

ing

SUL2

CN

V(a

mp

lifica

tio

n)

uva

557

218

62

37(S

anch

eze

tal

201

7)ch

rXV

I466

649

DIG

1N

on

syn

on

ymo

us

E49Q

chrV

188

548

Inte

rgen

icSu

2ch

rX1

7735

03

4568

096

gen

esin

clu

din

gSU

L2C

NV

(am

plifi

cati

on

)ch

rIV

803

704

KT

R3

5rsquo-u

pst

ream

chrI

I121

779

PIN

4N

on

syn

on

ymo

us

N26

3Sch

rVII

165

902

MP

T5

No

nsy

no

nym

ou

sQ

618K

chrI

I836

169

RSC

3Sy

no

nym

ou

sR

4Rch

rIV

107

948

UFD

2Sy

no

nym

ou

sG

691G

chrI

II2

8761

8In

terg

enic

Poin

tm

uta

tio

ns

cop

yn

um

ber

vari

ants

(CN

V)

and

loss

ofh

eter

ozy

gosi

tyev

ents

(LO

H)

are

reco

rded

for

each

evo

lved

par

enta

lclo

ne

Clo

nes

are

iden

tifi

edb

yn

utr

ien

t(G

glu

cose

limit

atio

nP

ph

osp

hat

elim

itat

ion

an

dS

sulfa

telim

itat

ion

)b

ysp

ecie

s(ldquo

crdquod

eno

tes

Sce

revi

siae

ldquou

rdquod

eno

tes

Suv

arum

)an

dth

en

um

ber

ind

icat

esit

sd

eriv

atio

nfr

om

ind

epen

den

tp

op

ula

tio

ns

No

teth

atm

uta

tio

ns

inth

eS

uvar

umge

no

me

use

Suv

arum

chro

mo

som

esan

dco

ord

inat

esA

llb

reak

po

ints

wer

eca

lled

by

visu

alin

spec

tio

no

fse

qu

enci

ng

read

san

dar

eth

us

app

roxi

mat

eR

elat

ive

fitn

ess

isre

po

rted

wit

hst

and

ard

erro

r(S

E)s

ever

alcl

on

esar

ere

po

rted

wit

ho

ut

SEd

ue

tote

chn

ical

diffi

cult

ies

wit

hre

plic

ates

Adaptation in Hybrids doi101093molbevmsx098 MBE

7

had an average of 15 CNV per clone and the evolvedS uvarum clones had an average of 1 CNV per clone (table 2supplementary figs S1 and S2 Supplementary Material online)The most common event across nutrient limitations in theinterspecific hybrids was an amplification of the S cerevisiaecopy of chromosome IV which occurred in four

independent hybrid clones (three in phosphate limitationone in glucose limitation supplementary fig S3Supplementary Material online) Several other characteris-tic rearrangements occurred in the evolved S cerevisiaeclones including the amplification of the left arm of chro-mosome 14 accompanied by segmental monosomy of the

FIG 1 Evolved hybrids exhibit changes in copy number and loss of heterozygosity Copy number variants are displayed for selected evolved hybridclones from three nutrient-limited conditions Gh2 glucose Ph4 phosphate and Sh4 sulfate See additional figures in supplementary fig S3Supplementary Material online Hybrid copy number determined by normalized sequencing read depth per open reading frame (ORF) is plottedacross the genome according to S cerevisiae ORF coordinates to account for three reciprocal translocations between S cerevisiae and S uvarumChromosomes are plotted in alternating light and dark purple red indicates a S cerevisiae copy number variant and blue indicates a S uvarumcopy number variant Gh2 has a whole chromosome amplification of S cerevisiae chrIV a small segmental deletion of S uvarum chrIV (non-copyneutral loss of heterozygosity) and an amplification of S uvarum HXT67 Ph4 has a small segmental deletion of S cerevisiae chrIII (non-copyneutral loss of heterozygosity) and an amplification of S cerevisiae chrXIII with corresponding deletion of S uvarum chrXIII (copy neutral loss ofheterozygosity) Sh4 has an amplification of S cerevisiae SUL1 and a whole chromosome amplification of S uvarum chrVIII (note there is areciprocal translocation between chrVIII and chrXV in S uvarum relative to S cerevisiae) Note that Sh4 is plotted on a different scale For specificcoordinates of copy number variants see table 1

Smukowski Heil et al doi101093molbevmsx098 MBE

8

right arm of chromosome 14 an event seen previously inother evolved populations (Dunham et al 2002 Greshamet al 2008 Sunshine et al 2015) All copy number events in

S cerevisiae had break points at repetitive elements knownas Ty elements except those located on chrII which maybe mediated by another mechanism (Brewer et al 2015) In

FIG 2 Repeated loss of heterozygosity at the PHO84 locus in intra- and interspecific hybrids (A) The 25-kb region extending from the left telomereof chromosome XIII to the high-affinity phosphate transporter gene PHO84 (B) Copy number is plotted across part of chromosome XIII in thehybrid ancestor and three evolved hybrid clones in phosphate limitation (clone indicated in upper right corner) Red shows the S cerevisiae alleleblue shows the S uvarum allele and purple shows where both species exhibit the same copy number Note 8 kb of telomere sequence is removeddue to repetitive sequence (C) Alternate allele frequency is plotted for a portion of chromosome XIII in the ancestor and four evolved S cerevisiaeclones in phosphate limitation (clone indicated in upper right corner) All evolved S cerevisiae clones exhibit a loss of heterozygosity at thetelomeric portion of chromosome XIII (loss of S288C amplification of GRF167) as illustrated by an allele frequency of zero compared with theancestor Regions of heterozygosity are interspersed with regions of homozygosity as one of the parents of the diploid was itself the product of across between strains FL100 and S288C and the other parent was S288C Regions of heterozygosity are due to FL100 haplotypes S cerevisiae copynumber for the four evolved clones is shown below the ancestor is diploid across the chromosome (also see table 2 supplementary fig S1Supplementary Material online)

Adaptation in Hybrids doi101093molbevmsx098 MBE

9

contrast copy number variants in the hybrid were rarely facil-itated by repetitive elements perhaps in part because S uva-rum has no full length Ty elements however why S cerevisiaeTy elements and long terminal repeat (LTR) sequences fromeither genome were not utilized remains unknown

Frequently in nutrient-limited evolution experimentscopy number variants involve amplification of the nutrient-specific transporter and indeed we also observed amplifica-tion of these transporters in many of the clones In sulfatelimitation the S cerevisiae allele of the high-affinity sulfatetransporter gene SUL1 is amplified in 77 hybrid clones and 44 S cerevisiae clones (fig 1 tables 1 and 2 supplementary figsS1 and S3 Supplementary Material online) InterestinglySUL2 is the preferred sulfate transporter in S uvarum(Sanchez et al 2017) and was not observed to be amplifiedin the evolved hybrids (supplementary fig S2 and table S2Supplementary Material online) In glucose limitation previ-ous S cerevisiae evolution experiments found frequent am-plification of the high-affinity glucose transporter genesHXT67 (Brown et al 1998 Dunham et al 2002 Greshamet al 2008 Kao and Sherlock 2008 Kvitek and Sherlock2011) In our experiments the S uvarum alleles of theHXT67 transporters are amplified in 33 hybrid clones andboth S uvarum clones but are not amplified in evolved Scerevisiae clones This is suggestive that the S uvarum HXT67alleles confer a greater fitness advantage compared with Scerevisiae alternatively the genomic context could be morepermissive to amplification in S uvarum (fig 1 tables 1 and 2supplementary figs S1ndashS3 Supplementary Material online)Finally in phosphate limitation the S cerevisiae copy of thehigh-affinity phosphate transporter gene PHO84 is amplifiedand the S uvarum allele is lost in 36 hybrid clones in an eventknown as LOH (figs 1 and 2 table 1 supplementary fig 3Supplementary Material online) Intriguingly the evolved Scerevisiae clones also display LOH and accompanied amplifi-cation favoring the allele derived from strain GRF167 over theS288C allele in 44 clones (fig 2 table 2) All hybrid clonescarry the ldquopreferredrdquo GRF167 S cerevisiae allele as this was theallele used to create the de novo hybrid

Loss of Heterozygosity Is a Common Event inHeterozygous Evolving PopulationsSelection on heterozygosity as a LOH event could representis an underappreciated source of adaptation in microbial ex-perimental evolution as typical experiments evolve a haploidor homozygous diploid strain asexually and as a result havelittle variation to select upon LOH is observed in natural andindustrial hybrids (Albertin and Marullo 2012 Louis et al2012 Pryszcz et al 2014 Wolfe 2015 Schroder et al 2016)but here we document its occurrence in both intra- andinterspecific hybrids in the laboratory as a result of short-termevolution (also see (Dunn et al 2013 Burke et al 2014)) LOHis observed across all nutrient conditions with 13 independentLOH events detected in S cerevisiae and 9 independent eventsdocumented in the hybrids (figs 1 and 2 tables 1 and 2 sup-plementary figs S1 and S3 Supplementary Material online) Itthus appears that this type of mutational event is both com-mon and can occur over short evolutionary timescales

The LOH event can result in copy-neutral (where one alleleis lost and the other allele is amplified) or non-neutral chro-mosomal segments (where one allele is lost rendering thestrain hemizygous at that locus) and can favor the retentionof either allele In S cerevisiae there is a bias in resolutionwhere LOH events favor retaining the GRF167 allele over theS288C allele (1013 events Pfrac14 00169 table 2 supplementaryfig S4 Supplementary Material online) One unique case inclone Sc4 has a small approximately 5 kb LOH event onchrXV favoring GRF167 which switches to favoring S288Cfor the rest of the chromosome The retention of S cerevisiaeis slightly more common in the hybrids (59 events Pfrac14 10table 1 supplementary fig S3 Supplementary Material on-line) though not as drastic as the observed genome resolu-tion in the hybrid S pastorianus where LOH favorsS cerevisiae over S eubayanus (Nakao et al 2009) The sizeof the event ranges from approximately 25 kb to the wholechromosome level in the hybrids and from 5 kb to 540 kbin S cerevisiae Where LOH is accompanied by an amplifi-cation event the LOH event always occurs first and doesnot share break points with the amplification event Unlikemany CNV events most LOH events do not appear to bemediated by existing repetitive sequence such as a trans-posable element in the hybrid or S cerevisiae and are mostlikely a product of break-induced replication or mitoticgene conversion with break-induced replication as the fa-vored method as all events extend to the telomere (Hoanget al 2010) The exceptions are in hybrid clones Ph4 Ph5 andSh1 where there is a non-copy neutral loss of S cerevisiaemediated by a Ty element or LTR and S cerevisiae clonesSc1 and Sc4 where there is a 65-kb deletion of the S288Callele flanked by two Ty elements

LOH events in hybrids could signify several ongoing pro-cesses in hybrid genome evolution LOH regions may repre-sent 1) loci with incompatibilities 2) selection on existingvariation or 3) genetic drift eroding genomic segments It isimpossible to definitively rule out any of these hypotheseswithout further experimentation however there are severalarguments disfavoring the incompatibility hypothesis Firstalthough our sample size is modest failing to see repeatedLOH events across nutrient conditions may indicate a lack ofgeneral incompatibility between species (although we cannotpreclude condition-specific incompatibility Hou et al 2015Piatkowska et al 2013) This is consistent with previous stud-ies in yeast which suggest that classic DobzhanksyndashMullerincompatibilities are rare (Liti et al 2006 Greig 2009 Houet al 2014) Furthermore LOH events observed inevolved S cerevisiae suggest that this mutation type isnot unique to interspecific hybrids Instead repeatedevents within a particular condition such as the repeatedLOH at PHO84 in phosphate limitation or the 65 kbsegment on chrXIII in sulfate limitation suggest thatthese events are beneficial and are indeed selection onone allele over the other

LOH Is Driven by Selection on One AlleleTo test the hypothesis that LOH events provide a selectiveadvantage we used allele replacement in which the allele of

Smukowski Heil et al doi101093molbevmsx098 MBE

10

one speciesstrain is replaced with the allele of the otherspeciesstrain in an otherwise isogenic background We testedthis hypothesis using the most commonly seen LOH eventLOH at PHO84 Although the region extends from 25 kb to234 kb in length in the hybrids and from 40 kb to 85 kb in Scerevisiae and thus includes many genes PHO84 was a primecandidate driving this event PHO84 is one of only ten genesencompassed in the region extending from the telomere tothe break point of the shortest LOH event and is included inevery other LOH event on chromosome XIII (fig 2) It is ahigh-affinity phosphate transporter responsible for inorganicphosphate uptake in high and low phosphate conditions(Wykoff and OrsquoShea 2001) and previous work identified apoint mutation in PHO84 (an alanine to valine substitution atthe 50 end of the gene) which increased fitness by 23 inphosphate-limited conditions (Sunshine et al 2015) Finallyprior work with other nutrient transporters has shown am-plification of nutrient transporters to be a key event in adapt-ing to nutrient-limited conditions

We thus selected a region of approximately 25 kb encom-passing the PHO84 ORF its promoter and 30-UTR(Nagalakshmi et al 2008 Yassour et al 2009 Cherry et al2012) We created allele replacement strains using the twoalleles of S cerevisiae in a S cerevisiae diploid background thetwo alleles are 991 identical in this region (supplementaryfig S5 Supplementary Material online) and each strain isidentical to the ancestral strain used in our evolution exper-iments except at the PHO84 locus The S cerevisiae ancestorcarries one copy of GRF167 (preferred) and one copy ofS288C (ldquoun-preferredrdquo) so named due to which allele wasretained and amplified in the evolved clones To measureany resultant changes in fitness we competed each strainindividually against a fluorescent ancestral strain and mea-sured which strain overtook the culture Relative fitness isthus defined as the growth advantage per generation Twocopies of the un-preferred allele decreased relative fitness by531 (6186) whereas two copies of the preferred alleleincreased relative fitness by 993 (6027) This displays anoverall difference in relative fitness of 1524 between the un-preferred and preferred alleles By comparing the fitness ofthese allele replacement strains with the evolved clones (table 2)the allele replacement does not fully recapitulate the fit-ness gain observed in the evolved clone One explanation isthat the additional mutations present in the evolvedstrains also contribute to their total fitness Another expla-nation could be the increased copy number of the PHO84region that we see in these evolved clones To further ex-plore this fitness difference we cloned the GRF167 alleleonto a low copy number plasmid and transformed theallele replacement strain carrying two preferred S cerevi-siae alleles to simulate increased copy number of PHO84and saw only a minimal fitness increase of 176 (6106note all fitness competitions involving plasmids were donein conjunction with empty plasmids to take into accountany fitness effects from the plasmid itself) This supportsthe conclusion that relative fitness gains in the evolvedclone are largely due to the loss of the S288C allele andselection and amplification of the GRF167 allele with little

additional benefit from further amplification It could also bethe case that co-amplification of other genes in the segmentis required to attain the full benefit as previously observedby the contribution of BSD2 to the SUL1 amplicon in sulfatelimitation (Sunshine et al 2015 Payen et al 2016)

To understand the fitness effects of LOH and amplificationin the hybrids we generated hybrid strains with varying num-bers of S cerevisiae GRF167 PHO84 alleles Unfortunately wewere unable to obtain a successful strain carrying the pre-ferred S cerevisiae allele in a S uvarum background howeverwe were able to generate a S uvarum PHO84 knockout strainthus creating a hybrid with one copy of S cerevisiae PHO84When combined with a low copy plasmid carrying the sameallele this strain effectively has two or more copies ofS cerevisiae PHO84 in a hybrid background This hybrid straincan serve as a proxy for the LOH event observed in the evo-lution experiments and has a relative fitness gain of 2557(6288) The ancestral hybrid with the same plasmid (effec-tively 1 S uvarum allele and 2 or more S cerevisiae alleles) hasa relative fitness gain of 1253 (6131) The difference be-tween these two hybrids suggests that while amplification isbeneficial the highest fitness is achieved with the loss of theS uvarum allele (Pfrac14 00061)

Together these results support the conclusion that theS cerevisiae GRF167 allele is preferred over the S288C alleleand that S cerevisiae alleles are preferred over the S uvarumallele in the hybrid and hence that the LOH events seen inboth intra- and interspecific hybrids are the product ofselection

DiscussionIn summary we sought to understand forces underlying ge-nome stabilization and evolution in interspecific and intra-specific hybrids as they adapt to novel environments Weevolved and sequenced clones from 16 hybrid populationsand 16 parental populations to reveal a variety of mutationalevents conferring adaptation to 3 nutrient-limited conditionsOf particular note we find LOH in both evolved intraspecificand interspecific hybrid clones in all nutrient environmentspotentially signifying areas where selection has acted on pre-existing variation present in the ancestral clone We used anallele replacement strategy to test this hypothesis for a com-monly repeated LOH event and show that selection is indeeddriving the homogenization of the genome at this locusAlthough other studies in natural industrial and lab-evolved isolates have observed LOH we present the first em-pirical test of the causal evolutionary forces influencing theseevents This work can begin to help us understand past hy-bridization events and subsequent genome resolution in hy-brids in natural and artificial systems

Similarities and Differences between Intra- andInterspecific HybridsAlthough our sample size is modest we observe several in-teresting trends when comparing S uvarum clones (homo-zygous) S cerevisiae clones (intraspecific hybrid andpreviously published homozygous) and interspecific hybrid

Adaptation in Hybrids doi101093molbevmsx098 MBE

11

clones First theory and previous research predict the inter-specific hybrid may experience more genome instability in theform of chromosomal rearrangements and CNV events(Xiong et al 2011 Lloyd et al 2014 Chester et al 2015Mason and Batley 2015) Instead in our work the interspecifichybrid and S cerevisiae clones experience the same numberof CNV events (both 15 CNVsclone) However the mech-anism of CNV formation seems to differ between the hybridand S cerevisiae clones Whereas S cerevisiae CNV breakpoints typically occur at transposable elements in our studyand previous studies (Dunham et al 2002 Gresham et al2008 Fedoroff 2012) the interspecific hybrid CNVs do notutilize transposable elements although they obviously sharethe same sequence background as the S cerevisiae clones(albeit in one copy) Whether this difference is due to theabsence of full-length transposable elements in S uvarum isunknown but this could potentially explain the lower num-ber of CNV events in S uvarum clones (1 CNVclone) andpresents an intriguing direction for future study

The Predictability of EvolutionWe now have many examples of predictable evolution innatural systems (Losos et al 1998 Rundle et al 2000 Elmerand Meyer 2011 Conte et al 2012 Jones et al 2012 Martinand Orgogozo 2013 Wessinger and Rausher 2014) and inlaboratory experimental evolution in which there often ap-pears to be a limited number of high fitness pathways thatstrains follow when adapting to a particular condition (Fereaet al 1999 Woods et al 2006 Gresham et al 2008 Burke et al2010 Salverda et al 2011 Kawecki et al 2012 Kvitek andSherlock 2013 Lang and Desai 2014) For example it is wellestablished that amplifications of nutrient transporters aredrivers of adaptation in evolution in nutrient-limited condi-tions Previous work in our group has particularly focused onthe amplification of the high-affinity sulfate transporter geneSUL1 in sulfate-limited conditions which occurs in almostevery sulfate-limited evolution experiment and confers a fit-ness advantage of as much as 40 compared with the un-evolved ancestor strain The amplification of phosphatetransporters has been markedly less common and thus driv-ers of adaptation in this condition have been less clearGresham et al (2008) identified a whole chromosome ampli-fication of chrXIII in one population In a follow-up studySunshine et al (2015) found whole or partial amplification ofchrXIII in 38 populations A genome-wide screen for segmen-tal amplifications found a slight increase in fitness for a smalltelomeric segment of chromosome XIII and a A49V pointmutation in PHO84 was observed to increase fitness by 23However screens by Payen et al (2016) showed that althoughPHO84 is recurrently mutated in various experiments itshowed no benefit when amplified or deleted in phos-phate-limited conditions Finally additional evolution exper-iments recapitulated the point mutation seen in Sunshineet al (2015) in 2432 populations and saw amplification ofPHO84 in 832 populations (Miller A Dunham MJ unpub-lished data) It is important to note that all these experimentsused a strain background derived from S288C or CENPKboth of which carry the same (un-preferred) PHO84 allele

In our work we observed amplification of the S cerevisiaeGRF167 allele of PHO84 in 44 S cerevisiae clones from 4populations and 36 hybrid clones from 6 populations Thisamplification was always preceded by the loss of the S288Callele in S cerevisiae clones and the LOH break points arenever shared with the amplification break points There is a15 fitness difference between carrying two copies of theS288C allele of PHO84 compared with carrying two copiesof the GRF167 allele of PHO84 and additional copies of theGRF167 allele do not provide substantial further fitness gainsThe two alleles differ by several noncoding changes and threenonsynonymous substitutions a mutation from glutamicacid to aspartic acid (E229D) leucine to proline (L259P)and leucine to glutamine (L556Q) the latter two of whichare considered ldquononconservativerdquo protein mutations due tochanges in hydrophobicity and structure (supplementary figS5 Supplementary Material online) Intriguingly the L259Pmutation has actually been previously identified as being re-sponsible for resistance to tetrachloroisophthalonitrile andpartial resistance to pentachlorophenol in a QTL study ofsmall-molecule drugs (Perlstein et al 2007) IndeedPerlstein et al found proline at residue 259 to be conservedacross fungal species and even in orthologous human xeno-biotic transporters likely because proline-induced kinks intransmembrane spans have been shown to be essential toprotein function (Cordes et al 2002 Perlstein et al 2007) Itthus appears that amplification of PHO84 has been less pre-dictable as the S288C allele does not confer a fitness advan-tage unless mutated per Sunshine et al (2015) Togetherthese results imply that strain background can constrainadaptive pathways

In hybrids the amplification of the S cerevisiae segmentoccurred in conjunction with the loss of the S uvarum alleleHybrid strains with the LOH had a 2557 relative fitness gainwhereas hybrid strains with amplification of the S cerevisiaePHO84 allele without the LOH had a 1253 fitness gain ThusLOH confers an additional 1304 fitness gain showing thatselection for LOH has a larger impact on fitness than ampli-fication alone Note that S uvarum does have proline at res-idue 259 like the preferred GRF167 allele and differs fromboth S cerevisiae alleles at the other two coding substitutions(229N and 556K) but the amino acid and noncoding diver-gence is too high to speculate what substitutions are respon-sible for the selection of the GRF167 allele (supplementary figS6 Supplementary Material online) Why the loss of one alleleis more beneficial remains unclear as PHO84 is thought tofunction as a monomer (Bun-Ya et al 1991) but it may bedue to competition for cell wall space or negative interactionswith other genes in the PHO pathway (Mouillon and Persson2006)

The infusion of variation created by hybridization providesnew templates for selection to act upon which can be moreimportant than either point mutations or copy number var-iants alone Our work shows that outcrossing need not becommon to have long-lasting effects on adaptation This im-plication is particularly relevant in yeast where outcrossingmay occur quite rarely followed by thousands of asexual gen-erations (Ruderfer et al 2006 Greig and Leu 2009 Liti 2015)

Smukowski Heil et al doi101093molbevmsx098 MBE

12

Applications to Other Hybrids and CancerThe observation that LOH occurs in hybrid genomes is in-creasingly documented (Louis et al 2012 Borneman et al2014 Soltis et al 2014 Marcet-Houben and Gabaldon 2015Pryszcz et al 2014 Schroder et al 2016) although the rea-son(s) for this type of mutation has been unresolved As mostexamples stem from allopolyploid events that occurred mil-lions of years ago understanding why LOH is important inhybrid genome evolution is difficult Cancer cells are alsoknown to experience LOH sometimes involved in the inac-tivation of tumor suppressor genes leaving only one copy ofthe gene that may be mutated or silenced (Thiagalingam et al2001 Tuna et al 2009 Lapunzina and Monk 2011) Datasupport the conclusion that LOH events are selected for dur-ing tumor development as many LOH events involve specificchromosomal segments (Thiagalingam et al 2001) althoughthe underlying molecular and genetic reasons for selection isan open debate (Ryland et al 2015)

Here we experimentally demonstrate that LOH can occurin homoploid interspecific hybrids as well as in intraspecifichybrids These events occur within a few hundred generationsand are common mutations more common on average inthe intraspecific hybrid (13 eventsclone) than the interspe-cific hybrid (056 eventsclone) Interestingly the LOH eventsdo not share break points with the CNV events in S cerevisiaeinstead they appear to occur independently and to precedeany subsequent amplification (amplification occurs following913 LOH events) Competition assays with the PHO84 locusprovide support that LOH itself may be more beneficial thanamplification or at least increase the selective benefit of am-plification events The observation that LOH in intraspecifichybrids occurs independently from copy number change pro-vides different opportunities for adaptation to novelconditions

Other cases of LOH like the copy number neutral eventsobserved in Ph4 Ph5 and Sh4 all of which favor the retentionof the S uvarum allele (supplementary fig S3 SupplementaryMaterial online) may be due to hybrid incompatibility withina particular protein complex other epistatic interactions(Piatkowska et al 2013) or neutral processes We furthermorediscover examples where one species allele appears to bepreferred over the other without LOH such as the repeatedamplification of the S uvarum high-affinity glucose transpor-ters HXT67 When one species allele is amplified and theother is not amplified one explanation is that the local se-quence context can permit or deny amplification In the caseof HXT67 previous experiments in S cerevisiae have shownthat amplification of HXT67 is quite common (Brown et al1998 Dunham et al 2002 Gresham et al 2008 Kao andSherlock 2008 Kvitek and Sherlock 2011) thus suggestingthat when given a choice between this locus in S cerevisiaeor S uvarum in the hybrid the preferred allele is indeed Suvarum though more subtle differences in rate cannot yet beruled out A similar scenario is observed with S cerevisiaeSUL1 (Sanchez et al 2017) Together our results show thatthe heterozygosity supplied by hybridization is an importantcontributor to adaptive routes explored by populations asthey adapt to novel conditions

Although we cannot generalize our results from thePHO84 locus across the many other LOH events discoveredin our hybrids and S cerevisiae in the future we can usesimilar methodology to explore whether positive selectionalways drives LOH or whether other explanations such asincompatibility resolution contribute as well Future experi-ments might also utilize a high throughput method to ex-plore segmental LOH in hybrids at a genome-wide scalesimilar to ongoing experiments at the gene level (LancasterS Dunham MJ unpublished data) Although our sample sizeis moderate this is a novel and necessary step in understand-ing forces underlying hybrid genome stabilization and high-lighting an underappreciated mechanism of hybridadaptation

ConclusionsThe mutation events we observe in our experimentallyevolved hybrids are in many ways quite representative ofmutations observed in ancient hybrid genomes suggestingthat hybrid genome stabilization and adaptation can occurquite rapidly (within several hundred generations)Furthermore our results illustrate that the infusion of varia-tion introduced by hybridization at both the intra- and inter-species level can increase fitness by providing choices of allelesfor selection to act upon even when sexual reproduction israre This may be particularly important for leveraging existingvariation for agricultural and industrial processes and as cli-mate change potentially increases natural hybridization (Kellyet al 2010 Hoffmann and Sgro 2011 Muhlfeld et al 2014)

Materials and Methods

StrainsA list of strains used in this study is included in supplementarytable S1 Supplementary Material online All interspecific hy-brids were created by crossing a ura3 LYS2 haploid parent to aURA3 lys2 haploid parent of the other mating type plating onmedia lacking both uracil and lysine and selecting for proto-trophs The S cerevisiae strain background known asldquoGRF167rdquo is itself a cross between FL100 and the genomictype strain S288C (data not shown) GRF167 was chosen as astrain background for simultaneous work investigating trans-posable elements during experimental evolution which willbe addressed in a future study

Evolution ExperimentsContinuous cultures were established using media and con-ditions previously described (Gresham et al 2008 Sanchezet al 2017) Detailed protocols and media recipes are availableat httpdunhamgswashingtoneduprotocolsshtml (lastaccessed March 6 2017) Samples were taken daily and mea-sured for optical density at 600 nm and cell count micros-copy was performed to check for contamination andarchival glycerol stocks were made daily An experimentwas terminated when contamination growth in tubing orclumping appeared (number of generations at the endpoint for each population are presented in tables 1 and

Adaptation in Hybrids doi101093molbevmsx098 MBE

13

2) Samples from each end point population were colonypurified to yield two clones for further study

Array Comparative Genomic HybridizationPopulations from the end point of each evolution were ana-lyzed for copy number changes using array comparative ge-nomic hybridization following the protocol used in Sanchezet al (2017)

SequencingDNA was extracted from overnight cultures using theHoffmanndashWinston protocol (Hoffman and Winston 1987)and cleaned using the Clean amp Concentrator kit (ZymoResearch) Nextera libraries were prepared following theNextera library kit protocol and sequenced using paired end150 bp reads on the Illumina NextSeq 500 machine (sequenc-ing coverage in supplementary table S2 SupplementaryMaterial online) The reference genomes used were S cerevi-siae v3 (Engel et al 2014) S uvarum (Scannell et al 2011) anda hybrid reference genome created by concatenating the twogenomes Sequence was aligned to the appropriate referencegenome using bwa v062 (Li and Durbin 2009) and mutationswere called using GATK (McKenna et al 2010) and samtools0119 (Li et al 2009) Mutations in evolved clones were filteredin comparison with the ancestor to obtain de novo mutationsAll mutations were first visually inspected using IntegrativeGenomics Viewer (Robinson et al 2011) Subsequently pointmutations in the hybrids were confirmed with Sanger se-quencing (supplementary table S3 Supplementary Materialonline) Copy number variants were visualized usingDNAcopy for S cerevisiae and S uvarum (Seshan andOlshen 2016) LOH events were called based on sequencingcoverage in the hybrids and by identifying homozygous variantcalls in S cerevisiae All break points were called by visual in-spection of sequencing reads and are thus approximate

Fitness AssaysThe pairwise competition experiments were performed in 20ml chemostats (Miller and Dunham 2013) Each competitorstrain was cultured individually until steady state was reachedand then was mixed 5050 with a GFP-tagged ancestor Eachcompetition was conducted in at least two biological repli-cates for approximately 15 generations after mixing Sampleswere collected and analyzed twice daily The proportion ofGFPthorn cells in the population was detected using a BD AccuriC6 flow cytometer (BD Biosciences) The data were plottedwith ln [dark cellsGFPthorn cells] versus generations The rela-tive fitness coefficient was determined from the slope of thelinear region

Strain ConstructionAllele replacements for the PHO84 locus were done followingthe protocol of the Caudy lab with further modifications de-scribed here The native locus was replaced withKluyveromyces lactis URA3 The pho84DURA3 strain wasgrown overnight in 5 ml of C-URA media then inoculatedin a flask of 100 ml yeast extract peptone dextrose (YPD) andgrown to an optical density of 06ndash08 Cells were washed then

aliquoted 275 ml of transformation mix (35 ml 1 M lithiumacetate 240 ml of 50 3500 polyethylene glycol) 10 ml ofsalmon sperm and approximately 3 mg of polymerase chainreaction product were added to the cell pellet It was incu-bated at 37 C (S uvarum) or 42 C (S cerevisiae) for 45 minthen plated to YPD It was replica plated to 5-fluorooroticacid the following day and colonies were tested for the gain ofthe appropriate species allele The GRF167 allele was clonedinto the pIL37 plasmid using Gibson assembly (Gibson et al2009) Correct assembly was verified by Sanger sequencing Allprimers used can be found in supplementary table S3Supplementary Material online

Supplementary MaterialSupplementary data are available at Molecular Biology andEvolution online

AcknowledgmentsWe thank Noah Hanson and Erica Alcantara for technicalassistance We thank Monica Sanchez and three anonymousreviewers for helpful comments on this manuscript JosephSchacherer kindly confirmed the contribution of FL100 to theGRF167 strain background Thanks to Yixian Zheng and DougKoshland for contributing to the initial experimental designcreating yeast strains and purchasing the oligonucleotidesused for the microarrays This work was supported by theNational Science Foundation (grant number 1516330) andthe National Institutes of Health (grant number R01GM094306) MD is a Senior Fellow in the GeneticNetworks program at the Canadian Institute for AdvancedResearch and a Rita Allen Foundation Scholar CSH wassupported in part by National Institutes of Health (grantnumber T32 HG00035) This work was also supported bythe National Institutes of Health (grant number P50GM071508) to the Lewis-Sigler Institute and from theHoward Hughes Medical Institute to Doug Koshland andYixian Zheng Sequencing read data have been deposited atthe NCBI under BioProject accession PRJNA374049Microarray data are deposited in the Gene ExpressionOmnibus (GEO) repository under accession numbersGSE95086 and GSE87401 and in the Princeton MicroarrayDatabase

ReferencesAbbott R Albach D Ansell S Arntzen JW Baird SJ Bierne N Boughman J

Brelsford A Buerkle CA Buggs R et al 2013 Hybridization andspeciation J Evol Biol 26229ndash246

Ainouche ML Jenczewski E 2010 Focus on polyploidy New Phytol1861ndash4

Albertin W Marullo P 2012 Polyploidy in fungi evolution after whole-genome duplication Proc Biol Sci 2792497ndash2509

Almeida P Goncalves C Teixeira S Libkind D Bontrager M Masneuf-Pomarede I Albertin W Durrens P Sherman DJ Marullo P et al2014 A Gondwanan imprint on global diversity and domesticationof wine and cider yeast Saccharomyces uvarum Nat Commun54044

Antunovics Z Nguyen HV Gaillardin C Sipiczki M 2005 Gradual ge-nome stabilisation by progressive reduction of the Saccharomyces

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14

uvarum genome in an interspecific hybrid with Saccharomyces cer-evisiae FEMS Yeast Res 51141ndash1150

Baker E Wang B Bellora N Peris D Hulfachor AB Koshalek JA AdamsM Libkind D Hittinger CT 2015 The Genome sequence ofSaccharomyces eubayanus and the domestication of lager-brewingyeasts Mol Biol Evol 322818ndash2831

Barbosa R Almeida P Safar SVB Santos RO Morais PB Nielly-Thibault LLeducq JB Landry CR Goncalves P Rosa CA Sampaio JP 2016Evidence of natural hybridization in Brazilian wild lineages ofSaccharomyces cerevisiae Genome Biol Evol 8317ndash329

Barton NH 2001 The role of hybridization in evolution Mol Ecol10551ndash568

Belloch C Orlic S Barrio E Querol A 2008 Fermentative stress adapta-tion of hybrids within the Saccharomyces sensu stricto complex Int JFood Microbiol 122188ndash195

Bellon JR Yang F Day MP Inglis DL Chambers PJ 2015 Designingand creating Saccharomyces interspecific hybrids for improvedindustry relevant phenotypes Appl Microbiol Biotechnol998597ndash8609

Borneman AR Zeppel R Chambers PJ Curtin CD 2014 Insights into theDekkera bruxellensis genomic landscape comparative genomics re-veals variations in ploidy and nutrient utilisation potential amongstwine isolates PLoS Genet 10e1004161

Brewer BJ Payen C Di Rienzi SC Higgins MM Ong G Dunham MJRaghuraman MK 2015 Origin-dependent inverted-repeat amplifi-cation tests of a model for inverted DNA amplification PLoS Genet11e1005699

Brown CJ Todd KM Rosenzweig RF 1998 Multiple duplications of yeasthexose transport genes in response to selection in a glucose-limitedenvironment Mol Biol Evol 15931ndash942

Bullini L 1994 Origin and evolution of animal hybrid species Trends EcolEvol 9422ndash426

Bun-Ya M Nishimura M Harashima S Oshima Y 1991 The PHO84 geneof Saccharomyces cerevisiae encodes an inorganic phosphate trans-porter Mol Cell Biol 113229ndash3238

Burke MK Dunham JP Shahrestani P Thornton KR Rose MR Long AD2010 Genome-wide analysis of a long-term evolution experimentwith Drosophila Nature 467587ndash590

Burke MK Liti G Long AD 2014 Standing genetic variation drives re-peatable experimental evolution in outcrossing populations ofSaccharomyces cerevisiae Mol Biol Evol 313228ndash3239

Cherry JM Hong EL Amundsen C Balakrishnan R Binkley G Chan ETChristie KR Costanzo MC Dwight SS Engel SR et al 2012Saccharomyces genome database the genomics resource of buddingyeast Nucleic Acids Res 40D700ndashD705

Chester M Riley RK Soltis PS Soltis DE 2015 Patterns of chromosomalvariation in natural populations of the neoallotetraploid Tragopogonmirus (Asteraceae) Heredity (Edinb) 114309ndash317

Chou JY Leu JY 2010 Speciation through cytonuclear incompatibilityinsights from yeast and implications for higher eukaryotes Bioessays32401ndash411

Cliften PF Fulton RS Wilson RK Johnston M 2006 After the duplicationgene loss and adaptation in Saccharomyces genomes Genetics172863ndash872

Conte GL Arnegard ME Peichel CL Schluter D 2012 The probability ofgenetic parallelism and convergence in natural populations Proc BiolSci 2795039ndash5047

Cordes FS Bright JN Sansom MS 2002 Proline-induced distortions oftransmembrane helices J Mol Biol 323951ndash960

Crow JF 1998 90 years ago the beginning of hybrid maize Genetics148923ndash928

Dasmahapatra KK Walters JR Briscoe AD Davey JW Whibley A NadeauNJ Zimin AV Hughes DST Ferguson LC Martin SH et al 2012Butterfly genome reveals promiscuous exchange of mimicry adap-tations among species Nature 48794ndash98

Dowling TE Smith GR Brown WM 1989 Reproductive isolation andintrogression between Notropis-Cornutus and Notropis-Chrysocephalus (Family Cyprinidae)mdashcomparison of morphologyallozymes and mitochondrial-DNA Evolution 43620ndash634

Doyle JJ Flagel LE Paterson AH Rapp RA Soltis DE Soltis PS Wendel JF2008 Evolutionary genetics of genome merger and doubling inplants Annu Rev Genet 42443ndash461

Dunham MJ Badrane H Ferea T Adams J Brown PO Rosenzweig FBotstein D 2002 Characteristic genome rearrangements in experi-mental evolution of Saccharomyces cerevisiae Proc Natl Acad Sci U SA 9916144ndash16149

Dunn B Paulish T Stanbery A Piotrowski J Koniges G Kroll E LouisEJ Liti G Sherlock G Rosenzweig F 2013 Recurrent rearrange-ment during adaptive evolution in an interspecific yeast hybridsuggests a model for rapid introgression PLoS Genet9e1003366

Dunn B Richter C Kvitek DJ Pugh T Sherlock G 2012 Analysis of theSaccharomyces cerevisiae pan-genome reveals a pool of copy num-ber variants distributed in diverse yeast strains from differing indus-trial environments Genome Res 22908ndash924

Dunn B Sherlock G 2008 Reconstruction of the genome origins andevolution of the hybrid lager yeast Saccharomyces pastorianusGenome Res 181610ndash1623

Elmer KR Meyer A 2011 Adaptation in the age of ecological genomicsinsights from parallelism and convergence Trends Ecol Evol26298ndash306

Engel SR Dietrich FS Fisk DG Binkley G Balakrishnan R Costanzo MCDwight SS Hitz BC Karra K Nash RS et al 2014 The referencegenome sequence of Saccharomyces cerevisiae then and now G3(Bethesda) 4389ndash398

Fedoroff NV 2012 Presidential address Transposable elements epige-netics and genome evolution Science 338758ndash767

Ferea TL Botstein D Brown PO Rosenzweig RF 1999 Systematicchanges in gene expression patterns following adaptive evolutionin yeast Proc Natl Acad Sci U S A 969721ndash9726

Gibson B Liti G 2015 Saccharomyces pastorianus genomic insightsinspiring innovation for industry Yeast 3217ndash27

Gibson DG Young L Chuang RY Venter JC Hutchison CA Smith HO2009 Enzymatic assembly of DNA molecules up to several hundredkilobases Nat Methods 6343ndash5

Gonzalez SS Barrio E Gafner J Querol A 2006 Natural hybrids fromSaccharomyces cerevisiae Saccharomyces bayanus andSaccharomyces kudriavzevii in wine fermentations FEMS Yeast Res61221ndash1234

Gonzalez SS Barrio E Querol A 2008 Molecular characterization of newnatural hybrids of Saccharomyces cerevisiae and S kudriavzevii inbrewing Appl Environ Microbiol 742314ndash2320

Grant PR Grant BR 1994 Phenotypic and genetic consequences ofhybridization in Darwinrsquos finches Evolution 48297ndash316

Grant PR Grant BR 2002 Unpredictable evolution in a 30-year study ofDarwinrsquos finches Science 296707ndash711

Grant PR Grant BR 2010 Natural selection speciation and Darwinrsquosfinches Proc Calif Acad Sci 61245ndash260

Grant PR Grant BR Petren K 2005 Hybridization in the recent past AmNat 16656ndash67

Greig D 2009 Reproductive isolation in Saccharomyces Heredity (Edinb)10239ndash44

Greig D Leu JY 2009 Natural history of budding yeast Current Biol19R886ndashR890

Greig D Louis EJ Borts RH Travisano M 2002 Hybrid speciation inexperimental populations of yeast Science 2981773ndash1775

Gresham D Desai MM Tucker CM Jenq HT Pai DA Ward A DeSevoCG Botstein D Dunham MJ 2008 The repertoire and dynamics ofevolutionary adaptations to controlled nutrient-limited environ-ments in yeast PLoS Genet 4e1000303

Hajjar R Hodgkin T 2007 The use of wild relatives in crop improvementa survey of developments over the last 20 years Euphytica 1561ndash13

Heiser CB 1954 Variation and subspeciation in the common sunflowerHelianthus annuus Am Midl Nat 51287ndash305

Hittinger CT 2013 Saccharomyces diversity and evolution a buddingmodel genus Trends Genet 29309ndash317

Hoang ML Tan FJ Lai DC Celniker SE Hoskins RA Dunham MJ ZhengY Koshland D 2010 Competitive repair by naturally dispersed

Adaptation in Hybrids doi101093molbevmsx098 MBE

15

repetitive DNA during non-allelic homologous recombination PLoSGenet 6e1001228

Hoffmann AA Sgro CM 2011 Climate change and evolutionary adap-tation Nature 470479ndash485

Hoffman CS Winston F 1987 A 10-minute DNA preparation from yeastefficiently releases autonomous plasmids for transformation ofEscherichia coli Gene 57267ndash272

Hou J Friedrich A de Montigny J Schacherer J 2014 Chromosomalrearrangements as a major mechanism in the onset of reproductiveisolation in Saccharomyces cerevisiae Curr Biol 241153ndash1159

Hou J Friedrich A Gounot JS Schacherer J 2015 Comprehensive surveyof condition-specific reproductive isolation reveals genetic incom-patibility in yeast Nat Commun 67214

Huerta-Sanchez E Casey FP 2015 Archaic inheritance supporting high-altitude life in Tibet J Appl Physiol (1985) 1191129ndash1134

Jones FC Grabherr MG Chan YF Russell P Mauceli E Johnson JSwofford R Pirun M Zody MC White S et al 2012 The genomicbasis of adaptive evolution in threespine sticklebacks Nature48455ndash61

Kao KC Sherlock G 2008 Molecular characterization of clonal interfer-ence during adaptive evolution in asexual populations ofSaccharomyces cerevisiae Nat Genet 401499ndash1504

Kawecki TJ Lenski RE Ebert D Hollis B Olivieri I Whitlock MC 2012Experimental evolution Trends Ecol Evol 27547ndash560

Kellis M Patterson N Endrizzi M Birren B Lander ES 2003 Sequencingand comparison of yeast species to identify genes and regulatoryelements Nature 423241ndash254

Kelly B Whiteley A Tallmon D 2010 The Arctic melting pot Nature468891ndash891

Kvitek DJ Sherlock G 2011 Reciprocal sign epistasis between frequentlyexperimentally evolved adaptive mutations causes a rugged fitnesslandscape PLoS Genet 7e1002056

Kvitek DJ Sherlock G 2013 Whole genome whole population sequenc-ing reveals that loss of signaling networks is the major adaptivestrategy in a constant environment PLoS Genet 9e1003972

Landry CR Hartl DL Ranz JM 2007 Genome clashes in hybrids insightsfrom gene expression Heredity (Edinb) 99483ndash493

Lang GI Desai MM 2014 The spectrum of adaptive mutations in ex-perimental evolution Genomics 104412ndash416

Lapunzina P Monk D 2011 The consequences of uniparental disomyand copy number neutral loss-of-heterozygosity during human de-velopment and cancer Biol Cell 103303ndash317

Leducq JB Nielly-Thibault L Charron G Eberlein C Verta JP Samani PSylvester K Hittinger CT Bell G Landry CR 2016 Speciation drivenby hybridization and chromosomal plasticity in a wild yeast NatMicrobiol 115003

Lee HY Chou JY Cheong L Chang NH Yang SY Leu JY 2008Incompatibility of nuclear and mitochondrial genomes causes hy-brid sterility between two yeast species Cell 1351065ndash1073

Li H Durbin R 2009 Fast and accurate short read alignment withBurrows-Wheeler transform Bioinformatics 251754ndash1760

Li H Handsaker B Wysoker A Fennell T Ruan J Homer N Marth GAbecasis G Durbin R 2009 The Sequence AlignmentMap formatand SAMtools Bioinformatics 252078ndash2079

Liti G 2015 The fascinating and secret wild life of the budding yeast Scerevisiae Elife 4e05835

Liti G Barton DB Louis EJ 2006 Sequence diversity reproductiveisolation and species concepts in Saccharomyces Genetics174839ndash850

Lloyd AH Ranoux M Vautrin S Glover N Fourment J Charif D ChouletF Lassalle G Marande W Tran J et al 2014 Meiotic gene evolutioncan you teach a new dog new tricks Mol Biol Evol 311724ndash1727

Losos JB Jackman TR Larson A de Queiroz K Rodriguez-Schettino L1998 Contingency and determinism in replicated adaptive radia-tions of island lizards Science 2792115ndash2118

Louis VL Despons L Friedrich A Martin T Durrens P Casaregola SNeuveglise C Fairhead C Marck C Cruz JA et al 2012 Pichia sorbi-tophila an interspecies yeast hybrid reveals early steps of genomeresolution after polyploidization G3 (Bethesda) 2299ndash311

Mallet J 2007 Hybrid speciation Nature 446279ndash283Marcet-Houben M Gabaldon T 2015 Beyond the whole-genome du-

plication phylogenetic evidence for an ancient interspecies hybrid-ization in the bakerrsquos yeast lineage PLoS Biol 13e1002220

Marinoni G Manuel M Petersen RF Hvidtfeldt J Sulo P Piskur J 1999Horizontal transfer of genetic material among Saccharomyces yeastsJ Bacteriol 1816488ndash6496

Martin A Orgogozo V 2013 The loci of repeated evolution a catalog ofgenetic hotspots of phenotypic variation Evolution 671235ndash1250

Masly JP Jones CD Noor MAF Locke J Orr HA 2006 Gene transpositionas a cause of hybrid sterility in Drosophila Science 3131448ndash1450

Mason AS Batley J 2015 Creating new interspecific hybrid and polyploidcrops Trends Biotechnol 33436ndash441

Mavarez J Salazar CA Bermingham E Salcedo C Jiggins CD Linares M2006 Speciation by hybridization in Heliconius butterflies Nature441868ndash871

McKenna A Hanna M Banks E Sivachenko A Cibulskis K Kernytsky AGarimella K Altshuler D Gabriel S Daly M et al 2010 The genomeanalysis toolkit a MapReduce framework for analyzing next-generation DNA sequencing data Genome Res 201297ndash1303

Meiklejohn CD Holmbeck MA Siddiq MA Abt DN Rand DMMontooth KL 2013 An Incompatibility between a mitochondrialtRNA and its nuclear-encoded tRNA synthetase compromises de-velopment and fitness in Drosophila PLoS Genet 9e1003238

Meyer A Salzburger W Schartl M 2006 Hybrid origin of a swordtailspecies (Teleostei Xiphophorus clemenciae) driven by sexual selec-tion Mol Ecol 15721ndash730

Michalak P 2009 Epigenetic transposon and small RNA determinants ofhybrid dysfunctions Heredity (Edinb) 10245ndash50

Miller AW Dunham MJ 2013 Design and use of multiplexed chemostatarrays J Vis Exp 72e50262

Monod J 1949 The growth of bacterial cultures Annu Rev Microbiol3371ndash394

Mouillon JM Persson BL 2006 New aspects on phosphate sensing andsignalling in Saccharomyces cerevisiae FEMS Yeast Res 6171ndash176

Muhlfeld CC Kovach RP Jones LA Al-Chokhachy R Boyer MC Leary RFLowe WH Luikart G Allendorf FW 2014 Invasive hybridization in athreatened species is accelerated by climate change Nat ClimChange 4620ndash624

Muller LAH McCusker JH 2009 A multispecies-based taxonomic mi-croarray reveals interspecies hybridization and introgression inSaccharomyces cerevisiae FEMS Yeast Res 9143ndash152

Nagalakshmi U Wang Z Waern K Shou C Raha D Gerstein M SnyderM 2008 The transcriptional landscape of the yeast genome definedby RNA sequencing Science 3201344ndash1349

Nakao Y Kanamori T Itoh T Kodama Y Rainieri S Nakamura NShimonaga T Hattori M Ashikari T 2009 Genome sequence ofthe lager brewing yeast an interspecies hybrid DNA Res 16115ndash129

Nolte AW Freyhof J Stemshorn KC Tautz D 2005 An invasive lineage ofsculpins Cottus sp (Pisces Teleostei) in the Rhine with new habitatadaptations has originated from hybridization between old phylo-geographic groups Proc Biol Sci 2722379ndash2387

Novick A Szilard L 1950a Description of the chemostat Science112715ndash716

Novick A Szilard L 1950b Experiments with the Chemostat on spon-taneous mutations of bacteria Proc Natl Acad Sci U S A 36708ndash719

Ouyang YD Liu YG Zhang QF 2010 Hybrid sterility in plant storiesfrom rice Curr Opin Plant Biol 13186ndash192

Payen C Di Rienzi SC Ong GT Pogachar JL Sanchez JC SunshineAB Raghuraman MK Brewer BJ Dunham MJ 2014 The dynam-ics of diverse segmental amplifications in populations ofSaccharomyces cerevisiae adapting to strong selection G3(Bethesda) 4399ndash409

Payen C Sunshine AB Ong GT Pogachar JL Zhao W Dunham MJ 2016High-throughput identification of adaptive mutations in experimen-tally evolved yeast populations PLoS Genet 12e1006339

Perez-Prat E van Lookeren Campagne MM 2002 Hybrid seed produc-tion and the challenge of propagating male-sterile plants TrendsPlant Sci 7199ndash203

Smukowski Heil et al doi101093molbevmsx098 MBE

16

Peris D Langdon QK Moriarty RV Sylvester K Bontrager M Charron GLeducq JB Landry CR Libkind D Hittinger CT 2016 Complex ances-tries of lager-brewing hybrids were shaped by standing variation inthe wild yeast Saccharomyces eubayanus PLoS Genet 12e1006155

Perlstein EO Ruderfer DM Roberts DC Schreiber SL Kruglyak L 2007Genetic basis of individual differences in the response to small-molecule drugs in yeast Nat Genet 39496ndash502

Piatkowska EM Naseeb S Knight D Delneri D 2013 Chimeric proteincomplexes in hybrid species generate novel phenotypes PLoS Genet9e1003836

Piotrowski JS Nagarajan S Kroll E Stanbery A Chiotti KE Kruckeberg ALDunn B Sherlock G Rosenzweig F 2012 Different selective pressureslead to different genomic outcomes as newly-formed hybrid yeastsevolve BMC Evol Biol 1246

Pryszcz LP Nemeth T Gacser A Gabaldon T 2014 Genome comparisonof Candida orthopsilosis clinical strains reveals the existence of hy-brids between two distinct subspecies Genome Biol Evol61069ndash1078

Racimo F Sankararaman S Nielsen R Huerta-Sanchez E 2015 Evidencefor archaic adaptive introgression in humans Nat Rev Genet16359ndash371

Rieseberg LH 1991 Homoploid reticulate evolution in Helianthus(Asteraceae)mdashevidence from ribosomal genes Am J Bot781218ndash1237

Rieseberg LH 1997 Hybrid origins of plant species Annu Rev EcolSystemat 28359ndash389

Robinson JT Thorvaldsdottir H Winckler W Guttman M Lander ESGetz G Mesirov JP 2011 Integrative genomics viewer NatBiotechnol 2924ndash26

Rosenthal DM Schwarzbach AE Donovan LA Raymond O RiesebergLH 2002 Phenotypic differentiation between three ancient hybridtaxa and their parental species Int J Plant Sci 163387ndash398

Ruderfer DM Pratt SC Seidel HS Kruglyak L 2006 Population genomicanalysis of outcrossing and recombination in yeast Nat Genet381077ndash1081

Rundle HD Nagel L Boughman JW Schluter D 2000 Natural selectionand parallel speciation in sympatric sticklebacks Science287306ndash308

Ryland GL Doyle MA Goode D Boyle SE Choong DY Rowley SM Li JBowtell DD Tothill RW Campbell IG et al 2015 Australian OvarianCancer Study Group Loss of heterozygosity what is it good for BMCMed Genomics 845

Salverda MLM Dellus E Gorter FA Debets AJM van der Oost J HoekstraRF Tawfik DS de Visser JAGM 2011 Initial mutations direct alter-native pathways of protein evolution PLoS Genet 7e1001321

Sanchez MR Miller AW Liachko I Sunshine AB Lynch B Huang MAlcantara E DeSevo CG Pai DA Tucker CM et al 2017 Differentialparalog divergence modulates genome evolution across yeast spe-cies PLoS Genet 13(2)e1006585

Scannell DR Zill OA Rokas A Payen C Dunham MJ Eisen MB Rine JJohnston M Hittinger CT 2011 The awesome power of yeast evo-lutionary genetics new genome sequences and strain resources forthe Saccharomyces sensu stricto genus G3 (Bethesda) 111ndash25

Schroder MS Martinez de San Vicente K Prandini TH Hammel SHiggins DG Bagagli E Wolfe KH Butler G 2016 Multiple originsof the pathogenic yeast Candida orthopsilosis by separate hybridiza-tions between two parental species PLoS Genet 12e1006404

Schumer M Rosenthal GG Andolfatto P 2014 How common is homo-ploid hybrid speciation Evolution 681553ndash1560

Schwarzbach AE Donovan LA Rieseberg LH 2001 Transgressivecharacter expression in a hybrid sunflower species Am J Bot88270ndash277

Selmecki AM Maruvka YE Richmond PA Guillet M Shoresh NSorenson AL De S Kishony R Michor F Dowell R et al 2015Polyploidy can drive rapid adaptation in yeast Nature 519349ndash52

Seshan VE Olshen A 2016 DNAcopy DNA copy number data analysisVersion R package version 1460

Soltis DE Visger CJ Soltis PS 2014 The polyploidy revolution then andnow Stebbins revisited Am J Bot 1011057ndash1078

Soltis PS 2013 Hybridization speciation and novelty J Evol Biol26291ndash293

Soltis PS Soltis DE 2009 The role of hybridization in plant speciationAnnu Rev Plant Biol 60561ndash588

Sunshine AB Payen C Ong GT Liachko I Tan KM Dunham MJ 2015The fitness consequences of aneuploidy are driven by condition-dependent gene effects PLoS Biol 13e1002155

Tamai Y Momma T Yoshimoto H Kaneko Y 1998 Co-existence of twotypes of chromosome in the bottom fermenting yeastSaccharomyces pastorianus Yeast 14923ndash933

Taylor DJ Hebert PDN 1993 Habitat-dependent hybrid parentage anddifferential introgression between neighboringly sympatric Daphniaspecies Proc Natl Acad Sci U S A 907079ndash7083

Thiagalingam S Laken S Willson JK Markowitz SD Kinzler KWVogelstein B Lengauer C 2001 Mechanisms underlying losses ofheterozygosity in human colorectal cancers Proc Natl Acad Sci US A 982698ndash2702

Tuna M Knuutila S Mills GB 2009 Uniparental disomy in cancer TrendsMol Med 15120ndash128

Walther A Hesselbart A Wendland J 2014 Genome sequence ofSaccharomyces carlsbergensis the worldrsquos first pure culture lageryeast G3 (Bethesda) 4783ndash793

Wayne RK 1993 Molecular evolution of the dog family Trends Genet9218ndash224

Wenger JW Schwartz K Sherlock G 2010 Bulk segregant analysis byhigh-throughput sequencing reveals a novel xylose utilization genefrom Saccharomyces cerevisiae PLoS Genet 6e1000942

Wessinger CA Rausher MD 2014 Predictability and irreversibility ofgenetic changes associated with flower color evolution inPenstemon barbatus Evolution 681058ndash1070

Wolfe KH 2015 Origin of the yeast whole-genome duplication PLoSBiol 13e1002221

Woods R Schneider D Winkworth CL Riley MA Lenski RE 2006 Testsof parallel molecular evolution in a long-term experiment withEscherichia coli Proc Natl Acad Sci U S A 1039107ndash9112

Wykoff DD OrsquoShea EK 2001 Phosphate transport and sensing inSaccharomyces cerevisiae Genetics 1591491ndash1499

Xiong Z Gaeta RT Pires JC 2011 Homoeologous shuffling andchromosome compensation maintain genome balance inresynthesized allopolyploid Brassica napus Proc Natl Acad SciU S A 1087908ndash7913

Yassour M Kapian T Fraser HB Levin JZ Pfiffner J Adiconis X Schroth GLuo SJ Khrebtukova I Gnirke A et al 2009 Ab initio construction ofa eukaryotic transcriptome by massively parallel mRNA sequencingProc Natl Acad Sci U S A 1063264ndash3269

Zhu YO Sherlock G Petrov DA 2016 Whole genome analysis of 132clinical Saccharomyces cerevisiae strains reveals extensive ploidy var-iation G3 (Bethesda) doi101534g3116029397

Adaptation in Hybrids doi101093molbevmsx098 MBE

17

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Page 2: Loss of Heterozygosity Drives Adaptation in Hybrid Yeastdunham.gs.washington.edu/SmukowskiHeil.pdf · 2017-05-20 · Loss of Heterozygosity Drives Adaptation in Hybrid Yeast Caiti

and dune habitats respectively and show traits such as in-creased drought or salt tolerance relative to their parents(Heiser 1954 Rieseberg 1991 Schwarzbach et al 2001Rosenthal et al 2002)

Agriculture and industry use both intra- and interspecifichybrids as a tool to increase yield or robustness introduceresistance to pests and create novel phenotype or flavorprofiles For example plant breeders have crossed domesti-cated species to wild species to introduce resistance to avariety of pathogens in wheat potato and canola (Masonand Batley 2015) and almost all maize grown in the UnitedStates is grown from intraspecific hybrid seeds which hasincreased yield and provided improved resistance to bioticand abiotic factors (Crow 1998) Vintners and brewers havecreated interspecific hybrids to select for traits such as loweracetic acid concentration (Bellon et al 2015) and many inci-dental fungal hybrids have been discovered in brewing andindustry including Pichia sorbitophila (Louis et al 2012) andvarious hybrids across the Saccharomyces clade (Gonzalezet al 2006 2008 Muller and McCusker 2009 Hittinger2013 Bellon et al 2015) most notably the lager-brewing yeastSaccharomyces pastorianus (Tamai et al 1998 Dunn andSherlock 2008 Walther et al 2014 Baker et al 2015 Gibsonand Liti 2015 Peris et al 2016) It is presumed that the severeselection pressures exerted during industrial processes haveselected for interspecific hybrid genomes that may be moreable to cope with the extreme environments

At the genomic level hybridization induces chromosomelossaneuploidy chromosomal rearrangements gene losschanges in gene expression changes in epigenetic modificationstransposable element mobilization and large-scale loss of het-erozygosity (LOH) in which the allele of one species is lost andthe allele of the other species is retained and may even beduplicated via gene conversion or break-induced replication(Masly et al 2006 Landry et al 2007 Doyle et al 2008Michalak 2009 Ainouche and Jenczewski 2010 Albertin andMarullo 2012 Abbott et al 2013 Soltis 2013 Borneman et al2014 Soltis et al 2014) These extensive changes can result in achimeric stabilized hybrid although the period of time for ge-nome stabilization to occur can range dramatically (Soltis et al2014) It is unknown whether there are structural and functionalbiases in the ways in which genesalleles are lost or modifiedBoth drift and selection influence the resolution of the hybridgenome but their contributions are difficult to untangle

Researchers have long been exploring the genetics of hy-brid traits in the lab particularly in agricultural crops al-though this is often slowed by infertility and reducedviability in many interspecific hybrids (Perez-Prat and vanLookeren Campagne 2002 Hajjar and Hodgkin 2007Ouyang et al 2010) The Saccharomyces genus which in-cludes the budding yeast Saccharomyces cerevisiae lends itselfparticularly well to experimental study Many hybrids of thisgenus have been discovered in brewing industrial and natu-ral environments indeed the genus itself is speculated tohave been founded by the product of an ancient hybridiza-tion event (Hittinger 2013 Marcet-Houben and Gabaldon2015 Barbosa et al 2016 Leducq et al 2016) Viable interspe-cific hybrids can be created de novo in the lab (Marinoni et al

1999 Greig et al 2002) and their ability to grow mitoticallymeans that the catastrophic postzygotic barriers to speciationthat generally doom other obligate sexually reproducing hy-brids can be avoided This experimental system allows us toobserve evolution in real time in the laboratory environmentand the genetic and genomic tools available in this modelgenus facilitate characterization of the connection betweengenotype and phenotype including fitness

Previous work in our lab group has utilized experimentalevolution to investigate adaptive events in haploid andhomozygous diploid S cerevisiae (Gresham et al 2008Payen et al 2014 Sunshine et al 2015) To investigate genomeevolution post hybridization we utilize an interspecific hybridS cerevisiae Saccharomyces uvarum and its parentals ahomozygous diploid S uvarum and an intraspecific hybridS cerevisiae GRF167 S cerevisiae S288C This allows us tounderstand the impact of varying levels of heterozygosity onadaptation and genome evolution ranging from none(S uvarum and previous S cerevisiae experiments) to intra-specific heterozygosity (S cerevisiae GRF167 S cerevisiaeS288C) to the most extreme case of interspecific hybridsSaccharomyces uvarum is one of the most distantly relatedspecies of S cerevisiae in the Saccharomyces clade separatedby 20 My and 20 sequence divergence at coding sites (Kelliset al 2003 Cliften et al 2006) Despite this extensive diver-gence S cerevisiae and S uvarum are largely syntenic andcreate hybrids though less than 1 of spores are viable(Greig 2009) The two species differ in their stress tolerancesfor example S cerevisiae being more thermotolerant S uva-rum being cryotolerant (Almeida et al 2014) Previous evolu-tion experiments using lab-derived hybrids have revealednovel andor transgressive phenotypes for ammonium limi-tation ethanol tolerance and growth on xylose (Belloch et al2008 Wenger et al 2010 Piotrowski et al 2012 Dunn et al2013) Notably Dunn et al (2013) revealed several LOHevents and a repeatable nonreciprocal translocation that pro-duces a gene fusion at the high-affinity ammonium permeaseMEP2 after selection in ammonium limitation offering insightinto potential mutational events in the adaptation andorstabilization of S cerevisiae S uvarum hybrids

Here we evolved these hybrids and diploids in replicate inthree nutrient-limited conditions for hundreds of genera-tions Using whole genome sequencing we found wholechromosome aneuploidy genome rearrangements copynumber variants de novo point mutations and LOH Wesought to determine how initial heterozygosity affects adap-tation to novel conditions and explore whether neutral orselective forces are influencing the resolution of the hybridgenome over time In particular we investigated a reoccur-ring LOH event observed in both intra- and interspecifichybrids and found support for the hypothesis that LOH atthis locus is due to selection

Results

Experimental Evolution of Hybrid and Parental SpeciesAn interspecific hybrid was created by crossing S cerevisiaeand S uvarum (strains in supplementary table S1

Smukowski Heil et al doi101093molbevmsx098 MBE

2

Supplementary Material online) and evolved in continu-ous culture in the chemostat (Monod 1949 Novick andSzilard 1950a 1950b) In parallel homozygous diploidS uvarum and heterozygous diploid S cerevisiae(GRF167 S288C) were also evolved Each strain wasgrown in two or more replicate independent culturesunder three different nutrient limitationsmdashglucose phos-phate and sulfatemdashfor 85ndash557 generations (median 158)at 30 C except for S uvarum which was unable toachieve steady state in all conditions at 30 C and sowas evolved at 25 C The population sizes were largelysimilar across strains species and conditions

Evolved clones were isolated from each population andsubsequently competed individually against the appropriategreen fluorescent protein (GFP)-tagged ancestor to gaugerelative fitness As expected evolved hybrid and parentalclones generally exhibit higher fitness than their unevolvedancestor with typical relative fitness gains between 20 and30 (tables 1 and 2) To explore whether these fitness gainsare general or condition specific we additionally competedeach hybrid clone in the two nutrient-limited conditions inwhich the clone was not evolved Results are variable withsome clones having negative or neutral fitness in the alternateconditions suggesting condition-specific adaptation andsome clones experiencing fitness gains in multiple conditionssuggesting more general growth benefits (table 1) Only oneclone exhibited fitness gains in all three nutrient environ-ments and no clones have a greater fitness gain in an alter-nate condition than the condition it was evolved in signifyingthat clones are largely specifically adapted to the particularcondition in which they were evolved

Mutations Are Recovered in Both Novel andPreviously Observed Gene Targets in InterspecificHybridsTo identify mutations in the evolved hybrids we generatedwhole genome sequencing data for 16 clones from the endpoints of the evolution experiments (table 1) We thus cap-tured data from a range of nutrient limitations (phosphate 6glucose 3 sulfate 7) and generations (100ndash285 median 154)Each clone had an average of 24 point mutations a numberof which have been previously identified in prior S cerevisiaeevolution experiments For example a nonsynonymous mu-tation in the S cerevisiae allele of the glucose-sensing geneSNF3 has been identified in glucose-limited experiments inS cerevisiae (Kvitek and Sherlock 2013 Selmecki et al 2015)To our knowledge 2027 coding point mutations are uniqueto these experiments (Payen et al 2016)

In evolved parentals we again sequenced one clone fromthe end point of each population In total we sequenced 16clones 6 from each of the 3 nutrients (2 S uvarum diploidsand 4 S cerevisiae diploids) except in glucose limitation inwhich only 2 S cerevisiae populations were sampled Thegenerations ranged from 234 to 557 (median 477) in S uva-rum with an average of 283 mutations per clone and from127 to 190 (median 1665) in S cerevisiae with an average of09 point mutations per clone (table 2) This discrepancy in

point mutations between S cerevisiae and S uvarum may beexplained by differences in generation number

With the limited number of samples we have from hybridand parental clones it is difficult to draw any conclusionsregarding unique point mutations in hybrids Furthermorewe have not tested the fitness of these mutations to provethey are adaptive However one class of mutations that maybe of particular interest in hybrids are genomic mutationsthat may interact with the mitochondria as previous workhas shown that nuclearndashmitochondria interactions can un-derlie hybrid incompatibility (Lee et al 2008 Chou and Leu2010 Meiklejohn et al 2013) Other studies have found thatonly the S cerevisiae mitochondria are retained in S cerevisiae S uvarum hybrids (Antunovics et al 2005) and we reca-pitulate these findings potentially setting the stage for con-flicting interactions between the S uvarum nuclear genomeand the foreign mitochondria We observe several mitochon-dria-related mutations in hybrids in both S cerevisiae andS uvarum alleles For example one point mutation a nonsyn-onymous mutation in the S cerevisiae allele of the mitochon-drial ribosomal protein gene MHR1 was seen in two separateclones independently evolved in phosphate limitation Thisgene may be of particular interest as it was discovered in aprevious screen as being haploproficient (increased fitness of19) in hybrids in which the S cerevisiae allele is missing andthe S uvarum allele is retained (Lancaster S Dunham MJunpublished data) suggesting that this mutation may alteror disable the S cerevisiae protein in some way Another ex-ample involves the gene IRC3 a helicase responsible for themaintenance of the mitochondrial genome which has a non-synonymous mutation in the S uvarum allele in clone Gh3and is deleted in clone Gh2 potentially suggesting that theS uvarum allele is deleterious in the hybrid backgroundAlthough our sample size is small 427 point mutations inhybrids are related to mitochondria function compared with026 in parentals and may represent interesting targets forfurther exploration

Copy Number Variants Frequently Involve theAmplification of Nutrient TransportersYeasts in both natural and artificial environments are knownto frequently experience changes in copy number rangingfrom single genes to whole chromosomes (Dunham et al2002 Gresham et al 2008 Dunn et al 2012 Kvitek andSherlock 2013 Payen et al 2014 Selmecki et al 2015Sunshine et al 2015 Zhu et al 2016) This holds true in ourevolution experiments We observe copy number changesacross all genetic backgrounds (fig 1 supplementary figsS1ndashS3 Supplementary Material online) Clones were com-pared with array comparative genomic hybridization of pop-ulations to confirm that clones are representative ofpopulations (see Materials and Methods) The evolved hybridclones displayed an average of 15 copy number variants(CNVs) per clone (fig 1 table 1 Supplementary fig S3Supplementary Material online) as defined by the numberof segmental or whole chromosome amplificationsdeletions(though it is likely that some of these CNVs were created inthe same mutational event) The evolved S cerevisiae clones

Adaptation in Hybrids doi101093molbevmsx098 MBE

3

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Mu

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356

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1866

917

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17

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18(G

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48

60

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GLK

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252T

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V8

8480

19

1211

913

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9121

439

1747

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Gh

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rII

8894

21IR

C3

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333I

uva

124

186

56

047

(G)

176

86

367

(S)

104

6(P

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rII

9124

169

1777

8H

XT

67

CN

V(a

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lifica

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rV2

6939

2In

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enic

cer

103

291

86

137

(P)

168

60

78(G

)0

086

043

(S)

chrX

IV7

4668

8In

terg

enic

cer

chrI

V1

0558

64M

HR

1N

on

syn

on

ymo

us

T21

8Rce

rch

rIX

241

gen

esLO

HC

NV

uva

lost

cer

amp

Ph

2ch

rV4

3277

8G

LC7

Intr

on

cer

124

253

46

024

(P)

151

26

466

(G)

245

61

16(S

)ch

rVII

952

4P

DR

11N

on

syn

on

ymo

us

L383

u

vach

rXV

I23

2879

MR

PL4

0N

on

syn

on

ymo

us

V14

9Eu

vach

rXII

I19

4496

YM

L037

CN

on

syn

on

ymo

us

P30

6Su

vach

rIV

244

399

YD

L114

WN

on

syn

on

ymo

us

G11

9Cu

vach

rIV

836

gen

esC

NV

(am

plifi

cati

on

)ce

rP

h3

chrI

V1

0558

64M

HR

1N

on

syn

on

ymo

us

T21

8Rce

r16

730

03

64

31(P

)21

39

66

25(G

)N

A(S

)ch

rIX

308

303

3084

YIL

166C

CN

V(a

mp

lifica

tio

n)

cer

chrX

III

02

4562

10ge

nes

incl

ud

ing

PH

O84

LOH

CN

Vu

valo

stc

eram

pch

rIV

836

gen

esC

NV

(am

plifi

cati

on

)ce

rP

h4

chrV

II5

5588

5R

PL2

6BIn

tro

nce

r13

127

02

63

62(P

)0

686

410

(G)

204

66

860

(S)

chrX

246

208

PH

S1N

on

syn

on

ymo

us

K20

6Nce

rch

rXII

I32

4121

EIS1

No

nsy

no

nym

ou

sE3

49

uva

chrI

II0

826

8749

gen

esLO

HC

NV

cer

lost

chrX

III0

221

753

112

gen

esi

ncl

ud

ing

PH

O84

LOH

CN

Vu

valo

stc

eram

pP

h5

chrX

III

2317

31P

PZ

1N

on

syn

on

ymo

us

A63

Su

va12

230

24

68

32(P

)

820

60

34(G

)18

20

62

91(S

)ch

rXII

I0

234

112

120

gen

esi

ncl

ud

ing

PH

O84

LOH

CN

Vu

valo

stc

eram

pch

rIX

370

117

439

888

45ge

nes

LOH

CN

Vce

rlo

stP

h6

chrV

II9

7281

3P

FK1

No

nsy

no

nym

ou

sG

308S

cer

111

255

26

332

(P)

522

62

81(G

)N

A(S

)ch

rIV

836

gen

esC

NV

(am

plifi

cati

on

)ce

rSh

1ch

rII5

1136

26

4497

469

6397

81

3184

74ge

nes

63

gen

esin

clu

din

gSU

L1LO

HC

NV

cer

lost

cer

amp

126

338

66

460

(S)

381

61

17(G

)

154

86

855

(P)

chIV

680

386

866

667

8666

67

9837

7410

4ge

nes

63

gen

esLO

HC

NV

uva

amp

uva

lost

chrX

VI

8470

00

9480

6649

gen

esLO

HC

NV

cer

lost

Sh2

chrV

II9

3638

4M

RP

L9N

on

syn

on

ymo

us

D16

7Gce

r26

819

64

64

30(S

)

619

61

21(G

)

143

66

37(P

)ch

rXV

I57

2308

ICL2

No

nsy

no

nym

ou

sM

247I

uva

chrV

III

1166

61ER

G11

No

nsy

no

nym

ou

sS2

86C

uva

chrI

I787

389

813

184

11ge

nes

incl

ud

ing

SUL1

CN

V(a

mp

lifica

tio

n)

cer

Sh3

chrV

I16

2998

GC

N20

No

nsy

no

nym

ou

sD

171Y

cer

132

218

46

153

(S)

607

61

11(G

)5

556

481

(P)

chrX

IV4

9589

0FK

H2

Syn

on

ymo

us

S418

Su

vach

rII7

8658

48

131

8411

gen

esin

clu

din

gSU

L1C

NV

(am

plifi

cati

on

)ce

r

(co

nti

nu

ed)

Smukowski Heil et al doi101093molbevmsx098 MBE

4

Tab

le1

Co

nti

nu

ed

Clo

ne

Loca

tio

nG

ene(

s)M

uta

tio

nSp

ecie

sG

ener

atio

ns

Rel

ativ

eFi

tnes

s6

SE(c

on

dit

ion

)

Sh4

chrX

IV6

6667

5A

RE2

No

nsy

no

nym

ou

sI4

46T

cer

285

271

96

433

(S)

617

60

51(G

)

205

56

330

(P)

chrX

V8

0083

2A

PC

55rsquo

-up

stre

amce

rch

rIV

259

17T

RM

3Sy

no

nym

ou

sS2

01S

cer

chrV

342

563

Inte

rgen

icu

vach

rX7

6976

8SP

O77

No

nsy

no

nym

ou

sD

418G

uva

chrX

990

873

LEU

35rsquo

-up

stre

amu

vach

rXII

192

491

Inte

rgen

icu

vach

rXIV

251

38EG

T2

Syn

on

ymo

us

T16

8Tu

vach

rII

7703

118

1318

422

gen

esi

ncl

ud

ing

SUL1

CN

V(a

mp

lifica

tio

n)

cer

chrV

III

321

gen

esC

NV

(am

plifi

cati

on

)u

vaSh

5ch

rIV

310

881

RX

T3

No

nsy

no

nym

ou

sP

87T

uva

263

465

26

494

(S)

679

61

35(G

)3

726

723

(P)

chrV

III

1691

1In

terg

enic

uva

chrI

I78

6040

813

184

11ge

nes

incl

ud

ing

SUL1

CN

V(a

mp

lifica

tio

n)

cer

Sh6

chrV

269

392

Inte

rgen

icce

r27

347

52

63

69(S

)2

606

125

(G)

447

66

04(P

)ch

rXIV

746

688

Inte

rgen

icce

rch

rIV

413

046

Inte

rgen

icu

vach

rII7

7894

28

131

8414

gen

esin

clu

din

gSU

L1C

NV

(am

plifi

cati

on

)ce

rSh

7ch

rII

2388

75In

terg

enic

cer

129

314

46

049

(S)

187

61

99(G

)8

746

922

(P)

chrX

VI

4906

31SV

L3N

on

syn

on

ymo

us

A24

5Vce

rch

rXV

I86

106

YP

L245

WN

on

syn

on

ymo

us

A17

4Dce

rch

rII

2732

96In

terg

enic

uva

chrI

I737

875

813

184

42ge

nes

in

clu

din

gSU

L1C

NV

(am

plifi

cati

on

)ce

r

Poin

tm

uta

tio

ns

cop

yn

um

ber

vari

ants

(CN

Vs)

an

dlo

sso

fhet

ero

zygo

sity

even

ts(L

OH

)ar

ere

cord

edfo

rea

chev

olv

edh

ybri

dcl

on

eC

lon

esar

eid

enti

fied

by

nu

trie

nt

(Gg

luco

selim

itat

ion

Pp

ho

sph

ate

limit

atio

na

nd

Ssu

lfate

limit

atio

n)

anldquoh

rdquod

eno

tes

hyb

rid

an

dth

en

um

ber

ind

icat

esit

sd

eriv

atio

nfr

om

ind

epen

den

tp

op

ula

tio

ns

Gen

esin

un

der

line

hav

eb

een

fou

nd

toh

ave

po

int

mu

tati

on

sin

pri

or

exp

erim

ents

No

teth

atm

uta

tio

ns

inth

eS

uvar

umge

no

me

use

Suv

arum

chro

mo

som

esan

dco

ord

inat

esA

llb

reak

po

ints

wer

eca

lled

by

visu

alin

spec

tio

no

fseq

uen

cin

gre

ads

and

are

thu

sap

pro

xim

ate

Rel

ativ

efi

tnes

sis

rep

ort

edw

ith

stan

dar

der

ror(

SE)a

nd

the

con

dit

ion

the

clo

ne

was

evo

lved

inlis

ted

firs

tfo

llow

edb

yth

etw

oal

tern

ativ

eco

nd

itio

ns

seve

ralc

lon

esar

ere

po

rted

wit

ho

ut

SEd

ue

tote

chn

ical

diffi

cult

ies

wit

hre

plic

ates

Adaptation in Hybrids doi101093molbevmsx098 MBE

5

Tab

le2

Mu

tati

on

san

dFi

tnes

so

fEv

olv

edPa

ren

talC

lon

es

Clo

ne

Loca

tio

nG

ene(

s)M

uta

tio

nSp

ecie

sG

ener

atio

ns

Rel

ativ

eFi

tnes

s6

SE

Gc1

chrX

IV0

561

000

6322

507

8433

329

8ge

nes

79

gen

esC

NV

(am

plifi

cati

on

of

chr

14L

favo

rin

gG

RF1

67

del

etio

no

fch

r14R

)ce

r16

316

42

63

42

chrV

160

000

576

874

220

gen

esLO

H(f

avo

rsG

RF1

67)

Gc2

chrV

431

750

576

874

71ge

nes

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

167

103

66

058

chrX

V7

1000

01

0912

9119

6ge

nes

LOH

CN

V(m

on

oso

my

favo

rin

gS2

88C

)G

u1

chrX

V59

7ge

nes

CN

V(w

ho

lech

rom

oso

me

amp

lifica

tio

n)

uva

468

180

36

212

chrI

I911

925

917

281

HX

T6

7C

NV

(am

plifi

cati

on

)ch

rXV

385

930

NEL

1N

on

syn

on

ymo

us

N12

9Ich

rII9

1190

9In

terg

enic

par

to

fth

eH

XT

67

amp

lifica

tio

nG

u2

chrX

V59

7ge

nes

CN

V(w

ho

lech

rom

oso

me

amp

lifica

tio

n)

uva

486

131

2ch

rII9

1192

59

1728

1H

XT

67

CN

V(a

mp

lifica

tio

n)

chrI

V1

0029

3R

GT

2N

on

syn

on

ymo

us

G10

7Vch

rV4

2093

FRD

1N

on

syn

on

ymo

us

G12

8Ach

rII9

1719

1H

XT

7Sy

no

nym

ou

sH

53H

chrX

I155

787

Inte

rgen

icP

c1ch

rXII

I03

9000

(LO

H)

01

9662

8(C

NV

3co

pie

s)

1966

283

7300

0(C

NV

2co

pie

s)LO

H1

5ge

nes

incl

ud

ing

PH

O84

C

NV

201

gen

esLO

HC

NV

(am

plifi

cati

on

fav

ori

ng

GR

F167

)ce

r15

221

22

60

81

Pc2

chrX

III0

411

00(L

OH

)0

196

628

(CN

V3

cop

ies)

19

6628

373

000

(CN

V2

cop

ies)

LOH

16

gen

esin

clu

din

gP

HO

84

CN

V2

01ge

nes

LOH

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

149

181

36

103

chrV

III5

2034

9In

terg

enic

Pc3

chrX

III0

390

00(L

OH

)0

196

628

(CN

V3

cop

ies)

19

6628

373

000

(CN

V2

cop

ies)

LOH

15

gen

esin

clu

din

gP

HO

84

CN

V2

01ge

nes

LOH

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

127

194

9

Pc4

chrX

III0

855

00(L

OH

)0

196

628

(CN

V3

cop

ies)

19

6628

373

000

(CN

V2

cop

ies)

LOH

40

gen

esin

clu

din

gP

HO

84

CN

V2

01ge

nes

LOH

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

132

209

66

141

chrX

II2

6400

01

0781

7743

7ge

nes

LOH

(fav

ori

ng

S288

C)

chrX

V1

0231

97P

IP2

No

nsy

no

nym

ou

sE6

QP

u1

uva

240

1

686

110

Pu

2ch

rIX

144

80Y

PS6

5rsquo-u

pst

ream

uva

234

213

06

073

chrI

X2

2531

4SE

C6

No

nsy

no

nym

ou

sI1

84L

chrX

III

1295

67T

CB

3N

on

syn

on

ymo

us

E625

GSc

1ch

rXIV

01

0200

0(C

NV

3co

pie

s)6

3200

07

8433

3(C

NV

1co

py)

LO

H1

0000

07

8433

348

gen

es7

9ge

nes

367

gen

esLO

HC

NV

(am

plifi

cati

on

of

chr

14L

del

etio

no

fch

r14R

LO

Hfa

vori

ng

S288

C)

cer

182

380

66

175

chrV

III2

0796

7SM

F2N

on

syn

on

ymo

us

W10

5Sch

rXII

I190

000

196

500

RR

N11

CA

T2

VP

S71

LOH

CN

V(d

elet

ion

fav

ori

ng

GR

F167

)ch

rII7

8718

07

9735

0V

BA

1SU

L1P

CA

1C

NV

(am

plifi

cati

on

)Sc

2ch

rXII

578

gen

esC

NV

(wh

ole

chro

mo

som

eam

plifi

cati

on

fa

vori

ng

GR

F167

)ce

r17

640

21

61

33

chrX

II6

9200

01

0781

7719

3ge

nes

LOH

(fav

ori

ng

GR

F167

)ch

rII7

7322

08

1318

418

gen

esin

clu

din

gSU

L1C

NV

(am

plifi

cati

on

)Sc

3ch

rVI9

4104

FRS2

No

nsy

no

nym

ou

sV

303I

cer

201

413

46

677

chrV

III3

0890

3T

RA

1N

on

syn

on

ymo

us

V20

48A

chrX

IV2

3226

6P

OP

1N

on

syn

on

ymo

us

S477

ch

rXV

291

219

TLG

2N

on

syn

on

ymo

us

D28

6Ych

rXV

309

86H

PF1

Syn

on

ymo

us

T20

7Tch

rII7

8180

07

9223

05

gen

esin

clu

din

gB

SD2

and

SUL1

CN

V(a

mp

lifica

tio

n)

(co

nti

nu

ed)

Smukowski Heil et al doi101093molbevmsx098 MBE

6

Tab

le2

Co

nti

nu

ed

Clo

ne

Loca

tio

nG

ene(

s)M

uta

tio

nSp

ecie

sG

ener

atio

ns

Rel

ativ

eFi

tnes

s6

SE

Sc4

chrI

I275

000

813

184

289

gen

esLO

H(f

avo

rin

gG

RF1

67)

cer

190

312

56

613

chrI

I788

608

795

833

SUL1

PC

A1

CN

V(a

mp

lifica

tio

n)

chrX

I517

650

666

816

68ge

nes

CN

V(a

mp

lifica

tio

n)

chrX

III1

9000

01

9650

0R

RN

11C

AT

2V

PS7

1LO

HC

NV

(del

etio

nf

avo

rin

gG

RF1

67)

chrX

IV6

3200

07

8433

379

gen

esLO

HC

NV

(del

etio

n)

chrX

V3

3670

03

4200

034

2000

109

1291

2ge

nes

384

gen

esLO

H(f

avo

rin

gG

RF1

67f

avo

rin

gS2

88C

)ch

rIX

233

67C

SS1

No

nsy

no

nym

ou

sD

914N

Su1

chrX

177

350

345

680

96ge

nes

incl

ud

ing

SUL2

CN

V(a

mp

lifica

tio

n)

uva

557

218

62

37(S

anch

eze

tal

201

7)ch

rXV

I466

649

DIG

1N

on

syn

on

ymo

us

E49Q

chrV

188

548

Inte

rgen

icSu

2ch

rX1

7735

03

4568

096

gen

esin

clu

din

gSU

L2C

NV

(am

plifi

cati

on

)ch

rIV

803

704

KT

R3

5rsquo-u

pst

ream

chrI

I121

779

PIN

4N

on

syn

on

ymo

us

N26

3Sch

rVII

165

902

MP

T5

No

nsy

no

nym

ou

sQ

618K

chrI

I836

169

RSC

3Sy

no

nym

ou

sR

4Rch

rIV

107

948

UFD

2Sy

no

nym

ou

sG

691G

chrI

II2

8761

8In

terg

enic

Poin

tm

uta

tio

ns

cop

yn

um

ber

vari

ants

(CN

V)

and

loss

ofh

eter

ozy

gosi

tyev

ents

(LO

H)

are

reco

rded

for

each

evo

lved

par

enta

lclo

ne

Clo

nes

are

iden

tifi

edb

yn

utr

ien

t(G

glu

cose

limit

atio

nP

ph

osp

hat

elim

itat

ion

an

dS

sulfa

telim

itat

ion

)b

ysp

ecie

s(ldquo

crdquod

eno

tes

Sce

revi

siae

ldquou

rdquod

eno

tes

Suv

arum

)an

dth

en

um

ber

ind

icat

esit

sd

eriv

atio

nfr

om

ind

epen

den

tp

op

ula

tio

ns

No

teth

atm

uta

tio

ns

inth

eS

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Adaptation in Hybrids doi101093molbevmsx098 MBE

7

had an average of 15 CNV per clone and the evolvedS uvarum clones had an average of 1 CNV per clone (table 2supplementary figs S1 and S2 Supplementary Material online)The most common event across nutrient limitations in theinterspecific hybrids was an amplification of the S cerevisiaecopy of chromosome IV which occurred in four

independent hybrid clones (three in phosphate limitationone in glucose limitation supplementary fig S3Supplementary Material online) Several other characteris-tic rearrangements occurred in the evolved S cerevisiaeclones including the amplification of the left arm of chro-mosome 14 accompanied by segmental monosomy of the

FIG 1 Evolved hybrids exhibit changes in copy number and loss of heterozygosity Copy number variants are displayed for selected evolved hybridclones from three nutrient-limited conditions Gh2 glucose Ph4 phosphate and Sh4 sulfate See additional figures in supplementary fig S3Supplementary Material online Hybrid copy number determined by normalized sequencing read depth per open reading frame (ORF) is plottedacross the genome according to S cerevisiae ORF coordinates to account for three reciprocal translocations between S cerevisiae and S uvarumChromosomes are plotted in alternating light and dark purple red indicates a S cerevisiae copy number variant and blue indicates a S uvarumcopy number variant Gh2 has a whole chromosome amplification of S cerevisiae chrIV a small segmental deletion of S uvarum chrIV (non-copyneutral loss of heterozygosity) and an amplification of S uvarum HXT67 Ph4 has a small segmental deletion of S cerevisiae chrIII (non-copyneutral loss of heterozygosity) and an amplification of S cerevisiae chrXIII with corresponding deletion of S uvarum chrXIII (copy neutral loss ofheterozygosity) Sh4 has an amplification of S cerevisiae SUL1 and a whole chromosome amplification of S uvarum chrVIII (note there is areciprocal translocation between chrVIII and chrXV in S uvarum relative to S cerevisiae) Note that Sh4 is plotted on a different scale For specificcoordinates of copy number variants see table 1

Smukowski Heil et al doi101093molbevmsx098 MBE

8

right arm of chromosome 14 an event seen previously inother evolved populations (Dunham et al 2002 Greshamet al 2008 Sunshine et al 2015) All copy number events in

S cerevisiae had break points at repetitive elements knownas Ty elements except those located on chrII which maybe mediated by another mechanism (Brewer et al 2015) In

FIG 2 Repeated loss of heterozygosity at the PHO84 locus in intra- and interspecific hybrids (A) The 25-kb region extending from the left telomereof chromosome XIII to the high-affinity phosphate transporter gene PHO84 (B) Copy number is plotted across part of chromosome XIII in thehybrid ancestor and three evolved hybrid clones in phosphate limitation (clone indicated in upper right corner) Red shows the S cerevisiae alleleblue shows the S uvarum allele and purple shows where both species exhibit the same copy number Note 8 kb of telomere sequence is removeddue to repetitive sequence (C) Alternate allele frequency is plotted for a portion of chromosome XIII in the ancestor and four evolved S cerevisiaeclones in phosphate limitation (clone indicated in upper right corner) All evolved S cerevisiae clones exhibit a loss of heterozygosity at thetelomeric portion of chromosome XIII (loss of S288C amplification of GRF167) as illustrated by an allele frequency of zero compared with theancestor Regions of heterozygosity are interspersed with regions of homozygosity as one of the parents of the diploid was itself the product of across between strains FL100 and S288C and the other parent was S288C Regions of heterozygosity are due to FL100 haplotypes S cerevisiae copynumber for the four evolved clones is shown below the ancestor is diploid across the chromosome (also see table 2 supplementary fig S1Supplementary Material online)

Adaptation in Hybrids doi101093molbevmsx098 MBE

9

contrast copy number variants in the hybrid were rarely facil-itated by repetitive elements perhaps in part because S uva-rum has no full length Ty elements however why S cerevisiaeTy elements and long terminal repeat (LTR) sequences fromeither genome were not utilized remains unknown

Frequently in nutrient-limited evolution experimentscopy number variants involve amplification of the nutrient-specific transporter and indeed we also observed amplifica-tion of these transporters in many of the clones In sulfatelimitation the S cerevisiae allele of the high-affinity sulfatetransporter gene SUL1 is amplified in 77 hybrid clones and 44 S cerevisiae clones (fig 1 tables 1 and 2 supplementary figsS1 and S3 Supplementary Material online) InterestinglySUL2 is the preferred sulfate transporter in S uvarum(Sanchez et al 2017) and was not observed to be amplifiedin the evolved hybrids (supplementary fig S2 and table S2Supplementary Material online) In glucose limitation previ-ous S cerevisiae evolution experiments found frequent am-plification of the high-affinity glucose transporter genesHXT67 (Brown et al 1998 Dunham et al 2002 Greshamet al 2008 Kao and Sherlock 2008 Kvitek and Sherlock2011) In our experiments the S uvarum alleles of theHXT67 transporters are amplified in 33 hybrid clones andboth S uvarum clones but are not amplified in evolved Scerevisiae clones This is suggestive that the S uvarum HXT67alleles confer a greater fitness advantage compared with Scerevisiae alternatively the genomic context could be morepermissive to amplification in S uvarum (fig 1 tables 1 and 2supplementary figs S1ndashS3 Supplementary Material online)Finally in phosphate limitation the S cerevisiae copy of thehigh-affinity phosphate transporter gene PHO84 is amplifiedand the S uvarum allele is lost in 36 hybrid clones in an eventknown as LOH (figs 1 and 2 table 1 supplementary fig 3Supplementary Material online) Intriguingly the evolved Scerevisiae clones also display LOH and accompanied amplifi-cation favoring the allele derived from strain GRF167 over theS288C allele in 44 clones (fig 2 table 2) All hybrid clonescarry the ldquopreferredrdquo GRF167 S cerevisiae allele as this was theallele used to create the de novo hybrid

Loss of Heterozygosity Is a Common Event inHeterozygous Evolving PopulationsSelection on heterozygosity as a LOH event could representis an underappreciated source of adaptation in microbial ex-perimental evolution as typical experiments evolve a haploidor homozygous diploid strain asexually and as a result havelittle variation to select upon LOH is observed in natural andindustrial hybrids (Albertin and Marullo 2012 Louis et al2012 Pryszcz et al 2014 Wolfe 2015 Schroder et al 2016)but here we document its occurrence in both intra- andinterspecific hybrids in the laboratory as a result of short-termevolution (also see (Dunn et al 2013 Burke et al 2014)) LOHis observed across all nutrient conditions with 13 independentLOH events detected in S cerevisiae and 9 independent eventsdocumented in the hybrids (figs 1 and 2 tables 1 and 2 sup-plementary figs S1 and S3 Supplementary Material online) Itthus appears that this type of mutational event is both com-mon and can occur over short evolutionary timescales

The LOH event can result in copy-neutral (where one alleleis lost and the other allele is amplified) or non-neutral chro-mosomal segments (where one allele is lost rendering thestrain hemizygous at that locus) and can favor the retentionof either allele In S cerevisiae there is a bias in resolutionwhere LOH events favor retaining the GRF167 allele over theS288C allele (1013 events Pfrac14 00169 table 2 supplementaryfig S4 Supplementary Material online) One unique case inclone Sc4 has a small approximately 5 kb LOH event onchrXV favoring GRF167 which switches to favoring S288Cfor the rest of the chromosome The retention of S cerevisiaeis slightly more common in the hybrids (59 events Pfrac14 10table 1 supplementary fig S3 Supplementary Material on-line) though not as drastic as the observed genome resolu-tion in the hybrid S pastorianus where LOH favorsS cerevisiae over S eubayanus (Nakao et al 2009) The sizeof the event ranges from approximately 25 kb to the wholechromosome level in the hybrids and from 5 kb to 540 kbin S cerevisiae Where LOH is accompanied by an amplifi-cation event the LOH event always occurs first and doesnot share break points with the amplification event Unlikemany CNV events most LOH events do not appear to bemediated by existing repetitive sequence such as a trans-posable element in the hybrid or S cerevisiae and are mostlikely a product of break-induced replication or mitoticgene conversion with break-induced replication as the fa-vored method as all events extend to the telomere (Hoanget al 2010) The exceptions are in hybrid clones Ph4 Ph5 andSh1 where there is a non-copy neutral loss of S cerevisiaemediated by a Ty element or LTR and S cerevisiae clonesSc1 and Sc4 where there is a 65-kb deletion of the S288Callele flanked by two Ty elements

LOH events in hybrids could signify several ongoing pro-cesses in hybrid genome evolution LOH regions may repre-sent 1) loci with incompatibilities 2) selection on existingvariation or 3) genetic drift eroding genomic segments It isimpossible to definitively rule out any of these hypotheseswithout further experimentation however there are severalarguments disfavoring the incompatibility hypothesis Firstalthough our sample size is modest failing to see repeatedLOH events across nutrient conditions may indicate a lack ofgeneral incompatibility between species (although we cannotpreclude condition-specific incompatibility Hou et al 2015Piatkowska et al 2013) This is consistent with previous stud-ies in yeast which suggest that classic DobzhanksyndashMullerincompatibilities are rare (Liti et al 2006 Greig 2009 Houet al 2014) Furthermore LOH events observed inevolved S cerevisiae suggest that this mutation type isnot unique to interspecific hybrids Instead repeatedevents within a particular condition such as the repeatedLOH at PHO84 in phosphate limitation or the 65 kbsegment on chrXIII in sulfate limitation suggest thatthese events are beneficial and are indeed selection onone allele over the other

LOH Is Driven by Selection on One AlleleTo test the hypothesis that LOH events provide a selectiveadvantage we used allele replacement in which the allele of

Smukowski Heil et al doi101093molbevmsx098 MBE

10

one speciesstrain is replaced with the allele of the otherspeciesstrain in an otherwise isogenic background We testedthis hypothesis using the most commonly seen LOH eventLOH at PHO84 Although the region extends from 25 kb to234 kb in length in the hybrids and from 40 kb to 85 kb in Scerevisiae and thus includes many genes PHO84 was a primecandidate driving this event PHO84 is one of only ten genesencompassed in the region extending from the telomere tothe break point of the shortest LOH event and is included inevery other LOH event on chromosome XIII (fig 2) It is ahigh-affinity phosphate transporter responsible for inorganicphosphate uptake in high and low phosphate conditions(Wykoff and OrsquoShea 2001) and previous work identified apoint mutation in PHO84 (an alanine to valine substitution atthe 50 end of the gene) which increased fitness by 23 inphosphate-limited conditions (Sunshine et al 2015) Finallyprior work with other nutrient transporters has shown am-plification of nutrient transporters to be a key event in adapt-ing to nutrient-limited conditions

We thus selected a region of approximately 25 kb encom-passing the PHO84 ORF its promoter and 30-UTR(Nagalakshmi et al 2008 Yassour et al 2009 Cherry et al2012) We created allele replacement strains using the twoalleles of S cerevisiae in a S cerevisiae diploid background thetwo alleles are 991 identical in this region (supplementaryfig S5 Supplementary Material online) and each strain isidentical to the ancestral strain used in our evolution exper-iments except at the PHO84 locus The S cerevisiae ancestorcarries one copy of GRF167 (preferred) and one copy ofS288C (ldquoun-preferredrdquo) so named due to which allele wasretained and amplified in the evolved clones To measureany resultant changes in fitness we competed each strainindividually against a fluorescent ancestral strain and mea-sured which strain overtook the culture Relative fitness isthus defined as the growth advantage per generation Twocopies of the un-preferred allele decreased relative fitness by531 (6186) whereas two copies of the preferred alleleincreased relative fitness by 993 (6027) This displays anoverall difference in relative fitness of 1524 between the un-preferred and preferred alleles By comparing the fitness ofthese allele replacement strains with the evolved clones (table 2)the allele replacement does not fully recapitulate the fit-ness gain observed in the evolved clone One explanation isthat the additional mutations present in the evolvedstrains also contribute to their total fitness Another expla-nation could be the increased copy number of the PHO84region that we see in these evolved clones To further ex-plore this fitness difference we cloned the GRF167 alleleonto a low copy number plasmid and transformed theallele replacement strain carrying two preferred S cerevi-siae alleles to simulate increased copy number of PHO84and saw only a minimal fitness increase of 176 (6106note all fitness competitions involving plasmids were donein conjunction with empty plasmids to take into accountany fitness effects from the plasmid itself) This supportsthe conclusion that relative fitness gains in the evolvedclone are largely due to the loss of the S288C allele andselection and amplification of the GRF167 allele with little

additional benefit from further amplification It could also bethe case that co-amplification of other genes in the segmentis required to attain the full benefit as previously observedby the contribution of BSD2 to the SUL1 amplicon in sulfatelimitation (Sunshine et al 2015 Payen et al 2016)

To understand the fitness effects of LOH and amplificationin the hybrids we generated hybrid strains with varying num-bers of S cerevisiae GRF167 PHO84 alleles Unfortunately wewere unable to obtain a successful strain carrying the pre-ferred S cerevisiae allele in a S uvarum background howeverwe were able to generate a S uvarum PHO84 knockout strainthus creating a hybrid with one copy of S cerevisiae PHO84When combined with a low copy plasmid carrying the sameallele this strain effectively has two or more copies ofS cerevisiae PHO84 in a hybrid background This hybrid straincan serve as a proxy for the LOH event observed in the evo-lution experiments and has a relative fitness gain of 2557(6288) The ancestral hybrid with the same plasmid (effec-tively 1 S uvarum allele and 2 or more S cerevisiae alleles) hasa relative fitness gain of 1253 (6131) The difference be-tween these two hybrids suggests that while amplification isbeneficial the highest fitness is achieved with the loss of theS uvarum allele (Pfrac14 00061)

Together these results support the conclusion that theS cerevisiae GRF167 allele is preferred over the S288C alleleand that S cerevisiae alleles are preferred over the S uvarumallele in the hybrid and hence that the LOH events seen inboth intra- and interspecific hybrids are the product ofselection

DiscussionIn summary we sought to understand forces underlying ge-nome stabilization and evolution in interspecific and intra-specific hybrids as they adapt to novel environments Weevolved and sequenced clones from 16 hybrid populationsand 16 parental populations to reveal a variety of mutationalevents conferring adaptation to 3 nutrient-limited conditionsOf particular note we find LOH in both evolved intraspecificand interspecific hybrid clones in all nutrient environmentspotentially signifying areas where selection has acted on pre-existing variation present in the ancestral clone We used anallele replacement strategy to test this hypothesis for a com-monly repeated LOH event and show that selection is indeeddriving the homogenization of the genome at this locusAlthough other studies in natural industrial and lab-evolved isolates have observed LOH we present the first em-pirical test of the causal evolutionary forces influencing theseevents This work can begin to help us understand past hy-bridization events and subsequent genome resolution in hy-brids in natural and artificial systems

Similarities and Differences between Intra- andInterspecific HybridsAlthough our sample size is modest we observe several in-teresting trends when comparing S uvarum clones (homo-zygous) S cerevisiae clones (intraspecific hybrid andpreviously published homozygous) and interspecific hybrid

Adaptation in Hybrids doi101093molbevmsx098 MBE

11

clones First theory and previous research predict the inter-specific hybrid may experience more genome instability in theform of chromosomal rearrangements and CNV events(Xiong et al 2011 Lloyd et al 2014 Chester et al 2015Mason and Batley 2015) Instead in our work the interspecifichybrid and S cerevisiae clones experience the same numberof CNV events (both 15 CNVsclone) However the mech-anism of CNV formation seems to differ between the hybridand S cerevisiae clones Whereas S cerevisiae CNV breakpoints typically occur at transposable elements in our studyand previous studies (Dunham et al 2002 Gresham et al2008 Fedoroff 2012) the interspecific hybrid CNVs do notutilize transposable elements although they obviously sharethe same sequence background as the S cerevisiae clones(albeit in one copy) Whether this difference is due to theabsence of full-length transposable elements in S uvarum isunknown but this could potentially explain the lower num-ber of CNV events in S uvarum clones (1 CNVclone) andpresents an intriguing direction for future study

The Predictability of EvolutionWe now have many examples of predictable evolution innatural systems (Losos et al 1998 Rundle et al 2000 Elmerand Meyer 2011 Conte et al 2012 Jones et al 2012 Martinand Orgogozo 2013 Wessinger and Rausher 2014) and inlaboratory experimental evolution in which there often ap-pears to be a limited number of high fitness pathways thatstrains follow when adapting to a particular condition (Fereaet al 1999 Woods et al 2006 Gresham et al 2008 Burke et al2010 Salverda et al 2011 Kawecki et al 2012 Kvitek andSherlock 2013 Lang and Desai 2014) For example it is wellestablished that amplifications of nutrient transporters aredrivers of adaptation in evolution in nutrient-limited condi-tions Previous work in our group has particularly focused onthe amplification of the high-affinity sulfate transporter geneSUL1 in sulfate-limited conditions which occurs in almostevery sulfate-limited evolution experiment and confers a fit-ness advantage of as much as 40 compared with the un-evolved ancestor strain The amplification of phosphatetransporters has been markedly less common and thus driv-ers of adaptation in this condition have been less clearGresham et al (2008) identified a whole chromosome ampli-fication of chrXIII in one population In a follow-up studySunshine et al (2015) found whole or partial amplification ofchrXIII in 38 populations A genome-wide screen for segmen-tal amplifications found a slight increase in fitness for a smalltelomeric segment of chromosome XIII and a A49V pointmutation in PHO84 was observed to increase fitness by 23However screens by Payen et al (2016) showed that althoughPHO84 is recurrently mutated in various experiments itshowed no benefit when amplified or deleted in phos-phate-limited conditions Finally additional evolution exper-iments recapitulated the point mutation seen in Sunshineet al (2015) in 2432 populations and saw amplification ofPHO84 in 832 populations (Miller A Dunham MJ unpub-lished data) It is important to note that all these experimentsused a strain background derived from S288C or CENPKboth of which carry the same (un-preferred) PHO84 allele

In our work we observed amplification of the S cerevisiaeGRF167 allele of PHO84 in 44 S cerevisiae clones from 4populations and 36 hybrid clones from 6 populations Thisamplification was always preceded by the loss of the S288Callele in S cerevisiae clones and the LOH break points arenever shared with the amplification break points There is a15 fitness difference between carrying two copies of theS288C allele of PHO84 compared with carrying two copiesof the GRF167 allele of PHO84 and additional copies of theGRF167 allele do not provide substantial further fitness gainsThe two alleles differ by several noncoding changes and threenonsynonymous substitutions a mutation from glutamicacid to aspartic acid (E229D) leucine to proline (L259P)and leucine to glutamine (L556Q) the latter two of whichare considered ldquononconservativerdquo protein mutations due tochanges in hydrophobicity and structure (supplementary figS5 Supplementary Material online) Intriguingly the L259Pmutation has actually been previously identified as being re-sponsible for resistance to tetrachloroisophthalonitrile andpartial resistance to pentachlorophenol in a QTL study ofsmall-molecule drugs (Perlstein et al 2007) IndeedPerlstein et al found proline at residue 259 to be conservedacross fungal species and even in orthologous human xeno-biotic transporters likely because proline-induced kinks intransmembrane spans have been shown to be essential toprotein function (Cordes et al 2002 Perlstein et al 2007) Itthus appears that amplification of PHO84 has been less pre-dictable as the S288C allele does not confer a fitness advan-tage unless mutated per Sunshine et al (2015) Togetherthese results imply that strain background can constrainadaptive pathways

In hybrids the amplification of the S cerevisiae segmentoccurred in conjunction with the loss of the S uvarum alleleHybrid strains with the LOH had a 2557 relative fitness gainwhereas hybrid strains with amplification of the S cerevisiaePHO84 allele without the LOH had a 1253 fitness gain ThusLOH confers an additional 1304 fitness gain showing thatselection for LOH has a larger impact on fitness than ampli-fication alone Note that S uvarum does have proline at res-idue 259 like the preferred GRF167 allele and differs fromboth S cerevisiae alleles at the other two coding substitutions(229N and 556K) but the amino acid and noncoding diver-gence is too high to speculate what substitutions are respon-sible for the selection of the GRF167 allele (supplementary figS6 Supplementary Material online) Why the loss of one alleleis more beneficial remains unclear as PHO84 is thought tofunction as a monomer (Bun-Ya et al 1991) but it may bedue to competition for cell wall space or negative interactionswith other genes in the PHO pathway (Mouillon and Persson2006)

The infusion of variation created by hybridization providesnew templates for selection to act upon which can be moreimportant than either point mutations or copy number var-iants alone Our work shows that outcrossing need not becommon to have long-lasting effects on adaptation This im-plication is particularly relevant in yeast where outcrossingmay occur quite rarely followed by thousands of asexual gen-erations (Ruderfer et al 2006 Greig and Leu 2009 Liti 2015)

Smukowski Heil et al doi101093molbevmsx098 MBE

12

Applications to Other Hybrids and CancerThe observation that LOH occurs in hybrid genomes is in-creasingly documented (Louis et al 2012 Borneman et al2014 Soltis et al 2014 Marcet-Houben and Gabaldon 2015Pryszcz et al 2014 Schroder et al 2016) although the rea-son(s) for this type of mutation has been unresolved As mostexamples stem from allopolyploid events that occurred mil-lions of years ago understanding why LOH is important inhybrid genome evolution is difficult Cancer cells are alsoknown to experience LOH sometimes involved in the inac-tivation of tumor suppressor genes leaving only one copy ofthe gene that may be mutated or silenced (Thiagalingam et al2001 Tuna et al 2009 Lapunzina and Monk 2011) Datasupport the conclusion that LOH events are selected for dur-ing tumor development as many LOH events involve specificchromosomal segments (Thiagalingam et al 2001) althoughthe underlying molecular and genetic reasons for selection isan open debate (Ryland et al 2015)

Here we experimentally demonstrate that LOH can occurin homoploid interspecific hybrids as well as in intraspecifichybrids These events occur within a few hundred generationsand are common mutations more common on average inthe intraspecific hybrid (13 eventsclone) than the interspe-cific hybrid (056 eventsclone) Interestingly the LOH eventsdo not share break points with the CNV events in S cerevisiaeinstead they appear to occur independently and to precedeany subsequent amplification (amplification occurs following913 LOH events) Competition assays with the PHO84 locusprovide support that LOH itself may be more beneficial thanamplification or at least increase the selective benefit of am-plification events The observation that LOH in intraspecifichybrids occurs independently from copy number change pro-vides different opportunities for adaptation to novelconditions

Other cases of LOH like the copy number neutral eventsobserved in Ph4 Ph5 and Sh4 all of which favor the retentionof the S uvarum allele (supplementary fig S3 SupplementaryMaterial online) may be due to hybrid incompatibility withina particular protein complex other epistatic interactions(Piatkowska et al 2013) or neutral processes We furthermorediscover examples where one species allele appears to bepreferred over the other without LOH such as the repeatedamplification of the S uvarum high-affinity glucose transpor-ters HXT67 When one species allele is amplified and theother is not amplified one explanation is that the local se-quence context can permit or deny amplification In the caseof HXT67 previous experiments in S cerevisiae have shownthat amplification of HXT67 is quite common (Brown et al1998 Dunham et al 2002 Gresham et al 2008 Kao andSherlock 2008 Kvitek and Sherlock 2011) thus suggestingthat when given a choice between this locus in S cerevisiaeor S uvarum in the hybrid the preferred allele is indeed Suvarum though more subtle differences in rate cannot yet beruled out A similar scenario is observed with S cerevisiaeSUL1 (Sanchez et al 2017) Together our results show thatthe heterozygosity supplied by hybridization is an importantcontributor to adaptive routes explored by populations asthey adapt to novel conditions

Although we cannot generalize our results from thePHO84 locus across the many other LOH events discoveredin our hybrids and S cerevisiae in the future we can usesimilar methodology to explore whether positive selectionalways drives LOH or whether other explanations such asincompatibility resolution contribute as well Future experi-ments might also utilize a high throughput method to ex-plore segmental LOH in hybrids at a genome-wide scalesimilar to ongoing experiments at the gene level (LancasterS Dunham MJ unpublished data) Although our sample sizeis moderate this is a novel and necessary step in understand-ing forces underlying hybrid genome stabilization and high-lighting an underappreciated mechanism of hybridadaptation

ConclusionsThe mutation events we observe in our experimentallyevolved hybrids are in many ways quite representative ofmutations observed in ancient hybrid genomes suggestingthat hybrid genome stabilization and adaptation can occurquite rapidly (within several hundred generations)Furthermore our results illustrate that the infusion of varia-tion introduced by hybridization at both the intra- and inter-species level can increase fitness by providing choices of allelesfor selection to act upon even when sexual reproduction israre This may be particularly important for leveraging existingvariation for agricultural and industrial processes and as cli-mate change potentially increases natural hybridization (Kellyet al 2010 Hoffmann and Sgro 2011 Muhlfeld et al 2014)

Materials and Methods

StrainsA list of strains used in this study is included in supplementarytable S1 Supplementary Material online All interspecific hy-brids were created by crossing a ura3 LYS2 haploid parent to aURA3 lys2 haploid parent of the other mating type plating onmedia lacking both uracil and lysine and selecting for proto-trophs The S cerevisiae strain background known asldquoGRF167rdquo is itself a cross between FL100 and the genomictype strain S288C (data not shown) GRF167 was chosen as astrain background for simultaneous work investigating trans-posable elements during experimental evolution which willbe addressed in a future study

Evolution ExperimentsContinuous cultures were established using media and con-ditions previously described (Gresham et al 2008 Sanchezet al 2017) Detailed protocols and media recipes are availableat httpdunhamgswashingtoneduprotocolsshtml (lastaccessed March 6 2017) Samples were taken daily and mea-sured for optical density at 600 nm and cell count micros-copy was performed to check for contamination andarchival glycerol stocks were made daily An experimentwas terminated when contamination growth in tubing orclumping appeared (number of generations at the endpoint for each population are presented in tables 1 and

Adaptation in Hybrids doi101093molbevmsx098 MBE

13

2) Samples from each end point population were colonypurified to yield two clones for further study

Array Comparative Genomic HybridizationPopulations from the end point of each evolution were ana-lyzed for copy number changes using array comparative ge-nomic hybridization following the protocol used in Sanchezet al (2017)

SequencingDNA was extracted from overnight cultures using theHoffmanndashWinston protocol (Hoffman and Winston 1987)and cleaned using the Clean amp Concentrator kit (ZymoResearch) Nextera libraries were prepared following theNextera library kit protocol and sequenced using paired end150 bp reads on the Illumina NextSeq 500 machine (sequenc-ing coverage in supplementary table S2 SupplementaryMaterial online) The reference genomes used were S cerevi-siae v3 (Engel et al 2014) S uvarum (Scannell et al 2011) anda hybrid reference genome created by concatenating the twogenomes Sequence was aligned to the appropriate referencegenome using bwa v062 (Li and Durbin 2009) and mutationswere called using GATK (McKenna et al 2010) and samtools0119 (Li et al 2009) Mutations in evolved clones were filteredin comparison with the ancestor to obtain de novo mutationsAll mutations were first visually inspected using IntegrativeGenomics Viewer (Robinson et al 2011) Subsequently pointmutations in the hybrids were confirmed with Sanger se-quencing (supplementary table S3 Supplementary Materialonline) Copy number variants were visualized usingDNAcopy for S cerevisiae and S uvarum (Seshan andOlshen 2016) LOH events were called based on sequencingcoverage in the hybrids and by identifying homozygous variantcalls in S cerevisiae All break points were called by visual in-spection of sequencing reads and are thus approximate

Fitness AssaysThe pairwise competition experiments were performed in 20ml chemostats (Miller and Dunham 2013) Each competitorstrain was cultured individually until steady state was reachedand then was mixed 5050 with a GFP-tagged ancestor Eachcompetition was conducted in at least two biological repli-cates for approximately 15 generations after mixing Sampleswere collected and analyzed twice daily The proportion ofGFPthorn cells in the population was detected using a BD AccuriC6 flow cytometer (BD Biosciences) The data were plottedwith ln [dark cellsGFPthorn cells] versus generations The rela-tive fitness coefficient was determined from the slope of thelinear region

Strain ConstructionAllele replacements for the PHO84 locus were done followingthe protocol of the Caudy lab with further modifications de-scribed here The native locus was replaced withKluyveromyces lactis URA3 The pho84DURA3 strain wasgrown overnight in 5 ml of C-URA media then inoculatedin a flask of 100 ml yeast extract peptone dextrose (YPD) andgrown to an optical density of 06ndash08 Cells were washed then

aliquoted 275 ml of transformation mix (35 ml 1 M lithiumacetate 240 ml of 50 3500 polyethylene glycol) 10 ml ofsalmon sperm and approximately 3 mg of polymerase chainreaction product were added to the cell pellet It was incu-bated at 37 C (S uvarum) or 42 C (S cerevisiae) for 45 minthen plated to YPD It was replica plated to 5-fluorooroticacid the following day and colonies were tested for the gain ofthe appropriate species allele The GRF167 allele was clonedinto the pIL37 plasmid using Gibson assembly (Gibson et al2009) Correct assembly was verified by Sanger sequencing Allprimers used can be found in supplementary table S3Supplementary Material online

Supplementary MaterialSupplementary data are available at Molecular Biology andEvolution online

AcknowledgmentsWe thank Noah Hanson and Erica Alcantara for technicalassistance We thank Monica Sanchez and three anonymousreviewers for helpful comments on this manuscript JosephSchacherer kindly confirmed the contribution of FL100 to theGRF167 strain background Thanks to Yixian Zheng and DougKoshland for contributing to the initial experimental designcreating yeast strains and purchasing the oligonucleotidesused for the microarrays This work was supported by theNational Science Foundation (grant number 1516330) andthe National Institutes of Health (grant number R01GM094306) MD is a Senior Fellow in the GeneticNetworks program at the Canadian Institute for AdvancedResearch and a Rita Allen Foundation Scholar CSH wassupported in part by National Institutes of Health (grantnumber T32 HG00035) This work was also supported bythe National Institutes of Health (grant number P50GM071508) to the Lewis-Sigler Institute and from theHoward Hughes Medical Institute to Doug Koshland andYixian Zheng Sequencing read data have been deposited atthe NCBI under BioProject accession PRJNA374049Microarray data are deposited in the Gene ExpressionOmnibus (GEO) repository under accession numbersGSE95086 and GSE87401 and in the Princeton MicroarrayDatabase

ReferencesAbbott R Albach D Ansell S Arntzen JW Baird SJ Bierne N Boughman J

Brelsford A Buerkle CA Buggs R et al 2013 Hybridization andspeciation J Evol Biol 26229ndash246

Ainouche ML Jenczewski E 2010 Focus on polyploidy New Phytol1861ndash4

Albertin W Marullo P 2012 Polyploidy in fungi evolution after whole-genome duplication Proc Biol Sci 2792497ndash2509

Almeida P Goncalves C Teixeira S Libkind D Bontrager M Masneuf-Pomarede I Albertin W Durrens P Sherman DJ Marullo P et al2014 A Gondwanan imprint on global diversity and domesticationof wine and cider yeast Saccharomyces uvarum Nat Commun54044

Antunovics Z Nguyen HV Gaillardin C Sipiczki M 2005 Gradual ge-nome stabilisation by progressive reduction of the Saccharomyces

Smukowski Heil et al doi101093molbevmsx098 MBE

14

uvarum genome in an interspecific hybrid with Saccharomyces cer-evisiae FEMS Yeast Res 51141ndash1150

Baker E Wang B Bellora N Peris D Hulfachor AB Koshalek JA AdamsM Libkind D Hittinger CT 2015 The Genome sequence ofSaccharomyces eubayanus and the domestication of lager-brewingyeasts Mol Biol Evol 322818ndash2831

Barbosa R Almeida P Safar SVB Santos RO Morais PB Nielly-Thibault LLeducq JB Landry CR Goncalves P Rosa CA Sampaio JP 2016Evidence of natural hybridization in Brazilian wild lineages ofSaccharomyces cerevisiae Genome Biol Evol 8317ndash329

Barton NH 2001 The role of hybridization in evolution Mol Ecol10551ndash568

Belloch C Orlic S Barrio E Querol A 2008 Fermentative stress adapta-tion of hybrids within the Saccharomyces sensu stricto complex Int JFood Microbiol 122188ndash195

Bellon JR Yang F Day MP Inglis DL Chambers PJ 2015 Designingand creating Saccharomyces interspecific hybrids for improvedindustry relevant phenotypes Appl Microbiol Biotechnol998597ndash8609

Borneman AR Zeppel R Chambers PJ Curtin CD 2014 Insights into theDekkera bruxellensis genomic landscape comparative genomics re-veals variations in ploidy and nutrient utilisation potential amongstwine isolates PLoS Genet 10e1004161

Brewer BJ Payen C Di Rienzi SC Higgins MM Ong G Dunham MJRaghuraman MK 2015 Origin-dependent inverted-repeat amplifi-cation tests of a model for inverted DNA amplification PLoS Genet11e1005699

Brown CJ Todd KM Rosenzweig RF 1998 Multiple duplications of yeasthexose transport genes in response to selection in a glucose-limitedenvironment Mol Biol Evol 15931ndash942

Bullini L 1994 Origin and evolution of animal hybrid species Trends EcolEvol 9422ndash426

Bun-Ya M Nishimura M Harashima S Oshima Y 1991 The PHO84 geneof Saccharomyces cerevisiae encodes an inorganic phosphate trans-porter Mol Cell Biol 113229ndash3238

Burke MK Dunham JP Shahrestani P Thornton KR Rose MR Long AD2010 Genome-wide analysis of a long-term evolution experimentwith Drosophila Nature 467587ndash590

Burke MK Liti G Long AD 2014 Standing genetic variation drives re-peatable experimental evolution in outcrossing populations ofSaccharomyces cerevisiae Mol Biol Evol 313228ndash3239

Cherry JM Hong EL Amundsen C Balakrishnan R Binkley G Chan ETChristie KR Costanzo MC Dwight SS Engel SR et al 2012Saccharomyces genome database the genomics resource of buddingyeast Nucleic Acids Res 40D700ndashD705

Chester M Riley RK Soltis PS Soltis DE 2015 Patterns of chromosomalvariation in natural populations of the neoallotetraploid Tragopogonmirus (Asteraceae) Heredity (Edinb) 114309ndash317

Chou JY Leu JY 2010 Speciation through cytonuclear incompatibilityinsights from yeast and implications for higher eukaryotes Bioessays32401ndash411

Cliften PF Fulton RS Wilson RK Johnston M 2006 After the duplicationgene loss and adaptation in Saccharomyces genomes Genetics172863ndash872

Conte GL Arnegard ME Peichel CL Schluter D 2012 The probability ofgenetic parallelism and convergence in natural populations Proc BiolSci 2795039ndash5047

Cordes FS Bright JN Sansom MS 2002 Proline-induced distortions oftransmembrane helices J Mol Biol 323951ndash960

Crow JF 1998 90 years ago the beginning of hybrid maize Genetics148923ndash928

Dasmahapatra KK Walters JR Briscoe AD Davey JW Whibley A NadeauNJ Zimin AV Hughes DST Ferguson LC Martin SH et al 2012Butterfly genome reveals promiscuous exchange of mimicry adap-tations among species Nature 48794ndash98

Dowling TE Smith GR Brown WM 1989 Reproductive isolation andintrogression between Notropis-Cornutus and Notropis-Chrysocephalus (Family Cyprinidae)mdashcomparison of morphologyallozymes and mitochondrial-DNA Evolution 43620ndash634

Doyle JJ Flagel LE Paterson AH Rapp RA Soltis DE Soltis PS Wendel JF2008 Evolutionary genetics of genome merger and doubling inplants Annu Rev Genet 42443ndash461

Dunham MJ Badrane H Ferea T Adams J Brown PO Rosenzweig FBotstein D 2002 Characteristic genome rearrangements in experi-mental evolution of Saccharomyces cerevisiae Proc Natl Acad Sci U SA 9916144ndash16149

Dunn B Paulish T Stanbery A Piotrowski J Koniges G Kroll E LouisEJ Liti G Sherlock G Rosenzweig F 2013 Recurrent rearrange-ment during adaptive evolution in an interspecific yeast hybridsuggests a model for rapid introgression PLoS Genet9e1003366

Dunn B Richter C Kvitek DJ Pugh T Sherlock G 2012 Analysis of theSaccharomyces cerevisiae pan-genome reveals a pool of copy num-ber variants distributed in diverse yeast strains from differing indus-trial environments Genome Res 22908ndash924

Dunn B Sherlock G 2008 Reconstruction of the genome origins andevolution of the hybrid lager yeast Saccharomyces pastorianusGenome Res 181610ndash1623

Elmer KR Meyer A 2011 Adaptation in the age of ecological genomicsinsights from parallelism and convergence Trends Ecol Evol26298ndash306

Engel SR Dietrich FS Fisk DG Binkley G Balakrishnan R Costanzo MCDwight SS Hitz BC Karra K Nash RS et al 2014 The referencegenome sequence of Saccharomyces cerevisiae then and now G3(Bethesda) 4389ndash398

Fedoroff NV 2012 Presidential address Transposable elements epige-netics and genome evolution Science 338758ndash767

Ferea TL Botstein D Brown PO Rosenzweig RF 1999 Systematicchanges in gene expression patterns following adaptive evolutionin yeast Proc Natl Acad Sci U S A 969721ndash9726

Gibson B Liti G 2015 Saccharomyces pastorianus genomic insightsinspiring innovation for industry Yeast 3217ndash27

Gibson DG Young L Chuang RY Venter JC Hutchison CA Smith HO2009 Enzymatic assembly of DNA molecules up to several hundredkilobases Nat Methods 6343ndash5

Gonzalez SS Barrio E Gafner J Querol A 2006 Natural hybrids fromSaccharomyces cerevisiae Saccharomyces bayanus andSaccharomyces kudriavzevii in wine fermentations FEMS Yeast Res61221ndash1234

Gonzalez SS Barrio E Querol A 2008 Molecular characterization of newnatural hybrids of Saccharomyces cerevisiae and S kudriavzevii inbrewing Appl Environ Microbiol 742314ndash2320

Grant PR Grant BR 1994 Phenotypic and genetic consequences ofhybridization in Darwinrsquos finches Evolution 48297ndash316

Grant PR Grant BR 2002 Unpredictable evolution in a 30-year study ofDarwinrsquos finches Science 296707ndash711

Grant PR Grant BR 2010 Natural selection speciation and Darwinrsquosfinches Proc Calif Acad Sci 61245ndash260

Grant PR Grant BR Petren K 2005 Hybridization in the recent past AmNat 16656ndash67

Greig D 2009 Reproductive isolation in Saccharomyces Heredity (Edinb)10239ndash44

Greig D Leu JY 2009 Natural history of budding yeast Current Biol19R886ndashR890

Greig D Louis EJ Borts RH Travisano M 2002 Hybrid speciation inexperimental populations of yeast Science 2981773ndash1775

Gresham D Desai MM Tucker CM Jenq HT Pai DA Ward A DeSevoCG Botstein D Dunham MJ 2008 The repertoire and dynamics ofevolutionary adaptations to controlled nutrient-limited environ-ments in yeast PLoS Genet 4e1000303

Hajjar R Hodgkin T 2007 The use of wild relatives in crop improvementa survey of developments over the last 20 years Euphytica 1561ndash13

Heiser CB 1954 Variation and subspeciation in the common sunflowerHelianthus annuus Am Midl Nat 51287ndash305

Hittinger CT 2013 Saccharomyces diversity and evolution a buddingmodel genus Trends Genet 29309ndash317

Hoang ML Tan FJ Lai DC Celniker SE Hoskins RA Dunham MJ ZhengY Koshland D 2010 Competitive repair by naturally dispersed

Adaptation in Hybrids doi101093molbevmsx098 MBE

15

repetitive DNA during non-allelic homologous recombination PLoSGenet 6e1001228

Hoffmann AA Sgro CM 2011 Climate change and evolutionary adap-tation Nature 470479ndash485

Hoffman CS Winston F 1987 A 10-minute DNA preparation from yeastefficiently releases autonomous plasmids for transformation ofEscherichia coli Gene 57267ndash272

Hou J Friedrich A de Montigny J Schacherer J 2014 Chromosomalrearrangements as a major mechanism in the onset of reproductiveisolation in Saccharomyces cerevisiae Curr Biol 241153ndash1159

Hou J Friedrich A Gounot JS Schacherer J 2015 Comprehensive surveyof condition-specific reproductive isolation reveals genetic incom-patibility in yeast Nat Commun 67214

Huerta-Sanchez E Casey FP 2015 Archaic inheritance supporting high-altitude life in Tibet J Appl Physiol (1985) 1191129ndash1134

Jones FC Grabherr MG Chan YF Russell P Mauceli E Johnson JSwofford R Pirun M Zody MC White S et al 2012 The genomicbasis of adaptive evolution in threespine sticklebacks Nature48455ndash61

Kao KC Sherlock G 2008 Molecular characterization of clonal interfer-ence during adaptive evolution in asexual populations ofSaccharomyces cerevisiae Nat Genet 401499ndash1504

Kawecki TJ Lenski RE Ebert D Hollis B Olivieri I Whitlock MC 2012Experimental evolution Trends Ecol Evol 27547ndash560

Kellis M Patterson N Endrizzi M Birren B Lander ES 2003 Sequencingand comparison of yeast species to identify genes and regulatoryelements Nature 423241ndash254

Kelly B Whiteley A Tallmon D 2010 The Arctic melting pot Nature468891ndash891

Kvitek DJ Sherlock G 2011 Reciprocal sign epistasis between frequentlyexperimentally evolved adaptive mutations causes a rugged fitnesslandscape PLoS Genet 7e1002056

Kvitek DJ Sherlock G 2013 Whole genome whole population sequenc-ing reveals that loss of signaling networks is the major adaptivestrategy in a constant environment PLoS Genet 9e1003972

Landry CR Hartl DL Ranz JM 2007 Genome clashes in hybrids insightsfrom gene expression Heredity (Edinb) 99483ndash493

Lang GI Desai MM 2014 The spectrum of adaptive mutations in ex-perimental evolution Genomics 104412ndash416

Lapunzina P Monk D 2011 The consequences of uniparental disomyand copy number neutral loss-of-heterozygosity during human de-velopment and cancer Biol Cell 103303ndash317

Leducq JB Nielly-Thibault L Charron G Eberlein C Verta JP Samani PSylvester K Hittinger CT Bell G Landry CR 2016 Speciation drivenby hybridization and chromosomal plasticity in a wild yeast NatMicrobiol 115003

Lee HY Chou JY Cheong L Chang NH Yang SY Leu JY 2008Incompatibility of nuclear and mitochondrial genomes causes hy-brid sterility between two yeast species Cell 1351065ndash1073

Li H Durbin R 2009 Fast and accurate short read alignment withBurrows-Wheeler transform Bioinformatics 251754ndash1760

Li H Handsaker B Wysoker A Fennell T Ruan J Homer N Marth GAbecasis G Durbin R 2009 The Sequence AlignmentMap formatand SAMtools Bioinformatics 252078ndash2079

Liti G 2015 The fascinating and secret wild life of the budding yeast Scerevisiae Elife 4e05835

Liti G Barton DB Louis EJ 2006 Sequence diversity reproductiveisolation and species concepts in Saccharomyces Genetics174839ndash850

Lloyd AH Ranoux M Vautrin S Glover N Fourment J Charif D ChouletF Lassalle G Marande W Tran J et al 2014 Meiotic gene evolutioncan you teach a new dog new tricks Mol Biol Evol 311724ndash1727

Losos JB Jackman TR Larson A de Queiroz K Rodriguez-Schettino L1998 Contingency and determinism in replicated adaptive radia-tions of island lizards Science 2792115ndash2118

Louis VL Despons L Friedrich A Martin T Durrens P Casaregola SNeuveglise C Fairhead C Marck C Cruz JA et al 2012 Pichia sorbi-tophila an interspecies yeast hybrid reveals early steps of genomeresolution after polyploidization G3 (Bethesda) 2299ndash311

Mallet J 2007 Hybrid speciation Nature 446279ndash283Marcet-Houben M Gabaldon T 2015 Beyond the whole-genome du-

plication phylogenetic evidence for an ancient interspecies hybrid-ization in the bakerrsquos yeast lineage PLoS Biol 13e1002220

Marinoni G Manuel M Petersen RF Hvidtfeldt J Sulo P Piskur J 1999Horizontal transfer of genetic material among Saccharomyces yeastsJ Bacteriol 1816488ndash6496

Martin A Orgogozo V 2013 The loci of repeated evolution a catalog ofgenetic hotspots of phenotypic variation Evolution 671235ndash1250

Masly JP Jones CD Noor MAF Locke J Orr HA 2006 Gene transpositionas a cause of hybrid sterility in Drosophila Science 3131448ndash1450

Mason AS Batley J 2015 Creating new interspecific hybrid and polyploidcrops Trends Biotechnol 33436ndash441

Mavarez J Salazar CA Bermingham E Salcedo C Jiggins CD Linares M2006 Speciation by hybridization in Heliconius butterflies Nature441868ndash871

McKenna A Hanna M Banks E Sivachenko A Cibulskis K Kernytsky AGarimella K Altshuler D Gabriel S Daly M et al 2010 The genomeanalysis toolkit a MapReduce framework for analyzing next-generation DNA sequencing data Genome Res 201297ndash1303

Meiklejohn CD Holmbeck MA Siddiq MA Abt DN Rand DMMontooth KL 2013 An Incompatibility between a mitochondrialtRNA and its nuclear-encoded tRNA synthetase compromises de-velopment and fitness in Drosophila PLoS Genet 9e1003238

Meyer A Salzburger W Schartl M 2006 Hybrid origin of a swordtailspecies (Teleostei Xiphophorus clemenciae) driven by sexual selec-tion Mol Ecol 15721ndash730

Michalak P 2009 Epigenetic transposon and small RNA determinants ofhybrid dysfunctions Heredity (Edinb) 10245ndash50

Miller AW Dunham MJ 2013 Design and use of multiplexed chemostatarrays J Vis Exp 72e50262

Monod J 1949 The growth of bacterial cultures Annu Rev Microbiol3371ndash394

Mouillon JM Persson BL 2006 New aspects on phosphate sensing andsignalling in Saccharomyces cerevisiae FEMS Yeast Res 6171ndash176

Muhlfeld CC Kovach RP Jones LA Al-Chokhachy R Boyer MC Leary RFLowe WH Luikart G Allendorf FW 2014 Invasive hybridization in athreatened species is accelerated by climate change Nat ClimChange 4620ndash624

Muller LAH McCusker JH 2009 A multispecies-based taxonomic mi-croarray reveals interspecies hybridization and introgression inSaccharomyces cerevisiae FEMS Yeast Res 9143ndash152

Nagalakshmi U Wang Z Waern K Shou C Raha D Gerstein M SnyderM 2008 The transcriptional landscape of the yeast genome definedby RNA sequencing Science 3201344ndash1349

Nakao Y Kanamori T Itoh T Kodama Y Rainieri S Nakamura NShimonaga T Hattori M Ashikari T 2009 Genome sequence ofthe lager brewing yeast an interspecies hybrid DNA Res 16115ndash129

Nolte AW Freyhof J Stemshorn KC Tautz D 2005 An invasive lineage ofsculpins Cottus sp (Pisces Teleostei) in the Rhine with new habitatadaptations has originated from hybridization between old phylo-geographic groups Proc Biol Sci 2722379ndash2387

Novick A Szilard L 1950a Description of the chemostat Science112715ndash716

Novick A Szilard L 1950b Experiments with the Chemostat on spon-taneous mutations of bacteria Proc Natl Acad Sci U S A 36708ndash719

Ouyang YD Liu YG Zhang QF 2010 Hybrid sterility in plant storiesfrom rice Curr Opin Plant Biol 13186ndash192

Payen C Di Rienzi SC Ong GT Pogachar JL Sanchez JC SunshineAB Raghuraman MK Brewer BJ Dunham MJ 2014 The dynam-ics of diverse segmental amplifications in populations ofSaccharomyces cerevisiae adapting to strong selection G3(Bethesda) 4399ndash409

Payen C Sunshine AB Ong GT Pogachar JL Zhao W Dunham MJ 2016High-throughput identification of adaptive mutations in experimen-tally evolved yeast populations PLoS Genet 12e1006339

Perez-Prat E van Lookeren Campagne MM 2002 Hybrid seed produc-tion and the challenge of propagating male-sterile plants TrendsPlant Sci 7199ndash203

Smukowski Heil et al doi101093molbevmsx098 MBE

16

Peris D Langdon QK Moriarty RV Sylvester K Bontrager M Charron GLeducq JB Landry CR Libkind D Hittinger CT 2016 Complex ances-tries of lager-brewing hybrids were shaped by standing variation inthe wild yeast Saccharomyces eubayanus PLoS Genet 12e1006155

Perlstein EO Ruderfer DM Roberts DC Schreiber SL Kruglyak L 2007Genetic basis of individual differences in the response to small-molecule drugs in yeast Nat Genet 39496ndash502

Piatkowska EM Naseeb S Knight D Delneri D 2013 Chimeric proteincomplexes in hybrid species generate novel phenotypes PLoS Genet9e1003836

Piotrowski JS Nagarajan S Kroll E Stanbery A Chiotti KE Kruckeberg ALDunn B Sherlock G Rosenzweig F 2012 Different selective pressureslead to different genomic outcomes as newly-formed hybrid yeastsevolve BMC Evol Biol 1246

Pryszcz LP Nemeth T Gacser A Gabaldon T 2014 Genome comparisonof Candida orthopsilosis clinical strains reveals the existence of hy-brids between two distinct subspecies Genome Biol Evol61069ndash1078

Racimo F Sankararaman S Nielsen R Huerta-Sanchez E 2015 Evidencefor archaic adaptive introgression in humans Nat Rev Genet16359ndash371

Rieseberg LH 1991 Homoploid reticulate evolution in Helianthus(Asteraceae)mdashevidence from ribosomal genes Am J Bot781218ndash1237

Rieseberg LH 1997 Hybrid origins of plant species Annu Rev EcolSystemat 28359ndash389

Robinson JT Thorvaldsdottir H Winckler W Guttman M Lander ESGetz G Mesirov JP 2011 Integrative genomics viewer NatBiotechnol 2924ndash26

Rosenthal DM Schwarzbach AE Donovan LA Raymond O RiesebergLH 2002 Phenotypic differentiation between three ancient hybridtaxa and their parental species Int J Plant Sci 163387ndash398

Ruderfer DM Pratt SC Seidel HS Kruglyak L 2006 Population genomicanalysis of outcrossing and recombination in yeast Nat Genet381077ndash1081

Rundle HD Nagel L Boughman JW Schluter D 2000 Natural selectionand parallel speciation in sympatric sticklebacks Science287306ndash308

Ryland GL Doyle MA Goode D Boyle SE Choong DY Rowley SM Li JBowtell DD Tothill RW Campbell IG et al 2015 Australian OvarianCancer Study Group Loss of heterozygosity what is it good for BMCMed Genomics 845

Salverda MLM Dellus E Gorter FA Debets AJM van der Oost J HoekstraRF Tawfik DS de Visser JAGM 2011 Initial mutations direct alter-native pathways of protein evolution PLoS Genet 7e1001321

Sanchez MR Miller AW Liachko I Sunshine AB Lynch B Huang MAlcantara E DeSevo CG Pai DA Tucker CM et al 2017 Differentialparalog divergence modulates genome evolution across yeast spe-cies PLoS Genet 13(2)e1006585

Scannell DR Zill OA Rokas A Payen C Dunham MJ Eisen MB Rine JJohnston M Hittinger CT 2011 The awesome power of yeast evo-lutionary genetics new genome sequences and strain resources forthe Saccharomyces sensu stricto genus G3 (Bethesda) 111ndash25

Schroder MS Martinez de San Vicente K Prandini TH Hammel SHiggins DG Bagagli E Wolfe KH Butler G 2016 Multiple originsof the pathogenic yeast Candida orthopsilosis by separate hybridiza-tions between two parental species PLoS Genet 12e1006404

Schumer M Rosenthal GG Andolfatto P 2014 How common is homo-ploid hybrid speciation Evolution 681553ndash1560

Schwarzbach AE Donovan LA Rieseberg LH 2001 Transgressivecharacter expression in a hybrid sunflower species Am J Bot88270ndash277

Selmecki AM Maruvka YE Richmond PA Guillet M Shoresh NSorenson AL De S Kishony R Michor F Dowell R et al 2015Polyploidy can drive rapid adaptation in yeast Nature 519349ndash52

Seshan VE Olshen A 2016 DNAcopy DNA copy number data analysisVersion R package version 1460

Soltis DE Visger CJ Soltis PS 2014 The polyploidy revolution then andnow Stebbins revisited Am J Bot 1011057ndash1078

Soltis PS 2013 Hybridization speciation and novelty J Evol Biol26291ndash293

Soltis PS Soltis DE 2009 The role of hybridization in plant speciationAnnu Rev Plant Biol 60561ndash588

Sunshine AB Payen C Ong GT Liachko I Tan KM Dunham MJ 2015The fitness consequences of aneuploidy are driven by condition-dependent gene effects PLoS Biol 13e1002155

Tamai Y Momma T Yoshimoto H Kaneko Y 1998 Co-existence of twotypes of chromosome in the bottom fermenting yeastSaccharomyces pastorianus Yeast 14923ndash933

Taylor DJ Hebert PDN 1993 Habitat-dependent hybrid parentage anddifferential introgression between neighboringly sympatric Daphniaspecies Proc Natl Acad Sci U S A 907079ndash7083

Thiagalingam S Laken S Willson JK Markowitz SD Kinzler KWVogelstein B Lengauer C 2001 Mechanisms underlying losses ofheterozygosity in human colorectal cancers Proc Natl Acad Sci US A 982698ndash2702

Tuna M Knuutila S Mills GB 2009 Uniparental disomy in cancer TrendsMol Med 15120ndash128

Walther A Hesselbart A Wendland J 2014 Genome sequence ofSaccharomyces carlsbergensis the worldrsquos first pure culture lageryeast G3 (Bethesda) 4783ndash793

Wayne RK 1993 Molecular evolution of the dog family Trends Genet9218ndash224

Wenger JW Schwartz K Sherlock G 2010 Bulk segregant analysis byhigh-throughput sequencing reveals a novel xylose utilization genefrom Saccharomyces cerevisiae PLoS Genet 6e1000942

Wessinger CA Rausher MD 2014 Predictability and irreversibility ofgenetic changes associated with flower color evolution inPenstemon barbatus Evolution 681058ndash1070

Wolfe KH 2015 Origin of the yeast whole-genome duplication PLoSBiol 13e1002221

Woods R Schneider D Winkworth CL Riley MA Lenski RE 2006 Testsof parallel molecular evolution in a long-term experiment withEscherichia coli Proc Natl Acad Sci U S A 1039107ndash9112

Wykoff DD OrsquoShea EK 2001 Phosphate transport and sensing inSaccharomyces cerevisiae Genetics 1591491ndash1499

Xiong Z Gaeta RT Pires JC 2011 Homoeologous shuffling andchromosome compensation maintain genome balance inresynthesized allopolyploid Brassica napus Proc Natl Acad SciU S A 1087908ndash7913

Yassour M Kapian T Fraser HB Levin JZ Pfiffner J Adiconis X Schroth GLuo SJ Khrebtukova I Gnirke A et al 2009 Ab initio construction ofa eukaryotic transcriptome by massively parallel mRNA sequencingProc Natl Acad Sci U S A 1063264ndash3269

Zhu YO Sherlock G Petrov DA 2016 Whole genome analysis of 132clinical Saccharomyces cerevisiae strains reveals extensive ploidy var-iation G3 (Bethesda) doi101534g3116029397

Adaptation in Hybrids doi101093molbevmsx098 MBE

17

  • msx098-TF1
  • msx098-TF2
Page 3: Loss of Heterozygosity Drives Adaptation in Hybrid Yeastdunham.gs.washington.edu/SmukowskiHeil.pdf · 2017-05-20 · Loss of Heterozygosity Drives Adaptation in Hybrid Yeast Caiti

Supplementary Material online) and evolved in continu-ous culture in the chemostat (Monod 1949 Novick andSzilard 1950a 1950b) In parallel homozygous diploidS uvarum and heterozygous diploid S cerevisiae(GRF167 S288C) were also evolved Each strain wasgrown in two or more replicate independent culturesunder three different nutrient limitationsmdashglucose phos-phate and sulfatemdashfor 85ndash557 generations (median 158)at 30 C except for S uvarum which was unable toachieve steady state in all conditions at 30 C and sowas evolved at 25 C The population sizes were largelysimilar across strains species and conditions

Evolved clones were isolated from each population andsubsequently competed individually against the appropriategreen fluorescent protein (GFP)-tagged ancestor to gaugerelative fitness As expected evolved hybrid and parentalclones generally exhibit higher fitness than their unevolvedancestor with typical relative fitness gains between 20 and30 (tables 1 and 2) To explore whether these fitness gainsare general or condition specific we additionally competedeach hybrid clone in the two nutrient-limited conditions inwhich the clone was not evolved Results are variable withsome clones having negative or neutral fitness in the alternateconditions suggesting condition-specific adaptation andsome clones experiencing fitness gains in multiple conditionssuggesting more general growth benefits (table 1) Only oneclone exhibited fitness gains in all three nutrient environ-ments and no clones have a greater fitness gain in an alter-nate condition than the condition it was evolved in signifyingthat clones are largely specifically adapted to the particularcondition in which they were evolved

Mutations Are Recovered in Both Novel andPreviously Observed Gene Targets in InterspecificHybridsTo identify mutations in the evolved hybrids we generatedwhole genome sequencing data for 16 clones from the endpoints of the evolution experiments (table 1) We thus cap-tured data from a range of nutrient limitations (phosphate 6glucose 3 sulfate 7) and generations (100ndash285 median 154)Each clone had an average of 24 point mutations a numberof which have been previously identified in prior S cerevisiaeevolution experiments For example a nonsynonymous mu-tation in the S cerevisiae allele of the glucose-sensing geneSNF3 has been identified in glucose-limited experiments inS cerevisiae (Kvitek and Sherlock 2013 Selmecki et al 2015)To our knowledge 2027 coding point mutations are uniqueto these experiments (Payen et al 2016)

In evolved parentals we again sequenced one clone fromthe end point of each population In total we sequenced 16clones 6 from each of the 3 nutrients (2 S uvarum diploidsand 4 S cerevisiae diploids) except in glucose limitation inwhich only 2 S cerevisiae populations were sampled Thegenerations ranged from 234 to 557 (median 477) in S uva-rum with an average of 283 mutations per clone and from127 to 190 (median 1665) in S cerevisiae with an average of09 point mutations per clone (table 2) This discrepancy in

point mutations between S cerevisiae and S uvarum may beexplained by differences in generation number

With the limited number of samples we have from hybridand parental clones it is difficult to draw any conclusionsregarding unique point mutations in hybrids Furthermorewe have not tested the fitness of these mutations to provethey are adaptive However one class of mutations that maybe of particular interest in hybrids are genomic mutationsthat may interact with the mitochondria as previous workhas shown that nuclearndashmitochondria interactions can un-derlie hybrid incompatibility (Lee et al 2008 Chou and Leu2010 Meiklejohn et al 2013) Other studies have found thatonly the S cerevisiae mitochondria are retained in S cerevisiae S uvarum hybrids (Antunovics et al 2005) and we reca-pitulate these findings potentially setting the stage for con-flicting interactions between the S uvarum nuclear genomeand the foreign mitochondria We observe several mitochon-dria-related mutations in hybrids in both S cerevisiae andS uvarum alleles For example one point mutation a nonsyn-onymous mutation in the S cerevisiae allele of the mitochon-drial ribosomal protein gene MHR1 was seen in two separateclones independently evolved in phosphate limitation Thisgene may be of particular interest as it was discovered in aprevious screen as being haploproficient (increased fitness of19) in hybrids in which the S cerevisiae allele is missing andthe S uvarum allele is retained (Lancaster S Dunham MJunpublished data) suggesting that this mutation may alteror disable the S cerevisiae protein in some way Another ex-ample involves the gene IRC3 a helicase responsible for themaintenance of the mitochondrial genome which has a non-synonymous mutation in the S uvarum allele in clone Gh3and is deleted in clone Gh2 potentially suggesting that theS uvarum allele is deleterious in the hybrid backgroundAlthough our sample size is small 427 point mutations inhybrids are related to mitochondria function compared with026 in parentals and may represent interesting targets forfurther exploration

Copy Number Variants Frequently Involve theAmplification of Nutrient TransportersYeasts in both natural and artificial environments are knownto frequently experience changes in copy number rangingfrom single genes to whole chromosomes (Dunham et al2002 Gresham et al 2008 Dunn et al 2012 Kvitek andSherlock 2013 Payen et al 2014 Selmecki et al 2015Sunshine et al 2015 Zhu et al 2016) This holds true in ourevolution experiments We observe copy number changesacross all genetic backgrounds (fig 1 supplementary figsS1ndashS3 Supplementary Material online) Clones were com-pared with array comparative genomic hybridization of pop-ulations to confirm that clones are representative ofpopulations (see Materials and Methods) The evolved hybridclones displayed an average of 15 copy number variants(CNVs) per clone (fig 1 table 1 Supplementary fig S3Supplementary Material online) as defined by the numberof segmental or whole chromosome amplificationsdeletions(though it is likely that some of these CNVs were created inthe same mutational event) The evolved S cerevisiae clones

Adaptation in Hybrids doi101093molbevmsx098 MBE

3

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Mu

tati

on

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dFi

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fEv

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Clo

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Loca

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s6

SE(c

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)

Gh

1ch

rXII

I85

2028

Inte

rgen

icce

r12

526

80

60

98(G

)0

356

160

(S)

118

(P)

chrI

I91

1866

917

272

HX

T6

7C

NV

(am

plifi

cati

on

)u

vaG

h2

chrI

V1

1191

9SN

F3N

on

syn

on

ymo

us

D11

4Yce

r10

028

17

62

18(G

)10

48

60

78(S

)11

23

(P)

chrI

II5

1593

GLK

1Sy

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nym

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sT

252T

cer

chrI

V8

8480

19

1211

913

gen

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din

gIR

C3

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CN

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valo

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rII

9121

439

1747

0H

XT

67

CN

V(a

mp

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chrI

V83

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CN

V(a

mp

lifica

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cer

Gh

3ch

rII

8894

21IR

C3

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nym

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sM

333I

uva

124

186

56

047

(G)

176

86

367

(S)

104

6(P

)ch

rII

9124

169

1777

8H

XT

67

CN

V(a

mp

lifica

tio

n)

uva

Ph

1ch

rV2

6939

2In

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enic

cer

103

291

86

137

(P)

168

60

78(G

)0

086

043

(S)

chrX

IV7

4668

8In

terg

enic

cer

chrI

V1

0558

64M

HR

1N

on

syn

on

ymo

us

T21

8Rce

rch

rIX

241

gen

esLO

HC

NV

uva

lost

cer

amp

Ph

2ch

rV4

3277

8G

LC7

Intr

on

cer

124

253

46

024

(P)

151

26

466

(G)

245

61

16(S

)ch

rVII

952

4P

DR

11N

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syn

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L383

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vach

rXV

I23

2879

MR

PL4

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syn

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ymo

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V14

9Eu

vach

rXII

I19

4496

YM

L037

CN

on

syn

on

ymo

us

P30

6Su

vach

rIV

244

399

YD

L114

WN

on

syn

on

ymo

us

G11

9Cu

vach

rIV

836

gen

esC

NV

(am

plifi

cati

on

)ce

rP

h3

chrI

V1

0558

64M

HR

1N

on

syn

on

ymo

us

T21

8Rce

r16

730

03

64

31(P

)21

39

66

25(G

)N

A(S

)ch

rIX

308

303

3084

YIL

166C

CN

V(a

mp

lifica

tio

n)

cer

chrX

III

02

4562

10ge

nes

incl

ud

ing

PH

O84

LOH

CN

Vu

valo

stc

eram

pch

rIV

836

gen

esC

NV

(am

plifi

cati

on

)ce

rP

h4

chrV

II5

5588

5R

PL2

6BIn

tro

nce

r13

127

02

63

62(P

)0

686

410

(G)

204

66

860

(S)

chrX

246

208

PH

S1N

on

syn

on

ymo

us

K20

6Nce

rch

rXII

I32

4121

EIS1

No

nsy

no

nym

ou

sE3

49

uva

chrI

II0

826

8749

gen

esLO

HC

NV

cer

lost

chrX

III0

221

753

112

gen

esi

ncl

ud

ing

PH

O84

LOH

CN

Vu

valo

stc

eram

pP

h5

chrX

III

2317

31P

PZ

1N

on

syn

on

ymo

us

A63

Su

va12

230

24

68

32(P

)

820

60

34(G

)18

20

62

91(S

)ch

rXII

I0

234

112

120

gen

esi

ncl

ud

ing

PH

O84

LOH

CN

Vu

valo

stc

eram

pch

rIX

370

117

439

888

45ge

nes

LOH

CN

Vce

rlo

stP

h6

chrV

II9

7281

3P

FK1

No

nsy

no

nym

ou

sG

308S

cer

111

255

26

332

(P)

522

62

81(G

)N

A(S

)ch

rIV

836

gen

esC

NV

(am

plifi

cati

on

)ce

rSh

1ch

rII5

1136

26

4497

469

6397

81

3184

74ge

nes

63

gen

esin

clu

din

gSU

L1LO

HC

NV

cer

lost

cer

amp

126

338

66

460

(S)

381

61

17(G

)

154

86

855

(P)

chIV

680

386

866

667

8666

67

9837

7410

4ge

nes

63

gen

esLO

HC

NV

uva

amp

uva

lost

chrX

VI

8470

00

9480

6649

gen

esLO

HC

NV

cer

lost

Sh2

chrV

II9

3638

4M

RP

L9N

on

syn

on

ymo

us

D16

7Gce

r26

819

64

64

30(S

)

619

61

21(G

)

143

66

37(P

)ch

rXV

I57

2308

ICL2

No

nsy

no

nym

ou

sM

247I

uva

chrV

III

1166

61ER

G11

No

nsy

no

nym

ou

sS2

86C

uva

chrI

I787

389

813

184

11ge

nes

incl

ud

ing

SUL1

CN

V(a

mp

lifica

tio

n)

cer

Sh3

chrV

I16

2998

GC

N20

No

nsy

no

nym

ou

sD

171Y

cer

132

218

46

153

(S)

607

61

11(G

)5

556

481

(P)

chrX

IV4

9589

0FK

H2

Syn

on

ymo

us

S418

Su

vach

rII7

8658

48

131

8411

gen

esin

clu

din

gSU

L1C

NV

(am

plifi

cati

on

)ce

r

(co

nti

nu

ed)

Smukowski Heil et al doi101093molbevmsx098 MBE

4

Tab

le1

Co

nti

nu

ed

Clo

ne

Loca

tio

nG

ene(

s)M

uta

tio

nSp

ecie

sG

ener

atio

ns

Rel

ativ

eFi

tnes

s6

SE(c

on

dit

ion

)

Sh4

chrX

IV6

6667

5A

RE2

No

nsy

no

nym

ou

sI4

46T

cer

285

271

96

433

(S)

617

60

51(G

)

205

56

330

(P)

chrX

V8

0083

2A

PC

55rsquo

-up

stre

amce

rch

rIV

259

17T

RM

3Sy

no

nym

ou

sS2

01S

cer

chrV

342

563

Inte

rgen

icu

vach

rX7

6976

8SP

O77

No

nsy

no

nym

ou

sD

418G

uva

chrX

990

873

LEU

35rsquo

-up

stre

amu

vach

rXII

192

491

Inte

rgen

icu

vach

rXIV

251

38EG

T2

Syn

on

ymo

us

T16

8Tu

vach

rII

7703

118

1318

422

gen

esi

ncl

ud

ing

SUL1

CN

V(a

mp

lifica

tio

n)

cer

chrV

III

321

gen

esC

NV

(am

plifi

cati

on

)u

vaSh

5ch

rIV

310

881

RX

T3

No

nsy

no

nym

ou

sP

87T

uva

263

465

26

494

(S)

679

61

35(G

)3

726

723

(P)

chrV

III

1691

1In

terg

enic

uva

chrI

I78

6040

813

184

11ge

nes

incl

ud

ing

SUL1

CN

V(a

mp

lifica

tio

n)

cer

Sh6

chrV

269

392

Inte

rgen

icce

r27

347

52

63

69(S

)2

606

125

(G)

447

66

04(P

)ch

rXIV

746

688

Inte

rgen

icce

rch

rIV

413

046

Inte

rgen

icu

vach

rII7

7894

28

131

8414

gen

esin

clu

din

gSU

L1C

NV

(am

plifi

cati

on

)ce

rSh

7ch

rII

2388

75In

terg

enic

cer

129

314

46

049

(S)

187

61

99(G

)8

746

922

(P)

chrX

VI

4906

31SV

L3N

on

syn

on

ymo

us

A24

5Vce

rch

rXV

I86

106

YP

L245

WN

on

syn

on

ymo

us

A17

4Dce

rch

rII

2732

96In

terg

enic

uva

chrI

I737

875

813

184

42ge

nes

in

clu

din

gSU

L1C

NV

(am

plifi

cati

on

)ce

r

Poin

tm

uta

tio

ns

cop

yn

um

ber

vari

ants

(CN

Vs)

an

dlo

sso

fhet

ero

zygo

sity

even

ts(L

OH

)ar

ere

cord

edfo

rea

chev

olv

edh

ybri

dcl

on

eC

lon

esar

eid

enti

fied

by

nu

trie

nt

(Gg

luco

selim

itat

ion

Pp

ho

sph

ate

limit

atio

na

nd

Ssu

lfate

limit

atio

n)

anldquoh

rdquod

eno

tes

hyb

rid

an

dth

en

um

ber

ind

icat

esit

sd

eriv

atio

nfr

om

ind

epen

den

tp

op

ula

tio

ns

Gen

esin

un

der

line

hav

eb

een

fou

nd

toh

ave

po

int

mu

tati

on

sin

pri

or

exp

erim

ents

No

teth

atm

uta

tio

ns

inth

eS

uvar

umge

no

me

use

Suv

arum

chro

mo

som

esan

dco

ord

inat

esA

llb

reak

po

ints

wer

eca

lled

by

visu

alin

spec

tio

no

fseq

uen

cin

gre

ads

and

are

thu

sap

pro

xim

ate

Rel

ativ

efi

tnes

sis

rep

ort

edw

ith

stan

dar

der

ror(

SE)a

nd

the

con

dit

ion

the

clo

ne

was

evo

lved

inlis

ted

firs

tfo

llow

edb

yth

etw

oal

tern

ativ

eco

nd

itio

ns

seve

ralc

lon

esar

ere

po

rted

wit

ho

ut

SEd

ue

tote

chn

ical

diffi

cult

ies

wit

hre

plic

ates

Adaptation in Hybrids doi101093molbevmsx098 MBE

5

Tab

le2

Mu

tati

on

san

dFi

tnes

so

fEv

olv

edPa

ren

talC

lon

es

Clo

ne

Loca

tio

nG

ene(

s)M

uta

tio

nSp

ecie

sG

ener

atio

ns

Rel

ativ

eFi

tnes

s6

SE

Gc1

chrX

IV0

561

000

6322

507

8433

329

8ge

nes

79

gen

esC

NV

(am

plifi

cati

on

of

chr

14L

favo

rin

gG

RF1

67

del

etio

no

fch

r14R

)ce

r16

316

42

63

42

chrV

160

000

576

874

220

gen

esLO

H(f

avo

rsG

RF1

67)

Gc2

chrV

431

750

576

874

71ge

nes

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

167

103

66

058

chrX

V7

1000

01

0912

9119

6ge

nes

LOH

CN

V(m

on

oso

my

favo

rin

gS2

88C

)G

u1

chrX

V59

7ge

nes

CN

V(w

ho

lech

rom

oso

me

amp

lifica

tio

n)

uva

468

180

36

212

chrI

I911

925

917

281

HX

T6

7C

NV

(am

plifi

cati

on

)ch

rXV

385

930

NEL

1N

on

syn

on

ymo

us

N12

9Ich

rII9

1190

9In

terg

enic

par

to

fth

eH

XT

67

amp

lifica

tio

nG

u2

chrX

V59

7ge

nes

CN

V(w

ho

lech

rom

oso

me

amp

lifica

tio

n)

uva

486

131

2ch

rII9

1192

59

1728

1H

XT

67

CN

V(a

mp

lifica

tio

n)

chrI

V1

0029

3R

GT

2N

on

syn

on

ymo

us

G10

7Vch

rV4

2093

FRD

1N

on

syn

on

ymo

us

G12

8Ach

rII9

1719

1H

XT

7Sy

no

nym

ou

sH

53H

chrX

I155

787

Inte

rgen

icP

c1ch

rXII

I03

9000

(LO

H)

01

9662

8(C

NV

3co

pie

s)

1966

283

7300

0(C

NV

2co

pie

s)LO

H1

5ge

nes

incl

ud

ing

PH

O84

C

NV

201

gen

esLO

HC

NV

(am

plifi

cati

on

fav

ori

ng

GR

F167

)ce

r15

221

22

60

81

Pc2

chrX

III0

411

00(L

OH

)0

196

628

(CN

V3

cop

ies)

19

6628

373

000

(CN

V2

cop

ies)

LOH

16

gen

esin

clu

din

gP

HO

84

CN

V2

01ge

nes

LOH

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

149

181

36

103

chrV

III5

2034

9In

terg

enic

Pc3

chrX

III0

390

00(L

OH

)0

196

628

(CN

V3

cop

ies)

19

6628

373

000

(CN

V2

cop

ies)

LOH

15

gen

esin

clu

din

gP

HO

84

CN

V2

01ge

nes

LOH

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

127

194

9

Pc4

chrX

III0

855

00(L

OH

)0

196

628

(CN

V3

cop

ies)

19

6628

373

000

(CN

V2

cop

ies)

LOH

40

gen

esin

clu

din

gP

HO

84

CN

V2

01ge

nes

LOH

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

132

209

66

141

chrX

II2

6400

01

0781

7743

7ge

nes

LOH

(fav

ori

ng

S288

C)

chrX

V1

0231

97P

IP2

No

nsy

no

nym

ou

sE6

QP

u1

uva

240

1

686

110

Pu

2ch

rIX

144

80Y

PS6

5rsquo-u

pst

ream

uva

234

213

06

073

chrI

X2

2531

4SE

C6

No

nsy

no

nym

ou

sI1

84L

chrX

III

1295

67T

CB

3N

on

syn

on

ymo

us

E625

GSc

1ch

rXIV

01

0200

0(C

NV

3co

pie

s)6

3200

07

8433

3(C

NV

1co

py)

LO

H1

0000

07

8433

348

gen

es7

9ge

nes

367

gen

esLO

HC

NV

(am

plifi

cati

on

of

chr

14L

del

etio

no

fch

r14R

LO

Hfa

vori

ng

S288

C)

cer

182

380

66

175

chrV

III2

0796

7SM

F2N

on

syn

on

ymo

us

W10

5Sch

rXII

I190

000

196

500

RR

N11

CA

T2

VP

S71

LOH

CN

V(d

elet

ion

fav

ori

ng

GR

F167

)ch

rII7

8718

07

9735

0V

BA

1SU

L1P

CA

1C

NV

(am

plifi

cati

on

)Sc

2ch

rXII

578

gen

esC

NV

(wh

ole

chro

mo

som

eam

plifi

cati

on

fa

vori

ng

GR

F167

)ce

r17

640

21

61

33

chrX

II6

9200

01

0781

7719

3ge

nes

LOH

(fav

ori

ng

GR

F167

)ch

rII7

7322

08

1318

418

gen

esin

clu

din

gSU

L1C

NV

(am

plifi

cati

on

)Sc

3ch

rVI9

4104

FRS2

No

nsy

no

nym

ou

sV

303I

cer

201

413

46

677

chrV

III3

0890

3T

RA

1N

on

syn

on

ymo

us

V20

48A

chrX

IV2

3226

6P

OP

1N

on

syn

on

ymo

us

S477

ch

rXV

291

219

TLG

2N

on

syn

on

ymo

us

D28

6Ych

rXV

309

86H

PF1

Syn

on

ymo

us

T20

7Tch

rII7

8180

07

9223

05

gen

esin

clu

din

gB

SD2

and

SUL1

CN

V(a

mp

lifica

tio

n)

(co

nti

nu

ed)

Smukowski Heil et al doi101093molbevmsx098 MBE

6

Tab

le2

Co

nti

nu

ed

Clo

ne

Loca

tio

nG

ene(

s)M

uta

tio

nSp

ecie

sG

ener

atio

ns

Rel

ativ

eFi

tnes

s6

SE

Sc4

chrI

I275

000

813

184

289

gen

esLO

H(f

avo

rin

gG

RF1

67)

cer

190

312

56

613

chrI

I788

608

795

833

SUL1

PC

A1

CN

V(a

mp

lifica

tio

n)

chrX

I517

650

666

816

68ge

nes

CN

V(a

mp

lifica

tio

n)

chrX

III1

9000

01

9650

0R

RN

11C

AT

2V

PS7

1LO

HC

NV

(del

etio

nf

avo

rin

gG

RF1

67)

chrX

IV6

3200

07

8433

379

gen

esLO

HC

NV

(del

etio

n)

chrX

V3

3670

03

4200

034

2000

109

1291

2ge

nes

384

gen

esLO

H(f

avo

rin

gG

RF1

67f

avo

rin

gS2

88C

)ch

rIX

233

67C

SS1

No

nsy

no

nym

ou

sD

914N

Su1

chrX

177

350

345

680

96ge

nes

incl

ud

ing

SUL2

CN

V(a

mp

lifica

tio

n)

uva

557

218

62

37(S

anch

eze

tal

201

7)ch

rXV

I466

649

DIG

1N

on

syn

on

ymo

us

E49Q

chrV

188

548

Inte

rgen

icSu

2ch

rX1

7735

03

4568

096

gen

esin

clu

din

gSU

L2C

NV

(am

plifi

cati

on

)ch

rIV

803

704

KT

R3

5rsquo-u

pst

ream

chrI

I121

779

PIN

4N

on

syn

on

ymo

us

N26

3Sch

rVII

165

902

MP

T5

No

nsy

no

nym

ou

sQ

618K

chrI

I836

169

RSC

3Sy

no

nym

ou

sR

4Rch

rIV

107

948

UFD

2Sy

no

nym

ou

sG

691G

chrI

II2

8761

8In

terg

enic

Poin

tm

uta

tio

ns

cop

yn

um

ber

vari

ants

(CN

V)

and

loss

ofh

eter

ozy

gosi

tyev

ents

(LO

H)

are

reco

rded

for

each

evo

lved

par

enta

lclo

ne

Clo

nes

are

iden

tifi

edb

yn

utr

ien

t(G

glu

cose

limit

atio

nP

ph

osp

hat

elim

itat

ion

an

dS

sulfa

telim

itat

ion

)b

ysp

ecie

s(ldquo

crdquod

eno

tes

Sce

revi

siae

ldquou

rdquod

eno

tes

Suv

arum

)an

dth

en

um

ber

ind

icat

esit

sd

eriv

atio

nfr

om

ind

epen

den

tp

op

ula

tio

ns

No

teth

atm

uta

tio

ns

inth

eS

uvar

umge

no

me

use

Suv

arum

chro

mo

som

esan

dco

ord

inat

esA

llb

reak

po

ints

wer

eca

lled

by

visu

alin

spec

tio

no

fse

qu

enci

ng

read

san

dar

eth

us

app

roxi

mat

eR

elat

ive

fitn

ess

isre

po

rted

wit

hst

and

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Adaptation in Hybrids doi101093molbevmsx098 MBE

7

had an average of 15 CNV per clone and the evolvedS uvarum clones had an average of 1 CNV per clone (table 2supplementary figs S1 and S2 Supplementary Material online)The most common event across nutrient limitations in theinterspecific hybrids was an amplification of the S cerevisiaecopy of chromosome IV which occurred in four

independent hybrid clones (three in phosphate limitationone in glucose limitation supplementary fig S3Supplementary Material online) Several other characteris-tic rearrangements occurred in the evolved S cerevisiaeclones including the amplification of the left arm of chro-mosome 14 accompanied by segmental monosomy of the

FIG 1 Evolved hybrids exhibit changes in copy number and loss of heterozygosity Copy number variants are displayed for selected evolved hybridclones from three nutrient-limited conditions Gh2 glucose Ph4 phosphate and Sh4 sulfate See additional figures in supplementary fig S3Supplementary Material online Hybrid copy number determined by normalized sequencing read depth per open reading frame (ORF) is plottedacross the genome according to S cerevisiae ORF coordinates to account for three reciprocal translocations between S cerevisiae and S uvarumChromosomes are plotted in alternating light and dark purple red indicates a S cerevisiae copy number variant and blue indicates a S uvarumcopy number variant Gh2 has a whole chromosome amplification of S cerevisiae chrIV a small segmental deletion of S uvarum chrIV (non-copyneutral loss of heterozygosity) and an amplification of S uvarum HXT67 Ph4 has a small segmental deletion of S cerevisiae chrIII (non-copyneutral loss of heterozygosity) and an amplification of S cerevisiae chrXIII with corresponding deletion of S uvarum chrXIII (copy neutral loss ofheterozygosity) Sh4 has an amplification of S cerevisiae SUL1 and a whole chromosome amplification of S uvarum chrVIII (note there is areciprocal translocation between chrVIII and chrXV in S uvarum relative to S cerevisiae) Note that Sh4 is plotted on a different scale For specificcoordinates of copy number variants see table 1

Smukowski Heil et al doi101093molbevmsx098 MBE

8

right arm of chromosome 14 an event seen previously inother evolved populations (Dunham et al 2002 Greshamet al 2008 Sunshine et al 2015) All copy number events in

S cerevisiae had break points at repetitive elements knownas Ty elements except those located on chrII which maybe mediated by another mechanism (Brewer et al 2015) In

FIG 2 Repeated loss of heterozygosity at the PHO84 locus in intra- and interspecific hybrids (A) The 25-kb region extending from the left telomereof chromosome XIII to the high-affinity phosphate transporter gene PHO84 (B) Copy number is plotted across part of chromosome XIII in thehybrid ancestor and three evolved hybrid clones in phosphate limitation (clone indicated in upper right corner) Red shows the S cerevisiae alleleblue shows the S uvarum allele and purple shows where both species exhibit the same copy number Note 8 kb of telomere sequence is removeddue to repetitive sequence (C) Alternate allele frequency is plotted for a portion of chromosome XIII in the ancestor and four evolved S cerevisiaeclones in phosphate limitation (clone indicated in upper right corner) All evolved S cerevisiae clones exhibit a loss of heterozygosity at thetelomeric portion of chromosome XIII (loss of S288C amplification of GRF167) as illustrated by an allele frequency of zero compared with theancestor Regions of heterozygosity are interspersed with regions of homozygosity as one of the parents of the diploid was itself the product of across between strains FL100 and S288C and the other parent was S288C Regions of heterozygosity are due to FL100 haplotypes S cerevisiae copynumber for the four evolved clones is shown below the ancestor is diploid across the chromosome (also see table 2 supplementary fig S1Supplementary Material online)

Adaptation in Hybrids doi101093molbevmsx098 MBE

9

contrast copy number variants in the hybrid were rarely facil-itated by repetitive elements perhaps in part because S uva-rum has no full length Ty elements however why S cerevisiaeTy elements and long terminal repeat (LTR) sequences fromeither genome were not utilized remains unknown

Frequently in nutrient-limited evolution experimentscopy number variants involve amplification of the nutrient-specific transporter and indeed we also observed amplifica-tion of these transporters in many of the clones In sulfatelimitation the S cerevisiae allele of the high-affinity sulfatetransporter gene SUL1 is amplified in 77 hybrid clones and 44 S cerevisiae clones (fig 1 tables 1 and 2 supplementary figsS1 and S3 Supplementary Material online) InterestinglySUL2 is the preferred sulfate transporter in S uvarum(Sanchez et al 2017) and was not observed to be amplifiedin the evolved hybrids (supplementary fig S2 and table S2Supplementary Material online) In glucose limitation previ-ous S cerevisiae evolution experiments found frequent am-plification of the high-affinity glucose transporter genesHXT67 (Brown et al 1998 Dunham et al 2002 Greshamet al 2008 Kao and Sherlock 2008 Kvitek and Sherlock2011) In our experiments the S uvarum alleles of theHXT67 transporters are amplified in 33 hybrid clones andboth S uvarum clones but are not amplified in evolved Scerevisiae clones This is suggestive that the S uvarum HXT67alleles confer a greater fitness advantage compared with Scerevisiae alternatively the genomic context could be morepermissive to amplification in S uvarum (fig 1 tables 1 and 2supplementary figs S1ndashS3 Supplementary Material online)Finally in phosphate limitation the S cerevisiae copy of thehigh-affinity phosphate transporter gene PHO84 is amplifiedand the S uvarum allele is lost in 36 hybrid clones in an eventknown as LOH (figs 1 and 2 table 1 supplementary fig 3Supplementary Material online) Intriguingly the evolved Scerevisiae clones also display LOH and accompanied amplifi-cation favoring the allele derived from strain GRF167 over theS288C allele in 44 clones (fig 2 table 2) All hybrid clonescarry the ldquopreferredrdquo GRF167 S cerevisiae allele as this was theallele used to create the de novo hybrid

Loss of Heterozygosity Is a Common Event inHeterozygous Evolving PopulationsSelection on heterozygosity as a LOH event could representis an underappreciated source of adaptation in microbial ex-perimental evolution as typical experiments evolve a haploidor homozygous diploid strain asexually and as a result havelittle variation to select upon LOH is observed in natural andindustrial hybrids (Albertin and Marullo 2012 Louis et al2012 Pryszcz et al 2014 Wolfe 2015 Schroder et al 2016)but here we document its occurrence in both intra- andinterspecific hybrids in the laboratory as a result of short-termevolution (also see (Dunn et al 2013 Burke et al 2014)) LOHis observed across all nutrient conditions with 13 independentLOH events detected in S cerevisiae and 9 independent eventsdocumented in the hybrids (figs 1 and 2 tables 1 and 2 sup-plementary figs S1 and S3 Supplementary Material online) Itthus appears that this type of mutational event is both com-mon and can occur over short evolutionary timescales

The LOH event can result in copy-neutral (where one alleleis lost and the other allele is amplified) or non-neutral chro-mosomal segments (where one allele is lost rendering thestrain hemizygous at that locus) and can favor the retentionof either allele In S cerevisiae there is a bias in resolutionwhere LOH events favor retaining the GRF167 allele over theS288C allele (1013 events Pfrac14 00169 table 2 supplementaryfig S4 Supplementary Material online) One unique case inclone Sc4 has a small approximately 5 kb LOH event onchrXV favoring GRF167 which switches to favoring S288Cfor the rest of the chromosome The retention of S cerevisiaeis slightly more common in the hybrids (59 events Pfrac14 10table 1 supplementary fig S3 Supplementary Material on-line) though not as drastic as the observed genome resolu-tion in the hybrid S pastorianus where LOH favorsS cerevisiae over S eubayanus (Nakao et al 2009) The sizeof the event ranges from approximately 25 kb to the wholechromosome level in the hybrids and from 5 kb to 540 kbin S cerevisiae Where LOH is accompanied by an amplifi-cation event the LOH event always occurs first and doesnot share break points with the amplification event Unlikemany CNV events most LOH events do not appear to bemediated by existing repetitive sequence such as a trans-posable element in the hybrid or S cerevisiae and are mostlikely a product of break-induced replication or mitoticgene conversion with break-induced replication as the fa-vored method as all events extend to the telomere (Hoanget al 2010) The exceptions are in hybrid clones Ph4 Ph5 andSh1 where there is a non-copy neutral loss of S cerevisiaemediated by a Ty element or LTR and S cerevisiae clonesSc1 and Sc4 where there is a 65-kb deletion of the S288Callele flanked by two Ty elements

LOH events in hybrids could signify several ongoing pro-cesses in hybrid genome evolution LOH regions may repre-sent 1) loci with incompatibilities 2) selection on existingvariation or 3) genetic drift eroding genomic segments It isimpossible to definitively rule out any of these hypotheseswithout further experimentation however there are severalarguments disfavoring the incompatibility hypothesis Firstalthough our sample size is modest failing to see repeatedLOH events across nutrient conditions may indicate a lack ofgeneral incompatibility between species (although we cannotpreclude condition-specific incompatibility Hou et al 2015Piatkowska et al 2013) This is consistent with previous stud-ies in yeast which suggest that classic DobzhanksyndashMullerincompatibilities are rare (Liti et al 2006 Greig 2009 Houet al 2014) Furthermore LOH events observed inevolved S cerevisiae suggest that this mutation type isnot unique to interspecific hybrids Instead repeatedevents within a particular condition such as the repeatedLOH at PHO84 in phosphate limitation or the 65 kbsegment on chrXIII in sulfate limitation suggest thatthese events are beneficial and are indeed selection onone allele over the other

LOH Is Driven by Selection on One AlleleTo test the hypothesis that LOH events provide a selectiveadvantage we used allele replacement in which the allele of

Smukowski Heil et al doi101093molbevmsx098 MBE

10

one speciesstrain is replaced with the allele of the otherspeciesstrain in an otherwise isogenic background We testedthis hypothesis using the most commonly seen LOH eventLOH at PHO84 Although the region extends from 25 kb to234 kb in length in the hybrids and from 40 kb to 85 kb in Scerevisiae and thus includes many genes PHO84 was a primecandidate driving this event PHO84 is one of only ten genesencompassed in the region extending from the telomere tothe break point of the shortest LOH event and is included inevery other LOH event on chromosome XIII (fig 2) It is ahigh-affinity phosphate transporter responsible for inorganicphosphate uptake in high and low phosphate conditions(Wykoff and OrsquoShea 2001) and previous work identified apoint mutation in PHO84 (an alanine to valine substitution atthe 50 end of the gene) which increased fitness by 23 inphosphate-limited conditions (Sunshine et al 2015) Finallyprior work with other nutrient transporters has shown am-plification of nutrient transporters to be a key event in adapt-ing to nutrient-limited conditions

We thus selected a region of approximately 25 kb encom-passing the PHO84 ORF its promoter and 30-UTR(Nagalakshmi et al 2008 Yassour et al 2009 Cherry et al2012) We created allele replacement strains using the twoalleles of S cerevisiae in a S cerevisiae diploid background thetwo alleles are 991 identical in this region (supplementaryfig S5 Supplementary Material online) and each strain isidentical to the ancestral strain used in our evolution exper-iments except at the PHO84 locus The S cerevisiae ancestorcarries one copy of GRF167 (preferred) and one copy ofS288C (ldquoun-preferredrdquo) so named due to which allele wasretained and amplified in the evolved clones To measureany resultant changes in fitness we competed each strainindividually against a fluorescent ancestral strain and mea-sured which strain overtook the culture Relative fitness isthus defined as the growth advantage per generation Twocopies of the un-preferred allele decreased relative fitness by531 (6186) whereas two copies of the preferred alleleincreased relative fitness by 993 (6027) This displays anoverall difference in relative fitness of 1524 between the un-preferred and preferred alleles By comparing the fitness ofthese allele replacement strains with the evolved clones (table 2)the allele replacement does not fully recapitulate the fit-ness gain observed in the evolved clone One explanation isthat the additional mutations present in the evolvedstrains also contribute to their total fitness Another expla-nation could be the increased copy number of the PHO84region that we see in these evolved clones To further ex-plore this fitness difference we cloned the GRF167 alleleonto a low copy number plasmid and transformed theallele replacement strain carrying two preferred S cerevi-siae alleles to simulate increased copy number of PHO84and saw only a minimal fitness increase of 176 (6106note all fitness competitions involving plasmids were donein conjunction with empty plasmids to take into accountany fitness effects from the plasmid itself) This supportsthe conclusion that relative fitness gains in the evolvedclone are largely due to the loss of the S288C allele andselection and amplification of the GRF167 allele with little

additional benefit from further amplification It could also bethe case that co-amplification of other genes in the segmentis required to attain the full benefit as previously observedby the contribution of BSD2 to the SUL1 amplicon in sulfatelimitation (Sunshine et al 2015 Payen et al 2016)

To understand the fitness effects of LOH and amplificationin the hybrids we generated hybrid strains with varying num-bers of S cerevisiae GRF167 PHO84 alleles Unfortunately wewere unable to obtain a successful strain carrying the pre-ferred S cerevisiae allele in a S uvarum background howeverwe were able to generate a S uvarum PHO84 knockout strainthus creating a hybrid with one copy of S cerevisiae PHO84When combined with a low copy plasmid carrying the sameallele this strain effectively has two or more copies ofS cerevisiae PHO84 in a hybrid background This hybrid straincan serve as a proxy for the LOH event observed in the evo-lution experiments and has a relative fitness gain of 2557(6288) The ancestral hybrid with the same plasmid (effec-tively 1 S uvarum allele and 2 or more S cerevisiae alleles) hasa relative fitness gain of 1253 (6131) The difference be-tween these two hybrids suggests that while amplification isbeneficial the highest fitness is achieved with the loss of theS uvarum allele (Pfrac14 00061)

Together these results support the conclusion that theS cerevisiae GRF167 allele is preferred over the S288C alleleand that S cerevisiae alleles are preferred over the S uvarumallele in the hybrid and hence that the LOH events seen inboth intra- and interspecific hybrids are the product ofselection

DiscussionIn summary we sought to understand forces underlying ge-nome stabilization and evolution in interspecific and intra-specific hybrids as they adapt to novel environments Weevolved and sequenced clones from 16 hybrid populationsand 16 parental populations to reveal a variety of mutationalevents conferring adaptation to 3 nutrient-limited conditionsOf particular note we find LOH in both evolved intraspecificand interspecific hybrid clones in all nutrient environmentspotentially signifying areas where selection has acted on pre-existing variation present in the ancestral clone We used anallele replacement strategy to test this hypothesis for a com-monly repeated LOH event and show that selection is indeeddriving the homogenization of the genome at this locusAlthough other studies in natural industrial and lab-evolved isolates have observed LOH we present the first em-pirical test of the causal evolutionary forces influencing theseevents This work can begin to help us understand past hy-bridization events and subsequent genome resolution in hy-brids in natural and artificial systems

Similarities and Differences between Intra- andInterspecific HybridsAlthough our sample size is modest we observe several in-teresting trends when comparing S uvarum clones (homo-zygous) S cerevisiae clones (intraspecific hybrid andpreviously published homozygous) and interspecific hybrid

Adaptation in Hybrids doi101093molbevmsx098 MBE

11

clones First theory and previous research predict the inter-specific hybrid may experience more genome instability in theform of chromosomal rearrangements and CNV events(Xiong et al 2011 Lloyd et al 2014 Chester et al 2015Mason and Batley 2015) Instead in our work the interspecifichybrid and S cerevisiae clones experience the same numberof CNV events (both 15 CNVsclone) However the mech-anism of CNV formation seems to differ between the hybridand S cerevisiae clones Whereas S cerevisiae CNV breakpoints typically occur at transposable elements in our studyand previous studies (Dunham et al 2002 Gresham et al2008 Fedoroff 2012) the interspecific hybrid CNVs do notutilize transposable elements although they obviously sharethe same sequence background as the S cerevisiae clones(albeit in one copy) Whether this difference is due to theabsence of full-length transposable elements in S uvarum isunknown but this could potentially explain the lower num-ber of CNV events in S uvarum clones (1 CNVclone) andpresents an intriguing direction for future study

The Predictability of EvolutionWe now have many examples of predictable evolution innatural systems (Losos et al 1998 Rundle et al 2000 Elmerand Meyer 2011 Conte et al 2012 Jones et al 2012 Martinand Orgogozo 2013 Wessinger and Rausher 2014) and inlaboratory experimental evolution in which there often ap-pears to be a limited number of high fitness pathways thatstrains follow when adapting to a particular condition (Fereaet al 1999 Woods et al 2006 Gresham et al 2008 Burke et al2010 Salverda et al 2011 Kawecki et al 2012 Kvitek andSherlock 2013 Lang and Desai 2014) For example it is wellestablished that amplifications of nutrient transporters aredrivers of adaptation in evolution in nutrient-limited condi-tions Previous work in our group has particularly focused onthe amplification of the high-affinity sulfate transporter geneSUL1 in sulfate-limited conditions which occurs in almostevery sulfate-limited evolution experiment and confers a fit-ness advantage of as much as 40 compared with the un-evolved ancestor strain The amplification of phosphatetransporters has been markedly less common and thus driv-ers of adaptation in this condition have been less clearGresham et al (2008) identified a whole chromosome ampli-fication of chrXIII in one population In a follow-up studySunshine et al (2015) found whole or partial amplification ofchrXIII in 38 populations A genome-wide screen for segmen-tal amplifications found a slight increase in fitness for a smalltelomeric segment of chromosome XIII and a A49V pointmutation in PHO84 was observed to increase fitness by 23However screens by Payen et al (2016) showed that althoughPHO84 is recurrently mutated in various experiments itshowed no benefit when amplified or deleted in phos-phate-limited conditions Finally additional evolution exper-iments recapitulated the point mutation seen in Sunshineet al (2015) in 2432 populations and saw amplification ofPHO84 in 832 populations (Miller A Dunham MJ unpub-lished data) It is important to note that all these experimentsused a strain background derived from S288C or CENPKboth of which carry the same (un-preferred) PHO84 allele

In our work we observed amplification of the S cerevisiaeGRF167 allele of PHO84 in 44 S cerevisiae clones from 4populations and 36 hybrid clones from 6 populations Thisamplification was always preceded by the loss of the S288Callele in S cerevisiae clones and the LOH break points arenever shared with the amplification break points There is a15 fitness difference between carrying two copies of theS288C allele of PHO84 compared with carrying two copiesof the GRF167 allele of PHO84 and additional copies of theGRF167 allele do not provide substantial further fitness gainsThe two alleles differ by several noncoding changes and threenonsynonymous substitutions a mutation from glutamicacid to aspartic acid (E229D) leucine to proline (L259P)and leucine to glutamine (L556Q) the latter two of whichare considered ldquononconservativerdquo protein mutations due tochanges in hydrophobicity and structure (supplementary figS5 Supplementary Material online) Intriguingly the L259Pmutation has actually been previously identified as being re-sponsible for resistance to tetrachloroisophthalonitrile andpartial resistance to pentachlorophenol in a QTL study ofsmall-molecule drugs (Perlstein et al 2007) IndeedPerlstein et al found proline at residue 259 to be conservedacross fungal species and even in orthologous human xeno-biotic transporters likely because proline-induced kinks intransmembrane spans have been shown to be essential toprotein function (Cordes et al 2002 Perlstein et al 2007) Itthus appears that amplification of PHO84 has been less pre-dictable as the S288C allele does not confer a fitness advan-tage unless mutated per Sunshine et al (2015) Togetherthese results imply that strain background can constrainadaptive pathways

In hybrids the amplification of the S cerevisiae segmentoccurred in conjunction with the loss of the S uvarum alleleHybrid strains with the LOH had a 2557 relative fitness gainwhereas hybrid strains with amplification of the S cerevisiaePHO84 allele without the LOH had a 1253 fitness gain ThusLOH confers an additional 1304 fitness gain showing thatselection for LOH has a larger impact on fitness than ampli-fication alone Note that S uvarum does have proline at res-idue 259 like the preferred GRF167 allele and differs fromboth S cerevisiae alleles at the other two coding substitutions(229N and 556K) but the amino acid and noncoding diver-gence is too high to speculate what substitutions are respon-sible for the selection of the GRF167 allele (supplementary figS6 Supplementary Material online) Why the loss of one alleleis more beneficial remains unclear as PHO84 is thought tofunction as a monomer (Bun-Ya et al 1991) but it may bedue to competition for cell wall space or negative interactionswith other genes in the PHO pathway (Mouillon and Persson2006)

The infusion of variation created by hybridization providesnew templates for selection to act upon which can be moreimportant than either point mutations or copy number var-iants alone Our work shows that outcrossing need not becommon to have long-lasting effects on adaptation This im-plication is particularly relevant in yeast where outcrossingmay occur quite rarely followed by thousands of asexual gen-erations (Ruderfer et al 2006 Greig and Leu 2009 Liti 2015)

Smukowski Heil et al doi101093molbevmsx098 MBE

12

Applications to Other Hybrids and CancerThe observation that LOH occurs in hybrid genomes is in-creasingly documented (Louis et al 2012 Borneman et al2014 Soltis et al 2014 Marcet-Houben and Gabaldon 2015Pryszcz et al 2014 Schroder et al 2016) although the rea-son(s) for this type of mutation has been unresolved As mostexamples stem from allopolyploid events that occurred mil-lions of years ago understanding why LOH is important inhybrid genome evolution is difficult Cancer cells are alsoknown to experience LOH sometimes involved in the inac-tivation of tumor suppressor genes leaving only one copy ofthe gene that may be mutated or silenced (Thiagalingam et al2001 Tuna et al 2009 Lapunzina and Monk 2011) Datasupport the conclusion that LOH events are selected for dur-ing tumor development as many LOH events involve specificchromosomal segments (Thiagalingam et al 2001) althoughthe underlying molecular and genetic reasons for selection isan open debate (Ryland et al 2015)

Here we experimentally demonstrate that LOH can occurin homoploid interspecific hybrids as well as in intraspecifichybrids These events occur within a few hundred generationsand are common mutations more common on average inthe intraspecific hybrid (13 eventsclone) than the interspe-cific hybrid (056 eventsclone) Interestingly the LOH eventsdo not share break points with the CNV events in S cerevisiaeinstead they appear to occur independently and to precedeany subsequent amplification (amplification occurs following913 LOH events) Competition assays with the PHO84 locusprovide support that LOH itself may be more beneficial thanamplification or at least increase the selective benefit of am-plification events The observation that LOH in intraspecifichybrids occurs independently from copy number change pro-vides different opportunities for adaptation to novelconditions

Other cases of LOH like the copy number neutral eventsobserved in Ph4 Ph5 and Sh4 all of which favor the retentionof the S uvarum allele (supplementary fig S3 SupplementaryMaterial online) may be due to hybrid incompatibility withina particular protein complex other epistatic interactions(Piatkowska et al 2013) or neutral processes We furthermorediscover examples where one species allele appears to bepreferred over the other without LOH such as the repeatedamplification of the S uvarum high-affinity glucose transpor-ters HXT67 When one species allele is amplified and theother is not amplified one explanation is that the local se-quence context can permit or deny amplification In the caseof HXT67 previous experiments in S cerevisiae have shownthat amplification of HXT67 is quite common (Brown et al1998 Dunham et al 2002 Gresham et al 2008 Kao andSherlock 2008 Kvitek and Sherlock 2011) thus suggestingthat when given a choice between this locus in S cerevisiaeor S uvarum in the hybrid the preferred allele is indeed Suvarum though more subtle differences in rate cannot yet beruled out A similar scenario is observed with S cerevisiaeSUL1 (Sanchez et al 2017) Together our results show thatthe heterozygosity supplied by hybridization is an importantcontributor to adaptive routes explored by populations asthey adapt to novel conditions

Although we cannot generalize our results from thePHO84 locus across the many other LOH events discoveredin our hybrids and S cerevisiae in the future we can usesimilar methodology to explore whether positive selectionalways drives LOH or whether other explanations such asincompatibility resolution contribute as well Future experi-ments might also utilize a high throughput method to ex-plore segmental LOH in hybrids at a genome-wide scalesimilar to ongoing experiments at the gene level (LancasterS Dunham MJ unpublished data) Although our sample sizeis moderate this is a novel and necessary step in understand-ing forces underlying hybrid genome stabilization and high-lighting an underappreciated mechanism of hybridadaptation

ConclusionsThe mutation events we observe in our experimentallyevolved hybrids are in many ways quite representative ofmutations observed in ancient hybrid genomes suggestingthat hybrid genome stabilization and adaptation can occurquite rapidly (within several hundred generations)Furthermore our results illustrate that the infusion of varia-tion introduced by hybridization at both the intra- and inter-species level can increase fitness by providing choices of allelesfor selection to act upon even when sexual reproduction israre This may be particularly important for leveraging existingvariation for agricultural and industrial processes and as cli-mate change potentially increases natural hybridization (Kellyet al 2010 Hoffmann and Sgro 2011 Muhlfeld et al 2014)

Materials and Methods

StrainsA list of strains used in this study is included in supplementarytable S1 Supplementary Material online All interspecific hy-brids were created by crossing a ura3 LYS2 haploid parent to aURA3 lys2 haploid parent of the other mating type plating onmedia lacking both uracil and lysine and selecting for proto-trophs The S cerevisiae strain background known asldquoGRF167rdquo is itself a cross between FL100 and the genomictype strain S288C (data not shown) GRF167 was chosen as astrain background for simultaneous work investigating trans-posable elements during experimental evolution which willbe addressed in a future study

Evolution ExperimentsContinuous cultures were established using media and con-ditions previously described (Gresham et al 2008 Sanchezet al 2017) Detailed protocols and media recipes are availableat httpdunhamgswashingtoneduprotocolsshtml (lastaccessed March 6 2017) Samples were taken daily and mea-sured for optical density at 600 nm and cell count micros-copy was performed to check for contamination andarchival glycerol stocks were made daily An experimentwas terminated when contamination growth in tubing orclumping appeared (number of generations at the endpoint for each population are presented in tables 1 and

Adaptation in Hybrids doi101093molbevmsx098 MBE

13

2) Samples from each end point population were colonypurified to yield two clones for further study

Array Comparative Genomic HybridizationPopulations from the end point of each evolution were ana-lyzed for copy number changes using array comparative ge-nomic hybridization following the protocol used in Sanchezet al (2017)

SequencingDNA was extracted from overnight cultures using theHoffmanndashWinston protocol (Hoffman and Winston 1987)and cleaned using the Clean amp Concentrator kit (ZymoResearch) Nextera libraries were prepared following theNextera library kit protocol and sequenced using paired end150 bp reads on the Illumina NextSeq 500 machine (sequenc-ing coverage in supplementary table S2 SupplementaryMaterial online) The reference genomes used were S cerevi-siae v3 (Engel et al 2014) S uvarum (Scannell et al 2011) anda hybrid reference genome created by concatenating the twogenomes Sequence was aligned to the appropriate referencegenome using bwa v062 (Li and Durbin 2009) and mutationswere called using GATK (McKenna et al 2010) and samtools0119 (Li et al 2009) Mutations in evolved clones were filteredin comparison with the ancestor to obtain de novo mutationsAll mutations were first visually inspected using IntegrativeGenomics Viewer (Robinson et al 2011) Subsequently pointmutations in the hybrids were confirmed with Sanger se-quencing (supplementary table S3 Supplementary Materialonline) Copy number variants were visualized usingDNAcopy for S cerevisiae and S uvarum (Seshan andOlshen 2016) LOH events were called based on sequencingcoverage in the hybrids and by identifying homozygous variantcalls in S cerevisiae All break points were called by visual in-spection of sequencing reads and are thus approximate

Fitness AssaysThe pairwise competition experiments were performed in 20ml chemostats (Miller and Dunham 2013) Each competitorstrain was cultured individually until steady state was reachedand then was mixed 5050 with a GFP-tagged ancestor Eachcompetition was conducted in at least two biological repli-cates for approximately 15 generations after mixing Sampleswere collected and analyzed twice daily The proportion ofGFPthorn cells in the population was detected using a BD AccuriC6 flow cytometer (BD Biosciences) The data were plottedwith ln [dark cellsGFPthorn cells] versus generations The rela-tive fitness coefficient was determined from the slope of thelinear region

Strain ConstructionAllele replacements for the PHO84 locus were done followingthe protocol of the Caudy lab with further modifications de-scribed here The native locus was replaced withKluyveromyces lactis URA3 The pho84DURA3 strain wasgrown overnight in 5 ml of C-URA media then inoculatedin a flask of 100 ml yeast extract peptone dextrose (YPD) andgrown to an optical density of 06ndash08 Cells were washed then

aliquoted 275 ml of transformation mix (35 ml 1 M lithiumacetate 240 ml of 50 3500 polyethylene glycol) 10 ml ofsalmon sperm and approximately 3 mg of polymerase chainreaction product were added to the cell pellet It was incu-bated at 37 C (S uvarum) or 42 C (S cerevisiae) for 45 minthen plated to YPD It was replica plated to 5-fluorooroticacid the following day and colonies were tested for the gain ofthe appropriate species allele The GRF167 allele was clonedinto the pIL37 plasmid using Gibson assembly (Gibson et al2009) Correct assembly was verified by Sanger sequencing Allprimers used can be found in supplementary table S3Supplementary Material online

Supplementary MaterialSupplementary data are available at Molecular Biology andEvolution online

AcknowledgmentsWe thank Noah Hanson and Erica Alcantara for technicalassistance We thank Monica Sanchez and three anonymousreviewers for helpful comments on this manuscript JosephSchacherer kindly confirmed the contribution of FL100 to theGRF167 strain background Thanks to Yixian Zheng and DougKoshland for contributing to the initial experimental designcreating yeast strains and purchasing the oligonucleotidesused for the microarrays This work was supported by theNational Science Foundation (grant number 1516330) andthe National Institutes of Health (grant number R01GM094306) MD is a Senior Fellow in the GeneticNetworks program at the Canadian Institute for AdvancedResearch and a Rita Allen Foundation Scholar CSH wassupported in part by National Institutes of Health (grantnumber T32 HG00035) This work was also supported bythe National Institutes of Health (grant number P50GM071508) to the Lewis-Sigler Institute and from theHoward Hughes Medical Institute to Doug Koshland andYixian Zheng Sequencing read data have been deposited atthe NCBI under BioProject accession PRJNA374049Microarray data are deposited in the Gene ExpressionOmnibus (GEO) repository under accession numbersGSE95086 and GSE87401 and in the Princeton MicroarrayDatabase

ReferencesAbbott R Albach D Ansell S Arntzen JW Baird SJ Bierne N Boughman J

Brelsford A Buerkle CA Buggs R et al 2013 Hybridization andspeciation J Evol Biol 26229ndash246

Ainouche ML Jenczewski E 2010 Focus on polyploidy New Phytol1861ndash4

Albertin W Marullo P 2012 Polyploidy in fungi evolution after whole-genome duplication Proc Biol Sci 2792497ndash2509

Almeida P Goncalves C Teixeira S Libkind D Bontrager M Masneuf-Pomarede I Albertin W Durrens P Sherman DJ Marullo P et al2014 A Gondwanan imprint on global diversity and domesticationof wine and cider yeast Saccharomyces uvarum Nat Commun54044

Antunovics Z Nguyen HV Gaillardin C Sipiczki M 2005 Gradual ge-nome stabilisation by progressive reduction of the Saccharomyces

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14

uvarum genome in an interspecific hybrid with Saccharomyces cer-evisiae FEMS Yeast Res 51141ndash1150

Baker E Wang B Bellora N Peris D Hulfachor AB Koshalek JA AdamsM Libkind D Hittinger CT 2015 The Genome sequence ofSaccharomyces eubayanus and the domestication of lager-brewingyeasts Mol Biol Evol 322818ndash2831

Barbosa R Almeida P Safar SVB Santos RO Morais PB Nielly-Thibault LLeducq JB Landry CR Goncalves P Rosa CA Sampaio JP 2016Evidence of natural hybridization in Brazilian wild lineages ofSaccharomyces cerevisiae Genome Biol Evol 8317ndash329

Barton NH 2001 The role of hybridization in evolution Mol Ecol10551ndash568

Belloch C Orlic S Barrio E Querol A 2008 Fermentative stress adapta-tion of hybrids within the Saccharomyces sensu stricto complex Int JFood Microbiol 122188ndash195

Bellon JR Yang F Day MP Inglis DL Chambers PJ 2015 Designingand creating Saccharomyces interspecific hybrids for improvedindustry relevant phenotypes Appl Microbiol Biotechnol998597ndash8609

Borneman AR Zeppel R Chambers PJ Curtin CD 2014 Insights into theDekkera bruxellensis genomic landscape comparative genomics re-veals variations in ploidy and nutrient utilisation potential amongstwine isolates PLoS Genet 10e1004161

Brewer BJ Payen C Di Rienzi SC Higgins MM Ong G Dunham MJRaghuraman MK 2015 Origin-dependent inverted-repeat amplifi-cation tests of a model for inverted DNA amplification PLoS Genet11e1005699

Brown CJ Todd KM Rosenzweig RF 1998 Multiple duplications of yeasthexose transport genes in response to selection in a glucose-limitedenvironment Mol Biol Evol 15931ndash942

Bullini L 1994 Origin and evolution of animal hybrid species Trends EcolEvol 9422ndash426

Bun-Ya M Nishimura M Harashima S Oshima Y 1991 The PHO84 geneof Saccharomyces cerevisiae encodes an inorganic phosphate trans-porter Mol Cell Biol 113229ndash3238

Burke MK Dunham JP Shahrestani P Thornton KR Rose MR Long AD2010 Genome-wide analysis of a long-term evolution experimentwith Drosophila Nature 467587ndash590

Burke MK Liti G Long AD 2014 Standing genetic variation drives re-peatable experimental evolution in outcrossing populations ofSaccharomyces cerevisiae Mol Biol Evol 313228ndash3239

Cherry JM Hong EL Amundsen C Balakrishnan R Binkley G Chan ETChristie KR Costanzo MC Dwight SS Engel SR et al 2012Saccharomyces genome database the genomics resource of buddingyeast Nucleic Acids Res 40D700ndashD705

Chester M Riley RK Soltis PS Soltis DE 2015 Patterns of chromosomalvariation in natural populations of the neoallotetraploid Tragopogonmirus (Asteraceae) Heredity (Edinb) 114309ndash317

Chou JY Leu JY 2010 Speciation through cytonuclear incompatibilityinsights from yeast and implications for higher eukaryotes Bioessays32401ndash411

Cliften PF Fulton RS Wilson RK Johnston M 2006 After the duplicationgene loss and adaptation in Saccharomyces genomes Genetics172863ndash872

Conte GL Arnegard ME Peichel CL Schluter D 2012 The probability ofgenetic parallelism and convergence in natural populations Proc BiolSci 2795039ndash5047

Cordes FS Bright JN Sansom MS 2002 Proline-induced distortions oftransmembrane helices J Mol Biol 323951ndash960

Crow JF 1998 90 years ago the beginning of hybrid maize Genetics148923ndash928

Dasmahapatra KK Walters JR Briscoe AD Davey JW Whibley A NadeauNJ Zimin AV Hughes DST Ferguson LC Martin SH et al 2012Butterfly genome reveals promiscuous exchange of mimicry adap-tations among species Nature 48794ndash98

Dowling TE Smith GR Brown WM 1989 Reproductive isolation andintrogression between Notropis-Cornutus and Notropis-Chrysocephalus (Family Cyprinidae)mdashcomparison of morphologyallozymes and mitochondrial-DNA Evolution 43620ndash634

Doyle JJ Flagel LE Paterson AH Rapp RA Soltis DE Soltis PS Wendel JF2008 Evolutionary genetics of genome merger and doubling inplants Annu Rev Genet 42443ndash461

Dunham MJ Badrane H Ferea T Adams J Brown PO Rosenzweig FBotstein D 2002 Characteristic genome rearrangements in experi-mental evolution of Saccharomyces cerevisiae Proc Natl Acad Sci U SA 9916144ndash16149

Dunn B Paulish T Stanbery A Piotrowski J Koniges G Kroll E LouisEJ Liti G Sherlock G Rosenzweig F 2013 Recurrent rearrange-ment during adaptive evolution in an interspecific yeast hybridsuggests a model for rapid introgression PLoS Genet9e1003366

Dunn B Richter C Kvitek DJ Pugh T Sherlock G 2012 Analysis of theSaccharomyces cerevisiae pan-genome reveals a pool of copy num-ber variants distributed in diverse yeast strains from differing indus-trial environments Genome Res 22908ndash924

Dunn B Sherlock G 2008 Reconstruction of the genome origins andevolution of the hybrid lager yeast Saccharomyces pastorianusGenome Res 181610ndash1623

Elmer KR Meyer A 2011 Adaptation in the age of ecological genomicsinsights from parallelism and convergence Trends Ecol Evol26298ndash306

Engel SR Dietrich FS Fisk DG Binkley G Balakrishnan R Costanzo MCDwight SS Hitz BC Karra K Nash RS et al 2014 The referencegenome sequence of Saccharomyces cerevisiae then and now G3(Bethesda) 4389ndash398

Fedoroff NV 2012 Presidential address Transposable elements epige-netics and genome evolution Science 338758ndash767

Ferea TL Botstein D Brown PO Rosenzweig RF 1999 Systematicchanges in gene expression patterns following adaptive evolutionin yeast Proc Natl Acad Sci U S A 969721ndash9726

Gibson B Liti G 2015 Saccharomyces pastorianus genomic insightsinspiring innovation for industry Yeast 3217ndash27

Gibson DG Young L Chuang RY Venter JC Hutchison CA Smith HO2009 Enzymatic assembly of DNA molecules up to several hundredkilobases Nat Methods 6343ndash5

Gonzalez SS Barrio E Gafner J Querol A 2006 Natural hybrids fromSaccharomyces cerevisiae Saccharomyces bayanus andSaccharomyces kudriavzevii in wine fermentations FEMS Yeast Res61221ndash1234

Gonzalez SS Barrio E Querol A 2008 Molecular characterization of newnatural hybrids of Saccharomyces cerevisiae and S kudriavzevii inbrewing Appl Environ Microbiol 742314ndash2320

Grant PR Grant BR 1994 Phenotypic and genetic consequences ofhybridization in Darwinrsquos finches Evolution 48297ndash316

Grant PR Grant BR 2002 Unpredictable evolution in a 30-year study ofDarwinrsquos finches Science 296707ndash711

Grant PR Grant BR 2010 Natural selection speciation and Darwinrsquosfinches Proc Calif Acad Sci 61245ndash260

Grant PR Grant BR Petren K 2005 Hybridization in the recent past AmNat 16656ndash67

Greig D 2009 Reproductive isolation in Saccharomyces Heredity (Edinb)10239ndash44

Greig D Leu JY 2009 Natural history of budding yeast Current Biol19R886ndashR890

Greig D Louis EJ Borts RH Travisano M 2002 Hybrid speciation inexperimental populations of yeast Science 2981773ndash1775

Gresham D Desai MM Tucker CM Jenq HT Pai DA Ward A DeSevoCG Botstein D Dunham MJ 2008 The repertoire and dynamics ofevolutionary adaptations to controlled nutrient-limited environ-ments in yeast PLoS Genet 4e1000303

Hajjar R Hodgkin T 2007 The use of wild relatives in crop improvementa survey of developments over the last 20 years Euphytica 1561ndash13

Heiser CB 1954 Variation and subspeciation in the common sunflowerHelianthus annuus Am Midl Nat 51287ndash305

Hittinger CT 2013 Saccharomyces diversity and evolution a buddingmodel genus Trends Genet 29309ndash317

Hoang ML Tan FJ Lai DC Celniker SE Hoskins RA Dunham MJ ZhengY Koshland D 2010 Competitive repair by naturally dispersed

Adaptation in Hybrids doi101093molbevmsx098 MBE

15

repetitive DNA during non-allelic homologous recombination PLoSGenet 6e1001228

Hoffmann AA Sgro CM 2011 Climate change and evolutionary adap-tation Nature 470479ndash485

Hoffman CS Winston F 1987 A 10-minute DNA preparation from yeastefficiently releases autonomous plasmids for transformation ofEscherichia coli Gene 57267ndash272

Hou J Friedrich A de Montigny J Schacherer J 2014 Chromosomalrearrangements as a major mechanism in the onset of reproductiveisolation in Saccharomyces cerevisiae Curr Biol 241153ndash1159

Hou J Friedrich A Gounot JS Schacherer J 2015 Comprehensive surveyof condition-specific reproductive isolation reveals genetic incom-patibility in yeast Nat Commun 67214

Huerta-Sanchez E Casey FP 2015 Archaic inheritance supporting high-altitude life in Tibet J Appl Physiol (1985) 1191129ndash1134

Jones FC Grabherr MG Chan YF Russell P Mauceli E Johnson JSwofford R Pirun M Zody MC White S et al 2012 The genomicbasis of adaptive evolution in threespine sticklebacks Nature48455ndash61

Kao KC Sherlock G 2008 Molecular characterization of clonal interfer-ence during adaptive evolution in asexual populations ofSaccharomyces cerevisiae Nat Genet 401499ndash1504

Kawecki TJ Lenski RE Ebert D Hollis B Olivieri I Whitlock MC 2012Experimental evolution Trends Ecol Evol 27547ndash560

Kellis M Patterson N Endrizzi M Birren B Lander ES 2003 Sequencingand comparison of yeast species to identify genes and regulatoryelements Nature 423241ndash254

Kelly B Whiteley A Tallmon D 2010 The Arctic melting pot Nature468891ndash891

Kvitek DJ Sherlock G 2011 Reciprocal sign epistasis between frequentlyexperimentally evolved adaptive mutations causes a rugged fitnesslandscape PLoS Genet 7e1002056

Kvitek DJ Sherlock G 2013 Whole genome whole population sequenc-ing reveals that loss of signaling networks is the major adaptivestrategy in a constant environment PLoS Genet 9e1003972

Landry CR Hartl DL Ranz JM 2007 Genome clashes in hybrids insightsfrom gene expression Heredity (Edinb) 99483ndash493

Lang GI Desai MM 2014 The spectrum of adaptive mutations in ex-perimental evolution Genomics 104412ndash416

Lapunzina P Monk D 2011 The consequences of uniparental disomyand copy number neutral loss-of-heterozygosity during human de-velopment and cancer Biol Cell 103303ndash317

Leducq JB Nielly-Thibault L Charron G Eberlein C Verta JP Samani PSylvester K Hittinger CT Bell G Landry CR 2016 Speciation drivenby hybridization and chromosomal plasticity in a wild yeast NatMicrobiol 115003

Lee HY Chou JY Cheong L Chang NH Yang SY Leu JY 2008Incompatibility of nuclear and mitochondrial genomes causes hy-brid sterility between two yeast species Cell 1351065ndash1073

Li H Durbin R 2009 Fast and accurate short read alignment withBurrows-Wheeler transform Bioinformatics 251754ndash1760

Li H Handsaker B Wysoker A Fennell T Ruan J Homer N Marth GAbecasis G Durbin R 2009 The Sequence AlignmentMap formatand SAMtools Bioinformatics 252078ndash2079

Liti G 2015 The fascinating and secret wild life of the budding yeast Scerevisiae Elife 4e05835

Liti G Barton DB Louis EJ 2006 Sequence diversity reproductiveisolation and species concepts in Saccharomyces Genetics174839ndash850

Lloyd AH Ranoux M Vautrin S Glover N Fourment J Charif D ChouletF Lassalle G Marande W Tran J et al 2014 Meiotic gene evolutioncan you teach a new dog new tricks Mol Biol Evol 311724ndash1727

Losos JB Jackman TR Larson A de Queiroz K Rodriguez-Schettino L1998 Contingency and determinism in replicated adaptive radia-tions of island lizards Science 2792115ndash2118

Louis VL Despons L Friedrich A Martin T Durrens P Casaregola SNeuveglise C Fairhead C Marck C Cruz JA et al 2012 Pichia sorbi-tophila an interspecies yeast hybrid reveals early steps of genomeresolution after polyploidization G3 (Bethesda) 2299ndash311

Mallet J 2007 Hybrid speciation Nature 446279ndash283Marcet-Houben M Gabaldon T 2015 Beyond the whole-genome du-

plication phylogenetic evidence for an ancient interspecies hybrid-ization in the bakerrsquos yeast lineage PLoS Biol 13e1002220

Marinoni G Manuel M Petersen RF Hvidtfeldt J Sulo P Piskur J 1999Horizontal transfer of genetic material among Saccharomyces yeastsJ Bacteriol 1816488ndash6496

Martin A Orgogozo V 2013 The loci of repeated evolution a catalog ofgenetic hotspots of phenotypic variation Evolution 671235ndash1250

Masly JP Jones CD Noor MAF Locke J Orr HA 2006 Gene transpositionas a cause of hybrid sterility in Drosophila Science 3131448ndash1450

Mason AS Batley J 2015 Creating new interspecific hybrid and polyploidcrops Trends Biotechnol 33436ndash441

Mavarez J Salazar CA Bermingham E Salcedo C Jiggins CD Linares M2006 Speciation by hybridization in Heliconius butterflies Nature441868ndash871

McKenna A Hanna M Banks E Sivachenko A Cibulskis K Kernytsky AGarimella K Altshuler D Gabriel S Daly M et al 2010 The genomeanalysis toolkit a MapReduce framework for analyzing next-generation DNA sequencing data Genome Res 201297ndash1303

Meiklejohn CD Holmbeck MA Siddiq MA Abt DN Rand DMMontooth KL 2013 An Incompatibility between a mitochondrialtRNA and its nuclear-encoded tRNA synthetase compromises de-velopment and fitness in Drosophila PLoS Genet 9e1003238

Meyer A Salzburger W Schartl M 2006 Hybrid origin of a swordtailspecies (Teleostei Xiphophorus clemenciae) driven by sexual selec-tion Mol Ecol 15721ndash730

Michalak P 2009 Epigenetic transposon and small RNA determinants ofhybrid dysfunctions Heredity (Edinb) 10245ndash50

Miller AW Dunham MJ 2013 Design and use of multiplexed chemostatarrays J Vis Exp 72e50262

Monod J 1949 The growth of bacterial cultures Annu Rev Microbiol3371ndash394

Mouillon JM Persson BL 2006 New aspects on phosphate sensing andsignalling in Saccharomyces cerevisiae FEMS Yeast Res 6171ndash176

Muhlfeld CC Kovach RP Jones LA Al-Chokhachy R Boyer MC Leary RFLowe WH Luikart G Allendorf FW 2014 Invasive hybridization in athreatened species is accelerated by climate change Nat ClimChange 4620ndash624

Muller LAH McCusker JH 2009 A multispecies-based taxonomic mi-croarray reveals interspecies hybridization and introgression inSaccharomyces cerevisiae FEMS Yeast Res 9143ndash152

Nagalakshmi U Wang Z Waern K Shou C Raha D Gerstein M SnyderM 2008 The transcriptional landscape of the yeast genome definedby RNA sequencing Science 3201344ndash1349

Nakao Y Kanamori T Itoh T Kodama Y Rainieri S Nakamura NShimonaga T Hattori M Ashikari T 2009 Genome sequence ofthe lager brewing yeast an interspecies hybrid DNA Res 16115ndash129

Nolte AW Freyhof J Stemshorn KC Tautz D 2005 An invasive lineage ofsculpins Cottus sp (Pisces Teleostei) in the Rhine with new habitatadaptations has originated from hybridization between old phylo-geographic groups Proc Biol Sci 2722379ndash2387

Novick A Szilard L 1950a Description of the chemostat Science112715ndash716

Novick A Szilard L 1950b Experiments with the Chemostat on spon-taneous mutations of bacteria Proc Natl Acad Sci U S A 36708ndash719

Ouyang YD Liu YG Zhang QF 2010 Hybrid sterility in plant storiesfrom rice Curr Opin Plant Biol 13186ndash192

Payen C Di Rienzi SC Ong GT Pogachar JL Sanchez JC SunshineAB Raghuraman MK Brewer BJ Dunham MJ 2014 The dynam-ics of diverse segmental amplifications in populations ofSaccharomyces cerevisiae adapting to strong selection G3(Bethesda) 4399ndash409

Payen C Sunshine AB Ong GT Pogachar JL Zhao W Dunham MJ 2016High-throughput identification of adaptive mutations in experimen-tally evolved yeast populations PLoS Genet 12e1006339

Perez-Prat E van Lookeren Campagne MM 2002 Hybrid seed produc-tion and the challenge of propagating male-sterile plants TrendsPlant Sci 7199ndash203

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16

Peris D Langdon QK Moriarty RV Sylvester K Bontrager M Charron GLeducq JB Landry CR Libkind D Hittinger CT 2016 Complex ances-tries of lager-brewing hybrids were shaped by standing variation inthe wild yeast Saccharomyces eubayanus PLoS Genet 12e1006155

Perlstein EO Ruderfer DM Roberts DC Schreiber SL Kruglyak L 2007Genetic basis of individual differences in the response to small-molecule drugs in yeast Nat Genet 39496ndash502

Piatkowska EM Naseeb S Knight D Delneri D 2013 Chimeric proteincomplexes in hybrid species generate novel phenotypes PLoS Genet9e1003836

Piotrowski JS Nagarajan S Kroll E Stanbery A Chiotti KE Kruckeberg ALDunn B Sherlock G Rosenzweig F 2012 Different selective pressureslead to different genomic outcomes as newly-formed hybrid yeastsevolve BMC Evol Biol 1246

Pryszcz LP Nemeth T Gacser A Gabaldon T 2014 Genome comparisonof Candida orthopsilosis clinical strains reveals the existence of hy-brids between two distinct subspecies Genome Biol Evol61069ndash1078

Racimo F Sankararaman S Nielsen R Huerta-Sanchez E 2015 Evidencefor archaic adaptive introgression in humans Nat Rev Genet16359ndash371

Rieseberg LH 1991 Homoploid reticulate evolution in Helianthus(Asteraceae)mdashevidence from ribosomal genes Am J Bot781218ndash1237

Rieseberg LH 1997 Hybrid origins of plant species Annu Rev EcolSystemat 28359ndash389

Robinson JT Thorvaldsdottir H Winckler W Guttman M Lander ESGetz G Mesirov JP 2011 Integrative genomics viewer NatBiotechnol 2924ndash26

Rosenthal DM Schwarzbach AE Donovan LA Raymond O RiesebergLH 2002 Phenotypic differentiation between three ancient hybridtaxa and their parental species Int J Plant Sci 163387ndash398

Ruderfer DM Pratt SC Seidel HS Kruglyak L 2006 Population genomicanalysis of outcrossing and recombination in yeast Nat Genet381077ndash1081

Rundle HD Nagel L Boughman JW Schluter D 2000 Natural selectionand parallel speciation in sympatric sticklebacks Science287306ndash308

Ryland GL Doyle MA Goode D Boyle SE Choong DY Rowley SM Li JBowtell DD Tothill RW Campbell IG et al 2015 Australian OvarianCancer Study Group Loss of heterozygosity what is it good for BMCMed Genomics 845

Salverda MLM Dellus E Gorter FA Debets AJM van der Oost J HoekstraRF Tawfik DS de Visser JAGM 2011 Initial mutations direct alter-native pathways of protein evolution PLoS Genet 7e1001321

Sanchez MR Miller AW Liachko I Sunshine AB Lynch B Huang MAlcantara E DeSevo CG Pai DA Tucker CM et al 2017 Differentialparalog divergence modulates genome evolution across yeast spe-cies PLoS Genet 13(2)e1006585

Scannell DR Zill OA Rokas A Payen C Dunham MJ Eisen MB Rine JJohnston M Hittinger CT 2011 The awesome power of yeast evo-lutionary genetics new genome sequences and strain resources forthe Saccharomyces sensu stricto genus G3 (Bethesda) 111ndash25

Schroder MS Martinez de San Vicente K Prandini TH Hammel SHiggins DG Bagagli E Wolfe KH Butler G 2016 Multiple originsof the pathogenic yeast Candida orthopsilosis by separate hybridiza-tions between two parental species PLoS Genet 12e1006404

Schumer M Rosenthal GG Andolfatto P 2014 How common is homo-ploid hybrid speciation Evolution 681553ndash1560

Schwarzbach AE Donovan LA Rieseberg LH 2001 Transgressivecharacter expression in a hybrid sunflower species Am J Bot88270ndash277

Selmecki AM Maruvka YE Richmond PA Guillet M Shoresh NSorenson AL De S Kishony R Michor F Dowell R et al 2015Polyploidy can drive rapid adaptation in yeast Nature 519349ndash52

Seshan VE Olshen A 2016 DNAcopy DNA copy number data analysisVersion R package version 1460

Soltis DE Visger CJ Soltis PS 2014 The polyploidy revolution then andnow Stebbins revisited Am J Bot 1011057ndash1078

Soltis PS 2013 Hybridization speciation and novelty J Evol Biol26291ndash293

Soltis PS Soltis DE 2009 The role of hybridization in plant speciationAnnu Rev Plant Biol 60561ndash588

Sunshine AB Payen C Ong GT Liachko I Tan KM Dunham MJ 2015The fitness consequences of aneuploidy are driven by condition-dependent gene effects PLoS Biol 13e1002155

Tamai Y Momma T Yoshimoto H Kaneko Y 1998 Co-existence of twotypes of chromosome in the bottom fermenting yeastSaccharomyces pastorianus Yeast 14923ndash933

Taylor DJ Hebert PDN 1993 Habitat-dependent hybrid parentage anddifferential introgression between neighboringly sympatric Daphniaspecies Proc Natl Acad Sci U S A 907079ndash7083

Thiagalingam S Laken S Willson JK Markowitz SD Kinzler KWVogelstein B Lengauer C 2001 Mechanisms underlying losses ofheterozygosity in human colorectal cancers Proc Natl Acad Sci US A 982698ndash2702

Tuna M Knuutila S Mills GB 2009 Uniparental disomy in cancer TrendsMol Med 15120ndash128

Walther A Hesselbart A Wendland J 2014 Genome sequence ofSaccharomyces carlsbergensis the worldrsquos first pure culture lageryeast G3 (Bethesda) 4783ndash793

Wayne RK 1993 Molecular evolution of the dog family Trends Genet9218ndash224

Wenger JW Schwartz K Sherlock G 2010 Bulk segregant analysis byhigh-throughput sequencing reveals a novel xylose utilization genefrom Saccharomyces cerevisiae PLoS Genet 6e1000942

Wessinger CA Rausher MD 2014 Predictability and irreversibility ofgenetic changes associated with flower color evolution inPenstemon barbatus Evolution 681058ndash1070

Wolfe KH 2015 Origin of the yeast whole-genome duplication PLoSBiol 13e1002221

Woods R Schneider D Winkworth CL Riley MA Lenski RE 2006 Testsof parallel molecular evolution in a long-term experiment withEscherichia coli Proc Natl Acad Sci U S A 1039107ndash9112

Wykoff DD OrsquoShea EK 2001 Phosphate transport and sensing inSaccharomyces cerevisiae Genetics 1591491ndash1499

Xiong Z Gaeta RT Pires JC 2011 Homoeologous shuffling andchromosome compensation maintain genome balance inresynthesized allopolyploid Brassica napus Proc Natl Acad SciU S A 1087908ndash7913

Yassour M Kapian T Fraser HB Levin JZ Pfiffner J Adiconis X Schroth GLuo SJ Khrebtukova I Gnirke A et al 2009 Ab initio construction ofa eukaryotic transcriptome by massively parallel mRNA sequencingProc Natl Acad Sci U S A 1063264ndash3269

Zhu YO Sherlock G Petrov DA 2016 Whole genome analysis of 132clinical Saccharomyces cerevisiae strains reveals extensive ploidy var-iation G3 (Bethesda) doi101534g3116029397

Adaptation in Hybrids doi101093molbevmsx098 MBE

17

  • msx098-TF1
  • msx098-TF2
Page 4: Loss of Heterozygosity Drives Adaptation in Hybrid Yeastdunham.gs.washington.edu/SmukowskiHeil.pdf · 2017-05-20 · Loss of Heterozygosity Drives Adaptation in Hybrid Yeast Caiti

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V(a

mp

lifica

tio

n)

cer

Sh3

chrV

I16

2998

GC

N20

No

nsy

no

nym

ou

sD

171Y

cer

132

218

46

153

(S)

607

61

11(G

)5

556

481

(P)

chrX

IV4

9589

0FK

H2

Syn

on

ymo

us

S418

Su

vach

rII7

8658

48

131

8411

gen

esin

clu

din

gSU

L1C

NV

(am

plifi

cati

on

)ce

r

(co

nti

nu

ed)

Smukowski Heil et al doi101093molbevmsx098 MBE

4

Tab

le1

Co

nti

nu

ed

Clo

ne

Loca

tio

nG

ene(

s)M

uta

tio

nSp

ecie

sG

ener

atio

ns

Rel

ativ

eFi

tnes

s6

SE(c

on

dit

ion

)

Sh4

chrX

IV6

6667

5A

RE2

No

nsy

no

nym

ou

sI4

46T

cer

285

271

96

433

(S)

617

60

51(G

)

205

56

330

(P)

chrX

V8

0083

2A

PC

55rsquo

-up

stre

amce

rch

rIV

259

17T

RM

3Sy

no

nym

ou

sS2

01S

cer

chrV

342

563

Inte

rgen

icu

vach

rX7

6976

8SP

O77

No

nsy

no

nym

ou

sD

418G

uva

chrX

990

873

LEU

35rsquo

-up

stre

amu

vach

rXII

192

491

Inte

rgen

icu

vach

rXIV

251

38EG

T2

Syn

on

ymo

us

T16

8Tu

vach

rII

7703

118

1318

422

gen

esi

ncl

ud

ing

SUL1

CN

V(a

mp

lifica

tio

n)

cer

chrV

III

321

gen

esC

NV

(am

plifi

cati

on

)u

vaSh

5ch

rIV

310

881

RX

T3

No

nsy

no

nym

ou

sP

87T

uva

263

465

26

494

(S)

679

61

35(G

)3

726

723

(P)

chrV

III

1691

1In

terg

enic

uva

chrI

I78

6040

813

184

11ge

nes

incl

ud

ing

SUL1

CN

V(a

mp

lifica

tio

n)

cer

Sh6

chrV

269

392

Inte

rgen

icce

r27

347

52

63

69(S

)2

606

125

(G)

447

66

04(P

)ch

rXIV

746

688

Inte

rgen

icce

rch

rIV

413

046

Inte

rgen

icu

vach

rII7

7894

28

131

8414

gen

esin

clu

din

gSU

L1C

NV

(am

plifi

cati

on

)ce

rSh

7ch

rII

2388

75In

terg

enic

cer

129

314

46

049

(S)

187

61

99(G

)8

746

922

(P)

chrX

VI

4906

31SV

L3N

on

syn

on

ymo

us

A24

5Vce

rch

rXV

I86

106

YP

L245

WN

on

syn

on

ymo

us

A17

4Dce

rch

rII

2732

96In

terg

enic

uva

chrI

I737

875

813

184

42ge

nes

in

clu

din

gSU

L1C

NV

(am

plifi

cati

on

)ce

r

Poin

tm

uta

tio

ns

cop

yn

um

ber

vari

ants

(CN

Vs)

an

dlo

sso

fhet

ero

zygo

sity

even

ts(L

OH

)ar

ere

cord

edfo

rea

chev

olv

edh

ybri

dcl

on

eC

lon

esar

eid

enti

fied

by

nu

trie

nt

(Gg

luco

selim

itat

ion

Pp

ho

sph

ate

limit

atio

na

nd

Ssu

lfate

limit

atio

n)

anldquoh

rdquod

eno

tes

hyb

rid

an

dth

en

um

ber

ind

icat

esit

sd

eriv

atio

nfr

om

ind

epen

den

tp

op

ula

tio

ns

Gen

esin

un

der

line

hav

eb

een

fou

nd

toh

ave

po

int

mu

tati

on

sin

pri

or

exp

erim

ents

No

teth

atm

uta

tio

ns

inth

eS

uvar

umge

no

me

use

Suv

arum

chro

mo

som

esan

dco

ord

inat

esA

llb

reak

po

ints

wer

eca

lled

by

visu

alin

spec

tio

no

fseq

uen

cin

gre

ads

and

are

thu

sap

pro

xim

ate

Rel

ativ

efi

tnes

sis

rep

ort

edw

ith

stan

dar

der

ror(

SE)a

nd

the

con

dit

ion

the

clo

ne

was

evo

lved

inlis

ted

firs

tfo

llow

edb

yth

etw

oal

tern

ativ

eco

nd

itio

ns

seve

ralc

lon

esar

ere

po

rted

wit

ho

ut

SEd

ue

tote

chn

ical

diffi

cult

ies

wit

hre

plic

ates

Adaptation in Hybrids doi101093molbevmsx098 MBE

5

Tab

le2

Mu

tati

on

san

dFi

tnes

so

fEv

olv

edPa

ren

talC

lon

es

Clo

ne

Loca

tio

nG

ene(

s)M

uta

tio

nSp

ecie

sG

ener

atio

ns

Rel

ativ

eFi

tnes

s6

SE

Gc1

chrX

IV0

561

000

6322

507

8433

329

8ge

nes

79

gen

esC

NV

(am

plifi

cati

on

of

chr

14L

favo

rin

gG

RF1

67

del

etio

no

fch

r14R

)ce

r16

316

42

63

42

chrV

160

000

576

874

220

gen

esLO

H(f

avo

rsG

RF1

67)

Gc2

chrV

431

750

576

874

71ge

nes

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

167

103

66

058

chrX

V7

1000

01

0912

9119

6ge

nes

LOH

CN

V(m

on

oso

my

favo

rin

gS2

88C

)G

u1

chrX

V59

7ge

nes

CN

V(w

ho

lech

rom

oso

me

amp

lifica

tio

n)

uva

468

180

36

212

chrI

I911

925

917

281

HX

T6

7C

NV

(am

plifi

cati

on

)ch

rXV

385

930

NEL

1N

on

syn

on

ymo

us

N12

9Ich

rII9

1190

9In

terg

enic

par

to

fth

eH

XT

67

amp

lifica

tio

nG

u2

chrX

V59

7ge

nes

CN

V(w

ho

lech

rom

oso

me

amp

lifica

tio

n)

uva

486

131

2ch

rII9

1192

59

1728

1H

XT

67

CN

V(a

mp

lifica

tio

n)

chrI

V1

0029

3R

GT

2N

on

syn

on

ymo

us

G10

7Vch

rV4

2093

FRD

1N

on

syn

on

ymo

us

G12

8Ach

rII9

1719

1H

XT

7Sy

no

nym

ou

sH

53H

chrX

I155

787

Inte

rgen

icP

c1ch

rXII

I03

9000

(LO

H)

01

9662

8(C

NV

3co

pie

s)

1966

283

7300

0(C

NV

2co

pie

s)LO

H1

5ge

nes

incl

ud

ing

PH

O84

C

NV

201

gen

esLO

HC

NV

(am

plifi

cati

on

fav

ori

ng

GR

F167

)ce

r15

221

22

60

81

Pc2

chrX

III0

411

00(L

OH

)0

196

628

(CN

V3

cop

ies)

19

6628

373

000

(CN

V2

cop

ies)

LOH

16

gen

esin

clu

din

gP

HO

84

CN

V2

01ge

nes

LOH

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

149

181

36

103

chrV

III5

2034

9In

terg

enic

Pc3

chrX

III0

390

00(L

OH

)0

196

628

(CN

V3

cop

ies)

19

6628

373

000

(CN

V2

cop

ies)

LOH

15

gen

esin

clu

din

gP

HO

84

CN

V2

01ge

nes

LOH

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

127

194

9

Pc4

chrX

III0

855

00(L

OH

)0

196

628

(CN

V3

cop

ies)

19

6628

373

000

(CN

V2

cop

ies)

LOH

40

gen

esin

clu

din

gP

HO

84

CN

V2

01ge

nes

LOH

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

132

209

66

141

chrX

II2

6400

01

0781

7743

7ge

nes

LOH

(fav

ori

ng

S288

C)

chrX

V1

0231

97P

IP2

No

nsy

no

nym

ou

sE6

QP

u1

uva

240

1

686

110

Pu

2ch

rIX

144

80Y

PS6

5rsquo-u

pst

ream

uva

234

213

06

073

chrI

X2

2531

4SE

C6

No

nsy

no

nym

ou

sI1

84L

chrX

III

1295

67T

CB

3N

on

syn

on

ymo

us

E625

GSc

1ch

rXIV

01

0200

0(C

NV

3co

pie

s)6

3200

07

8433

3(C

NV

1co

py)

LO

H1

0000

07

8433

348

gen

es7

9ge

nes

367

gen

esLO

HC

NV

(am

plifi

cati

on

of

chr

14L

del

etio

no

fch

r14R

LO

Hfa

vori

ng

S288

C)

cer

182

380

66

175

chrV

III2

0796

7SM

F2N

on

syn

on

ymo

us

W10

5Sch

rXII

I190

000

196

500

RR

N11

CA

T2

VP

S71

LOH

CN

V(d

elet

ion

fav

ori

ng

GR

F167

)ch

rII7

8718

07

9735

0V

BA

1SU

L1P

CA

1C

NV

(am

plifi

cati

on

)Sc

2ch

rXII

578

gen

esC

NV

(wh

ole

chro

mo

som

eam

plifi

cati

on

fa

vori

ng

GR

F167

)ce

r17

640

21

61

33

chrX

II6

9200

01

0781

7719

3ge

nes

LOH

(fav

ori

ng

GR

F167

)ch

rII7

7322

08

1318

418

gen

esin

clu

din

gSU

L1C

NV

(am

plifi

cati

on

)Sc

3ch

rVI9

4104

FRS2

No

nsy

no

nym

ou

sV

303I

cer

201

413

46

677

chrV

III3

0890

3T

RA

1N

on

syn

on

ymo

us

V20

48A

chrX

IV2

3226

6P

OP

1N

on

syn

on

ymo

us

S477

ch

rXV

291

219

TLG

2N

on

syn

on

ymo

us

D28

6Ych

rXV

309

86H

PF1

Syn

on

ymo

us

T20

7Tch

rII7

8180

07

9223

05

gen

esin

clu

din

gB

SD2

and

SUL1

CN

V(a

mp

lifica

tio

n)

(co

nti

nu

ed)

Smukowski Heil et al doi101093molbevmsx098 MBE

6

Tab

le2

Co

nti

nu

ed

Clo

ne

Loca

tio

nG

ene(

s)M

uta

tio

nSp

ecie

sG

ener

atio

ns

Rel

ativ

eFi

tnes

s6

SE

Sc4

chrI

I275

000

813

184

289

gen

esLO

H(f

avo

rin

gG

RF1

67)

cer

190

312

56

613

chrI

I788

608

795

833

SUL1

PC

A1

CN

V(a

mp

lifica

tio

n)

chrX

I517

650

666

816

68ge

nes

CN

V(a

mp

lifica

tio

n)

chrX

III1

9000

01

9650

0R

RN

11C

AT

2V

PS7

1LO

HC

NV

(del

etio

nf

avo

rin

gG

RF1

67)

chrX

IV6

3200

07

8433

379

gen

esLO

HC

NV

(del

etio

n)

chrX

V3

3670

03

4200

034

2000

109

1291

2ge

nes

384

gen

esLO

H(f

avo

rin

gG

RF1

67f

avo

rin

gS2

88C

)ch

rIX

233

67C

SS1

No

nsy

no

nym

ou

sD

914N

Su1

chrX

177

350

345

680

96ge

nes

incl

ud

ing

SUL2

CN

V(a

mp

lifica

tio

n)

uva

557

218

62

37(S

anch

eze

tal

201

7)ch

rXV

I466

649

DIG

1N

on

syn

on

ymo

us

E49Q

chrV

188

548

Inte

rgen

icSu

2ch

rX1

7735

03

4568

096

gen

esin

clu

din

gSU

L2C

NV

(am

plifi

cati

on

)ch

rIV

803

704

KT

R3

5rsquo-u

pst

ream

chrI

I121

779

PIN

4N

on

syn

on

ymo

us

N26

3Sch

rVII

165

902

MP

T5

No

nsy

no

nym

ou

sQ

618K

chrI

I836

169

RSC

3Sy

no

nym

ou

sR

4Rch

rIV

107

948

UFD

2Sy

no

nym

ou

sG

691G

chrI

II2

8761

8In

terg

enic

Poin

tm

uta

tio

ns

cop

yn

um

ber

vari

ants

(CN

V)

and

loss

ofh

eter

ozy

gosi

tyev

ents

(LO

H)

are

reco

rded

for

each

evo

lved

par

enta

lclo

ne

Clo

nes

are

iden

tifi

edb

yn

utr

ien

t(G

glu

cose

limit

atio

nP

ph

osp

hat

elim

itat

ion

an

dS

sulfa

telim

itat

ion

)b

ysp

ecie

s(ldquo

crdquod

eno

tes

Sce

revi

siae

ldquou

rdquod

eno

tes

Suv

arum

)an

dth

en

um

ber

ind

icat

esit

sd

eriv

atio

nfr

om

ind

epen

den

tp

op

ula

tio

ns

No

teth

atm

uta

tio

ns

inth

eS

uvar

umge

no

me

use

Suv

arum

chro

mo

som

esan

dco

ord

inat

esA

llb

reak

po

ints

wer

eca

lled

by

visu

alin

spec

tio

no

fse

qu

enci

ng

read

san

dar

eth

us

app

roxi

mat

eR

elat

ive

fitn

ess

isre

po

rted

wit

hst

and

ard

erro

r(S

E)s

ever

alcl

on

esar

ere

po

rted

wit

ho

ut

SEd

ue

tote

chn

ical

diffi

cult

ies

wit

hre

plic

ates

Adaptation in Hybrids doi101093molbevmsx098 MBE

7

had an average of 15 CNV per clone and the evolvedS uvarum clones had an average of 1 CNV per clone (table 2supplementary figs S1 and S2 Supplementary Material online)The most common event across nutrient limitations in theinterspecific hybrids was an amplification of the S cerevisiaecopy of chromosome IV which occurred in four

independent hybrid clones (three in phosphate limitationone in glucose limitation supplementary fig S3Supplementary Material online) Several other characteris-tic rearrangements occurred in the evolved S cerevisiaeclones including the amplification of the left arm of chro-mosome 14 accompanied by segmental monosomy of the

FIG 1 Evolved hybrids exhibit changes in copy number and loss of heterozygosity Copy number variants are displayed for selected evolved hybridclones from three nutrient-limited conditions Gh2 glucose Ph4 phosphate and Sh4 sulfate See additional figures in supplementary fig S3Supplementary Material online Hybrid copy number determined by normalized sequencing read depth per open reading frame (ORF) is plottedacross the genome according to S cerevisiae ORF coordinates to account for three reciprocal translocations between S cerevisiae and S uvarumChromosomes are plotted in alternating light and dark purple red indicates a S cerevisiae copy number variant and blue indicates a S uvarumcopy number variant Gh2 has a whole chromosome amplification of S cerevisiae chrIV a small segmental deletion of S uvarum chrIV (non-copyneutral loss of heterozygosity) and an amplification of S uvarum HXT67 Ph4 has a small segmental deletion of S cerevisiae chrIII (non-copyneutral loss of heterozygosity) and an amplification of S cerevisiae chrXIII with corresponding deletion of S uvarum chrXIII (copy neutral loss ofheterozygosity) Sh4 has an amplification of S cerevisiae SUL1 and a whole chromosome amplification of S uvarum chrVIII (note there is areciprocal translocation between chrVIII and chrXV in S uvarum relative to S cerevisiae) Note that Sh4 is plotted on a different scale For specificcoordinates of copy number variants see table 1

Smukowski Heil et al doi101093molbevmsx098 MBE

8

right arm of chromosome 14 an event seen previously inother evolved populations (Dunham et al 2002 Greshamet al 2008 Sunshine et al 2015) All copy number events in

S cerevisiae had break points at repetitive elements knownas Ty elements except those located on chrII which maybe mediated by another mechanism (Brewer et al 2015) In

FIG 2 Repeated loss of heterozygosity at the PHO84 locus in intra- and interspecific hybrids (A) The 25-kb region extending from the left telomereof chromosome XIII to the high-affinity phosphate transporter gene PHO84 (B) Copy number is plotted across part of chromosome XIII in thehybrid ancestor and three evolved hybrid clones in phosphate limitation (clone indicated in upper right corner) Red shows the S cerevisiae alleleblue shows the S uvarum allele and purple shows where both species exhibit the same copy number Note 8 kb of telomere sequence is removeddue to repetitive sequence (C) Alternate allele frequency is plotted for a portion of chromosome XIII in the ancestor and four evolved S cerevisiaeclones in phosphate limitation (clone indicated in upper right corner) All evolved S cerevisiae clones exhibit a loss of heterozygosity at thetelomeric portion of chromosome XIII (loss of S288C amplification of GRF167) as illustrated by an allele frequency of zero compared with theancestor Regions of heterozygosity are interspersed with regions of homozygosity as one of the parents of the diploid was itself the product of across between strains FL100 and S288C and the other parent was S288C Regions of heterozygosity are due to FL100 haplotypes S cerevisiae copynumber for the four evolved clones is shown below the ancestor is diploid across the chromosome (also see table 2 supplementary fig S1Supplementary Material online)

Adaptation in Hybrids doi101093molbevmsx098 MBE

9

contrast copy number variants in the hybrid were rarely facil-itated by repetitive elements perhaps in part because S uva-rum has no full length Ty elements however why S cerevisiaeTy elements and long terminal repeat (LTR) sequences fromeither genome were not utilized remains unknown

Frequently in nutrient-limited evolution experimentscopy number variants involve amplification of the nutrient-specific transporter and indeed we also observed amplifica-tion of these transporters in many of the clones In sulfatelimitation the S cerevisiae allele of the high-affinity sulfatetransporter gene SUL1 is amplified in 77 hybrid clones and 44 S cerevisiae clones (fig 1 tables 1 and 2 supplementary figsS1 and S3 Supplementary Material online) InterestinglySUL2 is the preferred sulfate transporter in S uvarum(Sanchez et al 2017) and was not observed to be amplifiedin the evolved hybrids (supplementary fig S2 and table S2Supplementary Material online) In glucose limitation previ-ous S cerevisiae evolution experiments found frequent am-plification of the high-affinity glucose transporter genesHXT67 (Brown et al 1998 Dunham et al 2002 Greshamet al 2008 Kao and Sherlock 2008 Kvitek and Sherlock2011) In our experiments the S uvarum alleles of theHXT67 transporters are amplified in 33 hybrid clones andboth S uvarum clones but are not amplified in evolved Scerevisiae clones This is suggestive that the S uvarum HXT67alleles confer a greater fitness advantage compared with Scerevisiae alternatively the genomic context could be morepermissive to amplification in S uvarum (fig 1 tables 1 and 2supplementary figs S1ndashS3 Supplementary Material online)Finally in phosphate limitation the S cerevisiae copy of thehigh-affinity phosphate transporter gene PHO84 is amplifiedand the S uvarum allele is lost in 36 hybrid clones in an eventknown as LOH (figs 1 and 2 table 1 supplementary fig 3Supplementary Material online) Intriguingly the evolved Scerevisiae clones also display LOH and accompanied amplifi-cation favoring the allele derived from strain GRF167 over theS288C allele in 44 clones (fig 2 table 2) All hybrid clonescarry the ldquopreferredrdquo GRF167 S cerevisiae allele as this was theallele used to create the de novo hybrid

Loss of Heterozygosity Is a Common Event inHeterozygous Evolving PopulationsSelection on heterozygosity as a LOH event could representis an underappreciated source of adaptation in microbial ex-perimental evolution as typical experiments evolve a haploidor homozygous diploid strain asexually and as a result havelittle variation to select upon LOH is observed in natural andindustrial hybrids (Albertin and Marullo 2012 Louis et al2012 Pryszcz et al 2014 Wolfe 2015 Schroder et al 2016)but here we document its occurrence in both intra- andinterspecific hybrids in the laboratory as a result of short-termevolution (also see (Dunn et al 2013 Burke et al 2014)) LOHis observed across all nutrient conditions with 13 independentLOH events detected in S cerevisiae and 9 independent eventsdocumented in the hybrids (figs 1 and 2 tables 1 and 2 sup-plementary figs S1 and S3 Supplementary Material online) Itthus appears that this type of mutational event is both com-mon and can occur over short evolutionary timescales

The LOH event can result in copy-neutral (where one alleleis lost and the other allele is amplified) or non-neutral chro-mosomal segments (where one allele is lost rendering thestrain hemizygous at that locus) and can favor the retentionof either allele In S cerevisiae there is a bias in resolutionwhere LOH events favor retaining the GRF167 allele over theS288C allele (1013 events Pfrac14 00169 table 2 supplementaryfig S4 Supplementary Material online) One unique case inclone Sc4 has a small approximately 5 kb LOH event onchrXV favoring GRF167 which switches to favoring S288Cfor the rest of the chromosome The retention of S cerevisiaeis slightly more common in the hybrids (59 events Pfrac14 10table 1 supplementary fig S3 Supplementary Material on-line) though not as drastic as the observed genome resolu-tion in the hybrid S pastorianus where LOH favorsS cerevisiae over S eubayanus (Nakao et al 2009) The sizeof the event ranges from approximately 25 kb to the wholechromosome level in the hybrids and from 5 kb to 540 kbin S cerevisiae Where LOH is accompanied by an amplifi-cation event the LOH event always occurs first and doesnot share break points with the amplification event Unlikemany CNV events most LOH events do not appear to bemediated by existing repetitive sequence such as a trans-posable element in the hybrid or S cerevisiae and are mostlikely a product of break-induced replication or mitoticgene conversion with break-induced replication as the fa-vored method as all events extend to the telomere (Hoanget al 2010) The exceptions are in hybrid clones Ph4 Ph5 andSh1 where there is a non-copy neutral loss of S cerevisiaemediated by a Ty element or LTR and S cerevisiae clonesSc1 and Sc4 where there is a 65-kb deletion of the S288Callele flanked by two Ty elements

LOH events in hybrids could signify several ongoing pro-cesses in hybrid genome evolution LOH regions may repre-sent 1) loci with incompatibilities 2) selection on existingvariation or 3) genetic drift eroding genomic segments It isimpossible to definitively rule out any of these hypotheseswithout further experimentation however there are severalarguments disfavoring the incompatibility hypothesis Firstalthough our sample size is modest failing to see repeatedLOH events across nutrient conditions may indicate a lack ofgeneral incompatibility between species (although we cannotpreclude condition-specific incompatibility Hou et al 2015Piatkowska et al 2013) This is consistent with previous stud-ies in yeast which suggest that classic DobzhanksyndashMullerincompatibilities are rare (Liti et al 2006 Greig 2009 Houet al 2014) Furthermore LOH events observed inevolved S cerevisiae suggest that this mutation type isnot unique to interspecific hybrids Instead repeatedevents within a particular condition such as the repeatedLOH at PHO84 in phosphate limitation or the 65 kbsegment on chrXIII in sulfate limitation suggest thatthese events are beneficial and are indeed selection onone allele over the other

LOH Is Driven by Selection on One AlleleTo test the hypothesis that LOH events provide a selectiveadvantage we used allele replacement in which the allele of

Smukowski Heil et al doi101093molbevmsx098 MBE

10

one speciesstrain is replaced with the allele of the otherspeciesstrain in an otherwise isogenic background We testedthis hypothesis using the most commonly seen LOH eventLOH at PHO84 Although the region extends from 25 kb to234 kb in length in the hybrids and from 40 kb to 85 kb in Scerevisiae and thus includes many genes PHO84 was a primecandidate driving this event PHO84 is one of only ten genesencompassed in the region extending from the telomere tothe break point of the shortest LOH event and is included inevery other LOH event on chromosome XIII (fig 2) It is ahigh-affinity phosphate transporter responsible for inorganicphosphate uptake in high and low phosphate conditions(Wykoff and OrsquoShea 2001) and previous work identified apoint mutation in PHO84 (an alanine to valine substitution atthe 50 end of the gene) which increased fitness by 23 inphosphate-limited conditions (Sunshine et al 2015) Finallyprior work with other nutrient transporters has shown am-plification of nutrient transporters to be a key event in adapt-ing to nutrient-limited conditions

We thus selected a region of approximately 25 kb encom-passing the PHO84 ORF its promoter and 30-UTR(Nagalakshmi et al 2008 Yassour et al 2009 Cherry et al2012) We created allele replacement strains using the twoalleles of S cerevisiae in a S cerevisiae diploid background thetwo alleles are 991 identical in this region (supplementaryfig S5 Supplementary Material online) and each strain isidentical to the ancestral strain used in our evolution exper-iments except at the PHO84 locus The S cerevisiae ancestorcarries one copy of GRF167 (preferred) and one copy ofS288C (ldquoun-preferredrdquo) so named due to which allele wasretained and amplified in the evolved clones To measureany resultant changes in fitness we competed each strainindividually against a fluorescent ancestral strain and mea-sured which strain overtook the culture Relative fitness isthus defined as the growth advantage per generation Twocopies of the un-preferred allele decreased relative fitness by531 (6186) whereas two copies of the preferred alleleincreased relative fitness by 993 (6027) This displays anoverall difference in relative fitness of 1524 between the un-preferred and preferred alleles By comparing the fitness ofthese allele replacement strains with the evolved clones (table 2)the allele replacement does not fully recapitulate the fit-ness gain observed in the evolved clone One explanation isthat the additional mutations present in the evolvedstrains also contribute to their total fitness Another expla-nation could be the increased copy number of the PHO84region that we see in these evolved clones To further ex-plore this fitness difference we cloned the GRF167 alleleonto a low copy number plasmid and transformed theallele replacement strain carrying two preferred S cerevi-siae alleles to simulate increased copy number of PHO84and saw only a minimal fitness increase of 176 (6106note all fitness competitions involving plasmids were donein conjunction with empty plasmids to take into accountany fitness effects from the plasmid itself) This supportsthe conclusion that relative fitness gains in the evolvedclone are largely due to the loss of the S288C allele andselection and amplification of the GRF167 allele with little

additional benefit from further amplification It could also bethe case that co-amplification of other genes in the segmentis required to attain the full benefit as previously observedby the contribution of BSD2 to the SUL1 amplicon in sulfatelimitation (Sunshine et al 2015 Payen et al 2016)

To understand the fitness effects of LOH and amplificationin the hybrids we generated hybrid strains with varying num-bers of S cerevisiae GRF167 PHO84 alleles Unfortunately wewere unable to obtain a successful strain carrying the pre-ferred S cerevisiae allele in a S uvarum background howeverwe were able to generate a S uvarum PHO84 knockout strainthus creating a hybrid with one copy of S cerevisiae PHO84When combined with a low copy plasmid carrying the sameallele this strain effectively has two or more copies ofS cerevisiae PHO84 in a hybrid background This hybrid straincan serve as a proxy for the LOH event observed in the evo-lution experiments and has a relative fitness gain of 2557(6288) The ancestral hybrid with the same plasmid (effec-tively 1 S uvarum allele and 2 or more S cerevisiae alleles) hasa relative fitness gain of 1253 (6131) The difference be-tween these two hybrids suggests that while amplification isbeneficial the highest fitness is achieved with the loss of theS uvarum allele (Pfrac14 00061)

Together these results support the conclusion that theS cerevisiae GRF167 allele is preferred over the S288C alleleand that S cerevisiae alleles are preferred over the S uvarumallele in the hybrid and hence that the LOH events seen inboth intra- and interspecific hybrids are the product ofselection

DiscussionIn summary we sought to understand forces underlying ge-nome stabilization and evolution in interspecific and intra-specific hybrids as they adapt to novel environments Weevolved and sequenced clones from 16 hybrid populationsand 16 parental populations to reveal a variety of mutationalevents conferring adaptation to 3 nutrient-limited conditionsOf particular note we find LOH in both evolved intraspecificand interspecific hybrid clones in all nutrient environmentspotentially signifying areas where selection has acted on pre-existing variation present in the ancestral clone We used anallele replacement strategy to test this hypothesis for a com-monly repeated LOH event and show that selection is indeeddriving the homogenization of the genome at this locusAlthough other studies in natural industrial and lab-evolved isolates have observed LOH we present the first em-pirical test of the causal evolutionary forces influencing theseevents This work can begin to help us understand past hy-bridization events and subsequent genome resolution in hy-brids in natural and artificial systems

Similarities and Differences between Intra- andInterspecific HybridsAlthough our sample size is modest we observe several in-teresting trends when comparing S uvarum clones (homo-zygous) S cerevisiae clones (intraspecific hybrid andpreviously published homozygous) and interspecific hybrid

Adaptation in Hybrids doi101093molbevmsx098 MBE

11

clones First theory and previous research predict the inter-specific hybrid may experience more genome instability in theform of chromosomal rearrangements and CNV events(Xiong et al 2011 Lloyd et al 2014 Chester et al 2015Mason and Batley 2015) Instead in our work the interspecifichybrid and S cerevisiae clones experience the same numberof CNV events (both 15 CNVsclone) However the mech-anism of CNV formation seems to differ between the hybridand S cerevisiae clones Whereas S cerevisiae CNV breakpoints typically occur at transposable elements in our studyand previous studies (Dunham et al 2002 Gresham et al2008 Fedoroff 2012) the interspecific hybrid CNVs do notutilize transposable elements although they obviously sharethe same sequence background as the S cerevisiae clones(albeit in one copy) Whether this difference is due to theabsence of full-length transposable elements in S uvarum isunknown but this could potentially explain the lower num-ber of CNV events in S uvarum clones (1 CNVclone) andpresents an intriguing direction for future study

The Predictability of EvolutionWe now have many examples of predictable evolution innatural systems (Losos et al 1998 Rundle et al 2000 Elmerand Meyer 2011 Conte et al 2012 Jones et al 2012 Martinand Orgogozo 2013 Wessinger and Rausher 2014) and inlaboratory experimental evolution in which there often ap-pears to be a limited number of high fitness pathways thatstrains follow when adapting to a particular condition (Fereaet al 1999 Woods et al 2006 Gresham et al 2008 Burke et al2010 Salverda et al 2011 Kawecki et al 2012 Kvitek andSherlock 2013 Lang and Desai 2014) For example it is wellestablished that amplifications of nutrient transporters aredrivers of adaptation in evolution in nutrient-limited condi-tions Previous work in our group has particularly focused onthe amplification of the high-affinity sulfate transporter geneSUL1 in sulfate-limited conditions which occurs in almostevery sulfate-limited evolution experiment and confers a fit-ness advantage of as much as 40 compared with the un-evolved ancestor strain The amplification of phosphatetransporters has been markedly less common and thus driv-ers of adaptation in this condition have been less clearGresham et al (2008) identified a whole chromosome ampli-fication of chrXIII in one population In a follow-up studySunshine et al (2015) found whole or partial amplification ofchrXIII in 38 populations A genome-wide screen for segmen-tal amplifications found a slight increase in fitness for a smalltelomeric segment of chromosome XIII and a A49V pointmutation in PHO84 was observed to increase fitness by 23However screens by Payen et al (2016) showed that althoughPHO84 is recurrently mutated in various experiments itshowed no benefit when amplified or deleted in phos-phate-limited conditions Finally additional evolution exper-iments recapitulated the point mutation seen in Sunshineet al (2015) in 2432 populations and saw amplification ofPHO84 in 832 populations (Miller A Dunham MJ unpub-lished data) It is important to note that all these experimentsused a strain background derived from S288C or CENPKboth of which carry the same (un-preferred) PHO84 allele

In our work we observed amplification of the S cerevisiaeGRF167 allele of PHO84 in 44 S cerevisiae clones from 4populations and 36 hybrid clones from 6 populations Thisamplification was always preceded by the loss of the S288Callele in S cerevisiae clones and the LOH break points arenever shared with the amplification break points There is a15 fitness difference between carrying two copies of theS288C allele of PHO84 compared with carrying two copiesof the GRF167 allele of PHO84 and additional copies of theGRF167 allele do not provide substantial further fitness gainsThe two alleles differ by several noncoding changes and threenonsynonymous substitutions a mutation from glutamicacid to aspartic acid (E229D) leucine to proline (L259P)and leucine to glutamine (L556Q) the latter two of whichare considered ldquononconservativerdquo protein mutations due tochanges in hydrophobicity and structure (supplementary figS5 Supplementary Material online) Intriguingly the L259Pmutation has actually been previously identified as being re-sponsible for resistance to tetrachloroisophthalonitrile andpartial resistance to pentachlorophenol in a QTL study ofsmall-molecule drugs (Perlstein et al 2007) IndeedPerlstein et al found proline at residue 259 to be conservedacross fungal species and even in orthologous human xeno-biotic transporters likely because proline-induced kinks intransmembrane spans have been shown to be essential toprotein function (Cordes et al 2002 Perlstein et al 2007) Itthus appears that amplification of PHO84 has been less pre-dictable as the S288C allele does not confer a fitness advan-tage unless mutated per Sunshine et al (2015) Togetherthese results imply that strain background can constrainadaptive pathways

In hybrids the amplification of the S cerevisiae segmentoccurred in conjunction with the loss of the S uvarum alleleHybrid strains with the LOH had a 2557 relative fitness gainwhereas hybrid strains with amplification of the S cerevisiaePHO84 allele without the LOH had a 1253 fitness gain ThusLOH confers an additional 1304 fitness gain showing thatselection for LOH has a larger impact on fitness than ampli-fication alone Note that S uvarum does have proline at res-idue 259 like the preferred GRF167 allele and differs fromboth S cerevisiae alleles at the other two coding substitutions(229N and 556K) but the amino acid and noncoding diver-gence is too high to speculate what substitutions are respon-sible for the selection of the GRF167 allele (supplementary figS6 Supplementary Material online) Why the loss of one alleleis more beneficial remains unclear as PHO84 is thought tofunction as a monomer (Bun-Ya et al 1991) but it may bedue to competition for cell wall space or negative interactionswith other genes in the PHO pathway (Mouillon and Persson2006)

The infusion of variation created by hybridization providesnew templates for selection to act upon which can be moreimportant than either point mutations or copy number var-iants alone Our work shows that outcrossing need not becommon to have long-lasting effects on adaptation This im-plication is particularly relevant in yeast where outcrossingmay occur quite rarely followed by thousands of asexual gen-erations (Ruderfer et al 2006 Greig and Leu 2009 Liti 2015)

Smukowski Heil et al doi101093molbevmsx098 MBE

12

Applications to Other Hybrids and CancerThe observation that LOH occurs in hybrid genomes is in-creasingly documented (Louis et al 2012 Borneman et al2014 Soltis et al 2014 Marcet-Houben and Gabaldon 2015Pryszcz et al 2014 Schroder et al 2016) although the rea-son(s) for this type of mutation has been unresolved As mostexamples stem from allopolyploid events that occurred mil-lions of years ago understanding why LOH is important inhybrid genome evolution is difficult Cancer cells are alsoknown to experience LOH sometimes involved in the inac-tivation of tumor suppressor genes leaving only one copy ofthe gene that may be mutated or silenced (Thiagalingam et al2001 Tuna et al 2009 Lapunzina and Monk 2011) Datasupport the conclusion that LOH events are selected for dur-ing tumor development as many LOH events involve specificchromosomal segments (Thiagalingam et al 2001) althoughthe underlying molecular and genetic reasons for selection isan open debate (Ryland et al 2015)

Here we experimentally demonstrate that LOH can occurin homoploid interspecific hybrids as well as in intraspecifichybrids These events occur within a few hundred generationsand are common mutations more common on average inthe intraspecific hybrid (13 eventsclone) than the interspe-cific hybrid (056 eventsclone) Interestingly the LOH eventsdo not share break points with the CNV events in S cerevisiaeinstead they appear to occur independently and to precedeany subsequent amplification (amplification occurs following913 LOH events) Competition assays with the PHO84 locusprovide support that LOH itself may be more beneficial thanamplification or at least increase the selective benefit of am-plification events The observation that LOH in intraspecifichybrids occurs independently from copy number change pro-vides different opportunities for adaptation to novelconditions

Other cases of LOH like the copy number neutral eventsobserved in Ph4 Ph5 and Sh4 all of which favor the retentionof the S uvarum allele (supplementary fig S3 SupplementaryMaterial online) may be due to hybrid incompatibility withina particular protein complex other epistatic interactions(Piatkowska et al 2013) or neutral processes We furthermorediscover examples where one species allele appears to bepreferred over the other without LOH such as the repeatedamplification of the S uvarum high-affinity glucose transpor-ters HXT67 When one species allele is amplified and theother is not amplified one explanation is that the local se-quence context can permit or deny amplification In the caseof HXT67 previous experiments in S cerevisiae have shownthat amplification of HXT67 is quite common (Brown et al1998 Dunham et al 2002 Gresham et al 2008 Kao andSherlock 2008 Kvitek and Sherlock 2011) thus suggestingthat when given a choice between this locus in S cerevisiaeor S uvarum in the hybrid the preferred allele is indeed Suvarum though more subtle differences in rate cannot yet beruled out A similar scenario is observed with S cerevisiaeSUL1 (Sanchez et al 2017) Together our results show thatthe heterozygosity supplied by hybridization is an importantcontributor to adaptive routes explored by populations asthey adapt to novel conditions

Although we cannot generalize our results from thePHO84 locus across the many other LOH events discoveredin our hybrids and S cerevisiae in the future we can usesimilar methodology to explore whether positive selectionalways drives LOH or whether other explanations such asincompatibility resolution contribute as well Future experi-ments might also utilize a high throughput method to ex-plore segmental LOH in hybrids at a genome-wide scalesimilar to ongoing experiments at the gene level (LancasterS Dunham MJ unpublished data) Although our sample sizeis moderate this is a novel and necessary step in understand-ing forces underlying hybrid genome stabilization and high-lighting an underappreciated mechanism of hybridadaptation

ConclusionsThe mutation events we observe in our experimentallyevolved hybrids are in many ways quite representative ofmutations observed in ancient hybrid genomes suggestingthat hybrid genome stabilization and adaptation can occurquite rapidly (within several hundred generations)Furthermore our results illustrate that the infusion of varia-tion introduced by hybridization at both the intra- and inter-species level can increase fitness by providing choices of allelesfor selection to act upon even when sexual reproduction israre This may be particularly important for leveraging existingvariation for agricultural and industrial processes and as cli-mate change potentially increases natural hybridization (Kellyet al 2010 Hoffmann and Sgro 2011 Muhlfeld et al 2014)

Materials and Methods

StrainsA list of strains used in this study is included in supplementarytable S1 Supplementary Material online All interspecific hy-brids were created by crossing a ura3 LYS2 haploid parent to aURA3 lys2 haploid parent of the other mating type plating onmedia lacking both uracil and lysine and selecting for proto-trophs The S cerevisiae strain background known asldquoGRF167rdquo is itself a cross between FL100 and the genomictype strain S288C (data not shown) GRF167 was chosen as astrain background for simultaneous work investigating trans-posable elements during experimental evolution which willbe addressed in a future study

Evolution ExperimentsContinuous cultures were established using media and con-ditions previously described (Gresham et al 2008 Sanchezet al 2017) Detailed protocols and media recipes are availableat httpdunhamgswashingtoneduprotocolsshtml (lastaccessed March 6 2017) Samples were taken daily and mea-sured for optical density at 600 nm and cell count micros-copy was performed to check for contamination andarchival glycerol stocks were made daily An experimentwas terminated when contamination growth in tubing orclumping appeared (number of generations at the endpoint for each population are presented in tables 1 and

Adaptation in Hybrids doi101093molbevmsx098 MBE

13

2) Samples from each end point population were colonypurified to yield two clones for further study

Array Comparative Genomic HybridizationPopulations from the end point of each evolution were ana-lyzed for copy number changes using array comparative ge-nomic hybridization following the protocol used in Sanchezet al (2017)

SequencingDNA was extracted from overnight cultures using theHoffmanndashWinston protocol (Hoffman and Winston 1987)and cleaned using the Clean amp Concentrator kit (ZymoResearch) Nextera libraries were prepared following theNextera library kit protocol and sequenced using paired end150 bp reads on the Illumina NextSeq 500 machine (sequenc-ing coverage in supplementary table S2 SupplementaryMaterial online) The reference genomes used were S cerevi-siae v3 (Engel et al 2014) S uvarum (Scannell et al 2011) anda hybrid reference genome created by concatenating the twogenomes Sequence was aligned to the appropriate referencegenome using bwa v062 (Li and Durbin 2009) and mutationswere called using GATK (McKenna et al 2010) and samtools0119 (Li et al 2009) Mutations in evolved clones were filteredin comparison with the ancestor to obtain de novo mutationsAll mutations were first visually inspected using IntegrativeGenomics Viewer (Robinson et al 2011) Subsequently pointmutations in the hybrids were confirmed with Sanger se-quencing (supplementary table S3 Supplementary Materialonline) Copy number variants were visualized usingDNAcopy for S cerevisiae and S uvarum (Seshan andOlshen 2016) LOH events were called based on sequencingcoverage in the hybrids and by identifying homozygous variantcalls in S cerevisiae All break points were called by visual in-spection of sequencing reads and are thus approximate

Fitness AssaysThe pairwise competition experiments were performed in 20ml chemostats (Miller and Dunham 2013) Each competitorstrain was cultured individually until steady state was reachedand then was mixed 5050 with a GFP-tagged ancestor Eachcompetition was conducted in at least two biological repli-cates for approximately 15 generations after mixing Sampleswere collected and analyzed twice daily The proportion ofGFPthorn cells in the population was detected using a BD AccuriC6 flow cytometer (BD Biosciences) The data were plottedwith ln [dark cellsGFPthorn cells] versus generations The rela-tive fitness coefficient was determined from the slope of thelinear region

Strain ConstructionAllele replacements for the PHO84 locus were done followingthe protocol of the Caudy lab with further modifications de-scribed here The native locus was replaced withKluyveromyces lactis URA3 The pho84DURA3 strain wasgrown overnight in 5 ml of C-URA media then inoculatedin a flask of 100 ml yeast extract peptone dextrose (YPD) andgrown to an optical density of 06ndash08 Cells were washed then

aliquoted 275 ml of transformation mix (35 ml 1 M lithiumacetate 240 ml of 50 3500 polyethylene glycol) 10 ml ofsalmon sperm and approximately 3 mg of polymerase chainreaction product were added to the cell pellet It was incu-bated at 37 C (S uvarum) or 42 C (S cerevisiae) for 45 minthen plated to YPD It was replica plated to 5-fluorooroticacid the following day and colonies were tested for the gain ofthe appropriate species allele The GRF167 allele was clonedinto the pIL37 plasmid using Gibson assembly (Gibson et al2009) Correct assembly was verified by Sanger sequencing Allprimers used can be found in supplementary table S3Supplementary Material online

Supplementary MaterialSupplementary data are available at Molecular Biology andEvolution online

AcknowledgmentsWe thank Noah Hanson and Erica Alcantara for technicalassistance We thank Monica Sanchez and three anonymousreviewers for helpful comments on this manuscript JosephSchacherer kindly confirmed the contribution of FL100 to theGRF167 strain background Thanks to Yixian Zheng and DougKoshland for contributing to the initial experimental designcreating yeast strains and purchasing the oligonucleotidesused for the microarrays This work was supported by theNational Science Foundation (grant number 1516330) andthe National Institutes of Health (grant number R01GM094306) MD is a Senior Fellow in the GeneticNetworks program at the Canadian Institute for AdvancedResearch and a Rita Allen Foundation Scholar CSH wassupported in part by National Institutes of Health (grantnumber T32 HG00035) This work was also supported bythe National Institutes of Health (grant number P50GM071508) to the Lewis-Sigler Institute and from theHoward Hughes Medical Institute to Doug Koshland andYixian Zheng Sequencing read data have been deposited atthe NCBI under BioProject accession PRJNA374049Microarray data are deposited in the Gene ExpressionOmnibus (GEO) repository under accession numbersGSE95086 and GSE87401 and in the Princeton MicroarrayDatabase

ReferencesAbbott R Albach D Ansell S Arntzen JW Baird SJ Bierne N Boughman J

Brelsford A Buerkle CA Buggs R et al 2013 Hybridization andspeciation J Evol Biol 26229ndash246

Ainouche ML Jenczewski E 2010 Focus on polyploidy New Phytol1861ndash4

Albertin W Marullo P 2012 Polyploidy in fungi evolution after whole-genome duplication Proc Biol Sci 2792497ndash2509

Almeida P Goncalves C Teixeira S Libkind D Bontrager M Masneuf-Pomarede I Albertin W Durrens P Sherman DJ Marullo P et al2014 A Gondwanan imprint on global diversity and domesticationof wine and cider yeast Saccharomyces uvarum Nat Commun54044

Antunovics Z Nguyen HV Gaillardin C Sipiczki M 2005 Gradual ge-nome stabilisation by progressive reduction of the Saccharomyces

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14

uvarum genome in an interspecific hybrid with Saccharomyces cer-evisiae FEMS Yeast Res 51141ndash1150

Baker E Wang B Bellora N Peris D Hulfachor AB Koshalek JA AdamsM Libkind D Hittinger CT 2015 The Genome sequence ofSaccharomyces eubayanus and the domestication of lager-brewingyeasts Mol Biol Evol 322818ndash2831

Barbosa R Almeida P Safar SVB Santos RO Morais PB Nielly-Thibault LLeducq JB Landry CR Goncalves P Rosa CA Sampaio JP 2016Evidence of natural hybridization in Brazilian wild lineages ofSaccharomyces cerevisiae Genome Biol Evol 8317ndash329

Barton NH 2001 The role of hybridization in evolution Mol Ecol10551ndash568

Belloch C Orlic S Barrio E Querol A 2008 Fermentative stress adapta-tion of hybrids within the Saccharomyces sensu stricto complex Int JFood Microbiol 122188ndash195

Bellon JR Yang F Day MP Inglis DL Chambers PJ 2015 Designingand creating Saccharomyces interspecific hybrids for improvedindustry relevant phenotypes Appl Microbiol Biotechnol998597ndash8609

Borneman AR Zeppel R Chambers PJ Curtin CD 2014 Insights into theDekkera bruxellensis genomic landscape comparative genomics re-veals variations in ploidy and nutrient utilisation potential amongstwine isolates PLoS Genet 10e1004161

Brewer BJ Payen C Di Rienzi SC Higgins MM Ong G Dunham MJRaghuraman MK 2015 Origin-dependent inverted-repeat amplifi-cation tests of a model for inverted DNA amplification PLoS Genet11e1005699

Brown CJ Todd KM Rosenzweig RF 1998 Multiple duplications of yeasthexose transport genes in response to selection in a glucose-limitedenvironment Mol Biol Evol 15931ndash942

Bullini L 1994 Origin and evolution of animal hybrid species Trends EcolEvol 9422ndash426

Bun-Ya M Nishimura M Harashima S Oshima Y 1991 The PHO84 geneof Saccharomyces cerevisiae encodes an inorganic phosphate trans-porter Mol Cell Biol 113229ndash3238

Burke MK Dunham JP Shahrestani P Thornton KR Rose MR Long AD2010 Genome-wide analysis of a long-term evolution experimentwith Drosophila Nature 467587ndash590

Burke MK Liti G Long AD 2014 Standing genetic variation drives re-peatable experimental evolution in outcrossing populations ofSaccharomyces cerevisiae Mol Biol Evol 313228ndash3239

Cherry JM Hong EL Amundsen C Balakrishnan R Binkley G Chan ETChristie KR Costanzo MC Dwight SS Engel SR et al 2012Saccharomyces genome database the genomics resource of buddingyeast Nucleic Acids Res 40D700ndashD705

Chester M Riley RK Soltis PS Soltis DE 2015 Patterns of chromosomalvariation in natural populations of the neoallotetraploid Tragopogonmirus (Asteraceae) Heredity (Edinb) 114309ndash317

Chou JY Leu JY 2010 Speciation through cytonuclear incompatibilityinsights from yeast and implications for higher eukaryotes Bioessays32401ndash411

Cliften PF Fulton RS Wilson RK Johnston M 2006 After the duplicationgene loss and adaptation in Saccharomyces genomes Genetics172863ndash872

Conte GL Arnegard ME Peichel CL Schluter D 2012 The probability ofgenetic parallelism and convergence in natural populations Proc BiolSci 2795039ndash5047

Cordes FS Bright JN Sansom MS 2002 Proline-induced distortions oftransmembrane helices J Mol Biol 323951ndash960

Crow JF 1998 90 years ago the beginning of hybrid maize Genetics148923ndash928

Dasmahapatra KK Walters JR Briscoe AD Davey JW Whibley A NadeauNJ Zimin AV Hughes DST Ferguson LC Martin SH et al 2012Butterfly genome reveals promiscuous exchange of mimicry adap-tations among species Nature 48794ndash98

Dowling TE Smith GR Brown WM 1989 Reproductive isolation andintrogression between Notropis-Cornutus and Notropis-Chrysocephalus (Family Cyprinidae)mdashcomparison of morphologyallozymes and mitochondrial-DNA Evolution 43620ndash634

Doyle JJ Flagel LE Paterson AH Rapp RA Soltis DE Soltis PS Wendel JF2008 Evolutionary genetics of genome merger and doubling inplants Annu Rev Genet 42443ndash461

Dunham MJ Badrane H Ferea T Adams J Brown PO Rosenzweig FBotstein D 2002 Characteristic genome rearrangements in experi-mental evolution of Saccharomyces cerevisiae Proc Natl Acad Sci U SA 9916144ndash16149

Dunn B Paulish T Stanbery A Piotrowski J Koniges G Kroll E LouisEJ Liti G Sherlock G Rosenzweig F 2013 Recurrent rearrange-ment during adaptive evolution in an interspecific yeast hybridsuggests a model for rapid introgression PLoS Genet9e1003366

Dunn B Richter C Kvitek DJ Pugh T Sherlock G 2012 Analysis of theSaccharomyces cerevisiae pan-genome reveals a pool of copy num-ber variants distributed in diverse yeast strains from differing indus-trial environments Genome Res 22908ndash924

Dunn B Sherlock G 2008 Reconstruction of the genome origins andevolution of the hybrid lager yeast Saccharomyces pastorianusGenome Res 181610ndash1623

Elmer KR Meyer A 2011 Adaptation in the age of ecological genomicsinsights from parallelism and convergence Trends Ecol Evol26298ndash306

Engel SR Dietrich FS Fisk DG Binkley G Balakrishnan R Costanzo MCDwight SS Hitz BC Karra K Nash RS et al 2014 The referencegenome sequence of Saccharomyces cerevisiae then and now G3(Bethesda) 4389ndash398

Fedoroff NV 2012 Presidential address Transposable elements epige-netics and genome evolution Science 338758ndash767

Ferea TL Botstein D Brown PO Rosenzweig RF 1999 Systematicchanges in gene expression patterns following adaptive evolutionin yeast Proc Natl Acad Sci U S A 969721ndash9726

Gibson B Liti G 2015 Saccharomyces pastorianus genomic insightsinspiring innovation for industry Yeast 3217ndash27

Gibson DG Young L Chuang RY Venter JC Hutchison CA Smith HO2009 Enzymatic assembly of DNA molecules up to several hundredkilobases Nat Methods 6343ndash5

Gonzalez SS Barrio E Gafner J Querol A 2006 Natural hybrids fromSaccharomyces cerevisiae Saccharomyces bayanus andSaccharomyces kudriavzevii in wine fermentations FEMS Yeast Res61221ndash1234

Gonzalez SS Barrio E Querol A 2008 Molecular characterization of newnatural hybrids of Saccharomyces cerevisiae and S kudriavzevii inbrewing Appl Environ Microbiol 742314ndash2320

Grant PR Grant BR 1994 Phenotypic and genetic consequences ofhybridization in Darwinrsquos finches Evolution 48297ndash316

Grant PR Grant BR 2002 Unpredictable evolution in a 30-year study ofDarwinrsquos finches Science 296707ndash711

Grant PR Grant BR 2010 Natural selection speciation and Darwinrsquosfinches Proc Calif Acad Sci 61245ndash260

Grant PR Grant BR Petren K 2005 Hybridization in the recent past AmNat 16656ndash67

Greig D 2009 Reproductive isolation in Saccharomyces Heredity (Edinb)10239ndash44

Greig D Leu JY 2009 Natural history of budding yeast Current Biol19R886ndashR890

Greig D Louis EJ Borts RH Travisano M 2002 Hybrid speciation inexperimental populations of yeast Science 2981773ndash1775

Gresham D Desai MM Tucker CM Jenq HT Pai DA Ward A DeSevoCG Botstein D Dunham MJ 2008 The repertoire and dynamics ofevolutionary adaptations to controlled nutrient-limited environ-ments in yeast PLoS Genet 4e1000303

Hajjar R Hodgkin T 2007 The use of wild relatives in crop improvementa survey of developments over the last 20 years Euphytica 1561ndash13

Heiser CB 1954 Variation and subspeciation in the common sunflowerHelianthus annuus Am Midl Nat 51287ndash305

Hittinger CT 2013 Saccharomyces diversity and evolution a buddingmodel genus Trends Genet 29309ndash317

Hoang ML Tan FJ Lai DC Celniker SE Hoskins RA Dunham MJ ZhengY Koshland D 2010 Competitive repair by naturally dispersed

Adaptation in Hybrids doi101093molbevmsx098 MBE

15

repetitive DNA during non-allelic homologous recombination PLoSGenet 6e1001228

Hoffmann AA Sgro CM 2011 Climate change and evolutionary adap-tation Nature 470479ndash485

Hoffman CS Winston F 1987 A 10-minute DNA preparation from yeastefficiently releases autonomous plasmids for transformation ofEscherichia coli Gene 57267ndash272

Hou J Friedrich A de Montigny J Schacherer J 2014 Chromosomalrearrangements as a major mechanism in the onset of reproductiveisolation in Saccharomyces cerevisiae Curr Biol 241153ndash1159

Hou J Friedrich A Gounot JS Schacherer J 2015 Comprehensive surveyof condition-specific reproductive isolation reveals genetic incom-patibility in yeast Nat Commun 67214

Huerta-Sanchez E Casey FP 2015 Archaic inheritance supporting high-altitude life in Tibet J Appl Physiol (1985) 1191129ndash1134

Jones FC Grabherr MG Chan YF Russell P Mauceli E Johnson JSwofford R Pirun M Zody MC White S et al 2012 The genomicbasis of adaptive evolution in threespine sticklebacks Nature48455ndash61

Kao KC Sherlock G 2008 Molecular characterization of clonal interfer-ence during adaptive evolution in asexual populations ofSaccharomyces cerevisiae Nat Genet 401499ndash1504

Kawecki TJ Lenski RE Ebert D Hollis B Olivieri I Whitlock MC 2012Experimental evolution Trends Ecol Evol 27547ndash560

Kellis M Patterson N Endrizzi M Birren B Lander ES 2003 Sequencingand comparison of yeast species to identify genes and regulatoryelements Nature 423241ndash254

Kelly B Whiteley A Tallmon D 2010 The Arctic melting pot Nature468891ndash891

Kvitek DJ Sherlock G 2011 Reciprocal sign epistasis between frequentlyexperimentally evolved adaptive mutations causes a rugged fitnesslandscape PLoS Genet 7e1002056

Kvitek DJ Sherlock G 2013 Whole genome whole population sequenc-ing reveals that loss of signaling networks is the major adaptivestrategy in a constant environment PLoS Genet 9e1003972

Landry CR Hartl DL Ranz JM 2007 Genome clashes in hybrids insightsfrom gene expression Heredity (Edinb) 99483ndash493

Lang GI Desai MM 2014 The spectrum of adaptive mutations in ex-perimental evolution Genomics 104412ndash416

Lapunzina P Monk D 2011 The consequences of uniparental disomyand copy number neutral loss-of-heterozygosity during human de-velopment and cancer Biol Cell 103303ndash317

Leducq JB Nielly-Thibault L Charron G Eberlein C Verta JP Samani PSylvester K Hittinger CT Bell G Landry CR 2016 Speciation drivenby hybridization and chromosomal plasticity in a wild yeast NatMicrobiol 115003

Lee HY Chou JY Cheong L Chang NH Yang SY Leu JY 2008Incompatibility of nuclear and mitochondrial genomes causes hy-brid sterility between two yeast species Cell 1351065ndash1073

Li H Durbin R 2009 Fast and accurate short read alignment withBurrows-Wheeler transform Bioinformatics 251754ndash1760

Li H Handsaker B Wysoker A Fennell T Ruan J Homer N Marth GAbecasis G Durbin R 2009 The Sequence AlignmentMap formatand SAMtools Bioinformatics 252078ndash2079

Liti G 2015 The fascinating and secret wild life of the budding yeast Scerevisiae Elife 4e05835

Liti G Barton DB Louis EJ 2006 Sequence diversity reproductiveisolation and species concepts in Saccharomyces Genetics174839ndash850

Lloyd AH Ranoux M Vautrin S Glover N Fourment J Charif D ChouletF Lassalle G Marande W Tran J et al 2014 Meiotic gene evolutioncan you teach a new dog new tricks Mol Biol Evol 311724ndash1727

Losos JB Jackman TR Larson A de Queiroz K Rodriguez-Schettino L1998 Contingency and determinism in replicated adaptive radia-tions of island lizards Science 2792115ndash2118

Louis VL Despons L Friedrich A Martin T Durrens P Casaregola SNeuveglise C Fairhead C Marck C Cruz JA et al 2012 Pichia sorbi-tophila an interspecies yeast hybrid reveals early steps of genomeresolution after polyploidization G3 (Bethesda) 2299ndash311

Mallet J 2007 Hybrid speciation Nature 446279ndash283Marcet-Houben M Gabaldon T 2015 Beyond the whole-genome du-

plication phylogenetic evidence for an ancient interspecies hybrid-ization in the bakerrsquos yeast lineage PLoS Biol 13e1002220

Marinoni G Manuel M Petersen RF Hvidtfeldt J Sulo P Piskur J 1999Horizontal transfer of genetic material among Saccharomyces yeastsJ Bacteriol 1816488ndash6496

Martin A Orgogozo V 2013 The loci of repeated evolution a catalog ofgenetic hotspots of phenotypic variation Evolution 671235ndash1250

Masly JP Jones CD Noor MAF Locke J Orr HA 2006 Gene transpositionas a cause of hybrid sterility in Drosophila Science 3131448ndash1450

Mason AS Batley J 2015 Creating new interspecific hybrid and polyploidcrops Trends Biotechnol 33436ndash441

Mavarez J Salazar CA Bermingham E Salcedo C Jiggins CD Linares M2006 Speciation by hybridization in Heliconius butterflies Nature441868ndash871

McKenna A Hanna M Banks E Sivachenko A Cibulskis K Kernytsky AGarimella K Altshuler D Gabriel S Daly M et al 2010 The genomeanalysis toolkit a MapReduce framework for analyzing next-generation DNA sequencing data Genome Res 201297ndash1303

Meiklejohn CD Holmbeck MA Siddiq MA Abt DN Rand DMMontooth KL 2013 An Incompatibility between a mitochondrialtRNA and its nuclear-encoded tRNA synthetase compromises de-velopment and fitness in Drosophila PLoS Genet 9e1003238

Meyer A Salzburger W Schartl M 2006 Hybrid origin of a swordtailspecies (Teleostei Xiphophorus clemenciae) driven by sexual selec-tion Mol Ecol 15721ndash730

Michalak P 2009 Epigenetic transposon and small RNA determinants ofhybrid dysfunctions Heredity (Edinb) 10245ndash50

Miller AW Dunham MJ 2013 Design and use of multiplexed chemostatarrays J Vis Exp 72e50262

Monod J 1949 The growth of bacterial cultures Annu Rev Microbiol3371ndash394

Mouillon JM Persson BL 2006 New aspects on phosphate sensing andsignalling in Saccharomyces cerevisiae FEMS Yeast Res 6171ndash176

Muhlfeld CC Kovach RP Jones LA Al-Chokhachy R Boyer MC Leary RFLowe WH Luikart G Allendorf FW 2014 Invasive hybridization in athreatened species is accelerated by climate change Nat ClimChange 4620ndash624

Muller LAH McCusker JH 2009 A multispecies-based taxonomic mi-croarray reveals interspecies hybridization and introgression inSaccharomyces cerevisiae FEMS Yeast Res 9143ndash152

Nagalakshmi U Wang Z Waern K Shou C Raha D Gerstein M SnyderM 2008 The transcriptional landscape of the yeast genome definedby RNA sequencing Science 3201344ndash1349

Nakao Y Kanamori T Itoh T Kodama Y Rainieri S Nakamura NShimonaga T Hattori M Ashikari T 2009 Genome sequence ofthe lager brewing yeast an interspecies hybrid DNA Res 16115ndash129

Nolte AW Freyhof J Stemshorn KC Tautz D 2005 An invasive lineage ofsculpins Cottus sp (Pisces Teleostei) in the Rhine with new habitatadaptations has originated from hybridization between old phylo-geographic groups Proc Biol Sci 2722379ndash2387

Novick A Szilard L 1950a Description of the chemostat Science112715ndash716

Novick A Szilard L 1950b Experiments with the Chemostat on spon-taneous mutations of bacteria Proc Natl Acad Sci U S A 36708ndash719

Ouyang YD Liu YG Zhang QF 2010 Hybrid sterility in plant storiesfrom rice Curr Opin Plant Biol 13186ndash192

Payen C Di Rienzi SC Ong GT Pogachar JL Sanchez JC SunshineAB Raghuraman MK Brewer BJ Dunham MJ 2014 The dynam-ics of diverse segmental amplifications in populations ofSaccharomyces cerevisiae adapting to strong selection G3(Bethesda) 4399ndash409

Payen C Sunshine AB Ong GT Pogachar JL Zhao W Dunham MJ 2016High-throughput identification of adaptive mutations in experimen-tally evolved yeast populations PLoS Genet 12e1006339

Perez-Prat E van Lookeren Campagne MM 2002 Hybrid seed produc-tion and the challenge of propagating male-sterile plants TrendsPlant Sci 7199ndash203

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16

Peris D Langdon QK Moriarty RV Sylvester K Bontrager M Charron GLeducq JB Landry CR Libkind D Hittinger CT 2016 Complex ances-tries of lager-brewing hybrids were shaped by standing variation inthe wild yeast Saccharomyces eubayanus PLoS Genet 12e1006155

Perlstein EO Ruderfer DM Roberts DC Schreiber SL Kruglyak L 2007Genetic basis of individual differences in the response to small-molecule drugs in yeast Nat Genet 39496ndash502

Piatkowska EM Naseeb S Knight D Delneri D 2013 Chimeric proteincomplexes in hybrid species generate novel phenotypes PLoS Genet9e1003836

Piotrowski JS Nagarajan S Kroll E Stanbery A Chiotti KE Kruckeberg ALDunn B Sherlock G Rosenzweig F 2012 Different selective pressureslead to different genomic outcomes as newly-formed hybrid yeastsevolve BMC Evol Biol 1246

Pryszcz LP Nemeth T Gacser A Gabaldon T 2014 Genome comparisonof Candida orthopsilosis clinical strains reveals the existence of hy-brids between two distinct subspecies Genome Biol Evol61069ndash1078

Racimo F Sankararaman S Nielsen R Huerta-Sanchez E 2015 Evidencefor archaic adaptive introgression in humans Nat Rev Genet16359ndash371

Rieseberg LH 1991 Homoploid reticulate evolution in Helianthus(Asteraceae)mdashevidence from ribosomal genes Am J Bot781218ndash1237

Rieseberg LH 1997 Hybrid origins of plant species Annu Rev EcolSystemat 28359ndash389

Robinson JT Thorvaldsdottir H Winckler W Guttman M Lander ESGetz G Mesirov JP 2011 Integrative genomics viewer NatBiotechnol 2924ndash26

Rosenthal DM Schwarzbach AE Donovan LA Raymond O RiesebergLH 2002 Phenotypic differentiation between three ancient hybridtaxa and their parental species Int J Plant Sci 163387ndash398

Ruderfer DM Pratt SC Seidel HS Kruglyak L 2006 Population genomicanalysis of outcrossing and recombination in yeast Nat Genet381077ndash1081

Rundle HD Nagel L Boughman JW Schluter D 2000 Natural selectionand parallel speciation in sympatric sticklebacks Science287306ndash308

Ryland GL Doyle MA Goode D Boyle SE Choong DY Rowley SM Li JBowtell DD Tothill RW Campbell IG et al 2015 Australian OvarianCancer Study Group Loss of heterozygosity what is it good for BMCMed Genomics 845

Salverda MLM Dellus E Gorter FA Debets AJM van der Oost J HoekstraRF Tawfik DS de Visser JAGM 2011 Initial mutations direct alter-native pathways of protein evolution PLoS Genet 7e1001321

Sanchez MR Miller AW Liachko I Sunshine AB Lynch B Huang MAlcantara E DeSevo CG Pai DA Tucker CM et al 2017 Differentialparalog divergence modulates genome evolution across yeast spe-cies PLoS Genet 13(2)e1006585

Scannell DR Zill OA Rokas A Payen C Dunham MJ Eisen MB Rine JJohnston M Hittinger CT 2011 The awesome power of yeast evo-lutionary genetics new genome sequences and strain resources forthe Saccharomyces sensu stricto genus G3 (Bethesda) 111ndash25

Schroder MS Martinez de San Vicente K Prandini TH Hammel SHiggins DG Bagagli E Wolfe KH Butler G 2016 Multiple originsof the pathogenic yeast Candida orthopsilosis by separate hybridiza-tions between two parental species PLoS Genet 12e1006404

Schumer M Rosenthal GG Andolfatto P 2014 How common is homo-ploid hybrid speciation Evolution 681553ndash1560

Schwarzbach AE Donovan LA Rieseberg LH 2001 Transgressivecharacter expression in a hybrid sunflower species Am J Bot88270ndash277

Selmecki AM Maruvka YE Richmond PA Guillet M Shoresh NSorenson AL De S Kishony R Michor F Dowell R et al 2015Polyploidy can drive rapid adaptation in yeast Nature 519349ndash52

Seshan VE Olshen A 2016 DNAcopy DNA copy number data analysisVersion R package version 1460

Soltis DE Visger CJ Soltis PS 2014 The polyploidy revolution then andnow Stebbins revisited Am J Bot 1011057ndash1078

Soltis PS 2013 Hybridization speciation and novelty J Evol Biol26291ndash293

Soltis PS Soltis DE 2009 The role of hybridization in plant speciationAnnu Rev Plant Biol 60561ndash588

Sunshine AB Payen C Ong GT Liachko I Tan KM Dunham MJ 2015The fitness consequences of aneuploidy are driven by condition-dependent gene effects PLoS Biol 13e1002155

Tamai Y Momma T Yoshimoto H Kaneko Y 1998 Co-existence of twotypes of chromosome in the bottom fermenting yeastSaccharomyces pastorianus Yeast 14923ndash933

Taylor DJ Hebert PDN 1993 Habitat-dependent hybrid parentage anddifferential introgression between neighboringly sympatric Daphniaspecies Proc Natl Acad Sci U S A 907079ndash7083

Thiagalingam S Laken S Willson JK Markowitz SD Kinzler KWVogelstein B Lengauer C 2001 Mechanisms underlying losses ofheterozygosity in human colorectal cancers Proc Natl Acad Sci US A 982698ndash2702

Tuna M Knuutila S Mills GB 2009 Uniparental disomy in cancer TrendsMol Med 15120ndash128

Walther A Hesselbart A Wendland J 2014 Genome sequence ofSaccharomyces carlsbergensis the worldrsquos first pure culture lageryeast G3 (Bethesda) 4783ndash793

Wayne RK 1993 Molecular evolution of the dog family Trends Genet9218ndash224

Wenger JW Schwartz K Sherlock G 2010 Bulk segregant analysis byhigh-throughput sequencing reveals a novel xylose utilization genefrom Saccharomyces cerevisiae PLoS Genet 6e1000942

Wessinger CA Rausher MD 2014 Predictability and irreversibility ofgenetic changes associated with flower color evolution inPenstemon barbatus Evolution 681058ndash1070

Wolfe KH 2015 Origin of the yeast whole-genome duplication PLoSBiol 13e1002221

Woods R Schneider D Winkworth CL Riley MA Lenski RE 2006 Testsof parallel molecular evolution in a long-term experiment withEscherichia coli Proc Natl Acad Sci U S A 1039107ndash9112

Wykoff DD OrsquoShea EK 2001 Phosphate transport and sensing inSaccharomyces cerevisiae Genetics 1591491ndash1499

Xiong Z Gaeta RT Pires JC 2011 Homoeologous shuffling andchromosome compensation maintain genome balance inresynthesized allopolyploid Brassica napus Proc Natl Acad SciU S A 1087908ndash7913

Yassour M Kapian T Fraser HB Levin JZ Pfiffner J Adiconis X Schroth GLuo SJ Khrebtukova I Gnirke A et al 2009 Ab initio construction ofa eukaryotic transcriptome by massively parallel mRNA sequencingProc Natl Acad Sci U S A 1063264ndash3269

Zhu YO Sherlock G Petrov DA 2016 Whole genome analysis of 132clinical Saccharomyces cerevisiae strains reveals extensive ploidy var-iation G3 (Bethesda) doi101534g3116029397

Adaptation in Hybrids doi101093molbevmsx098 MBE

17

  • msx098-TF1
  • msx098-TF2
Page 5: Loss of Heterozygosity Drives Adaptation in Hybrid Yeastdunham.gs.washington.edu/SmukowskiHeil.pdf · 2017-05-20 · Loss of Heterozygosity Drives Adaptation in Hybrid Yeast Caiti

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gG

RF1

67)

cer

127

194

9

Pc4

chrX

III0

855

00(L

OH

)0

196

628

(CN

V3

cop

ies)

19

6628

373

000

(CN

V2

cop

ies)

LOH

40

gen

esin

clu

din

gP

HO

84

CN

V2

01ge

nes

LOH

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

132

209

66

141

chrX

II2

6400

01

0781

7743

7ge

nes

LOH

(fav

ori

ng

S288

C)

chrX

V1

0231

97P

IP2

No

nsy

no

nym

ou

sE6

QP

u1

uva

240

1

686

110

Pu

2ch

rIX

144

80Y

PS6

5rsquo-u

pst

ream

uva

234

213

06

073

chrI

X2

2531

4SE

C6

No

nsy

no

nym

ou

sI1

84L

chrX

III

1295

67T

CB

3N

on

syn

on

ymo

us

E625

GSc

1ch

rXIV

01

0200

0(C

NV

3co

pie

s)6

3200

07

8433

3(C

NV

1co

py)

LO

H1

0000

07

8433

348

gen

es7

9ge

nes

367

gen

esLO

HC

NV

(am

plifi

cati

on

of

chr

14L

del

etio

no

fch

r14R

LO

Hfa

vori

ng

S288

C)

cer

182

380

66

175

chrV

III2

0796

7SM

F2N

on

syn

on

ymo

us

W10

5Sch

rXII

I190

000

196

500

RR

N11

CA

T2

VP

S71

LOH

CN

V(d

elet

ion

fav

ori

ng

GR

F167

)ch

rII7

8718

07

9735

0V

BA

1SU

L1P

CA

1C

NV

(am

plifi

cati

on

)Sc

2ch

rXII

578

gen

esC

NV

(wh

ole

chro

mo

som

eam

plifi

cati

on

fa

vori

ng

GR

F167

)ce

r17

640

21

61

33

chrX

II6

9200

01

0781

7719

3ge

nes

LOH

(fav

ori

ng

GR

F167

)ch

rII7

7322

08

1318

418

gen

esin

clu

din

gSU

L1C

NV

(am

plifi

cati

on

)Sc

3ch

rVI9

4104

FRS2

No

nsy

no

nym

ou

sV

303I

cer

201

413

46

677

chrV

III3

0890

3T

RA

1N

on

syn

on

ymo

us

V20

48A

chrX

IV2

3226

6P

OP

1N

on

syn

on

ymo

us

S477

ch

rXV

291

219

TLG

2N

on

syn

on

ymo

us

D28

6Ych

rXV

309

86H

PF1

Syn

on

ymo

us

T20

7Tch

rII7

8180

07

9223

05

gen

esin

clu

din

gB

SD2

and

SUL1

CN

V(a

mp

lifica

tio

n)

(co

nti

nu

ed)

Smukowski Heil et al doi101093molbevmsx098 MBE

6

Tab

le2

Co

nti

nu

ed

Clo

ne

Loca

tio

nG

ene(

s)M

uta

tio

nSp

ecie

sG

ener

atio

ns

Rel

ativ

eFi

tnes

s6

SE

Sc4

chrI

I275

000

813

184

289

gen

esLO

H(f

avo

rin

gG

RF1

67)

cer

190

312

56

613

chrI

I788

608

795

833

SUL1

PC

A1

CN

V(a

mp

lifica

tio

n)

chrX

I517

650

666

816

68ge

nes

CN

V(a

mp

lifica

tio

n)

chrX

III1

9000

01

9650

0R

RN

11C

AT

2V

PS7

1LO

HC

NV

(del

etio

nf

avo

rin

gG

RF1

67)

chrX

IV6

3200

07

8433

379

gen

esLO

HC

NV

(del

etio

n)

chrX

V3

3670

03

4200

034

2000

109

1291

2ge

nes

384

gen

esLO

H(f

avo

rin

gG

RF1

67f

avo

rin

gS2

88C

)ch

rIX

233

67C

SS1

No

nsy

no

nym

ou

sD

914N

Su1

chrX

177

350

345

680

96ge

nes

incl

ud

ing

SUL2

CN

V(a

mp

lifica

tio

n)

uva

557

218

62

37(S

anch

eze

tal

201

7)ch

rXV

I466

649

DIG

1N

on

syn

on

ymo

us

E49Q

chrV

188

548

Inte

rgen

icSu

2ch

rX1

7735

03

4568

096

gen

esin

clu

din

gSU

L2C

NV

(am

plifi

cati

on

)ch

rIV

803

704

KT

R3

5rsquo-u

pst

ream

chrI

I121

779

PIN

4N

on

syn

on

ymo

us

N26

3Sch

rVII

165

902

MP

T5

No

nsy

no

nym

ou

sQ

618K

chrI

I836

169

RSC

3Sy

no

nym

ou

sR

4Rch

rIV

107

948

UFD

2Sy

no

nym

ou

sG

691G

chrI

II2

8761

8In

terg

enic

Poin

tm

uta

tio

ns

cop

yn

um

ber

vari

ants

(CN

V)

and

loss

ofh

eter

ozy

gosi

tyev

ents

(LO

H)

are

reco

rded

for

each

evo

lved

par

enta

lclo

ne

Clo

nes

are

iden

tifi

edb

yn

utr

ien

t(G

glu

cose

limit

atio

nP

ph

osp

hat

elim

itat

ion

an

dS

sulfa

telim

itat

ion

)b

ysp

ecie

s(ldquo

crdquod

eno

tes

Sce

revi

siae

ldquou

rdquod

eno

tes

Suv

arum

)an

dth

en

um

ber

ind

icat

esit

sd

eriv

atio

nfr

om

ind

epen

den

tp

op

ula

tio

ns

No

teth

atm

uta

tio

ns

inth

eS

uvar

umge

no

me

use

Suv

arum

chro

mo

som

esan

dco

ord

inat

esA

llb

reak

po

ints

wer

eca

lled

by

visu

alin

spec

tio

no

fse

qu

enci

ng

read

san

dar

eth

us

app

roxi

mat

eR

elat

ive

fitn

ess

isre

po

rted

wit

hst

and

ard

erro

r(S

E)s

ever

alcl

on

esar

ere

po

rted

wit

ho

ut

SEd

ue

tote

chn

ical

diffi

cult

ies

wit

hre

plic

ates

Adaptation in Hybrids doi101093molbevmsx098 MBE

7

had an average of 15 CNV per clone and the evolvedS uvarum clones had an average of 1 CNV per clone (table 2supplementary figs S1 and S2 Supplementary Material online)The most common event across nutrient limitations in theinterspecific hybrids was an amplification of the S cerevisiaecopy of chromosome IV which occurred in four

independent hybrid clones (three in phosphate limitationone in glucose limitation supplementary fig S3Supplementary Material online) Several other characteris-tic rearrangements occurred in the evolved S cerevisiaeclones including the amplification of the left arm of chro-mosome 14 accompanied by segmental monosomy of the

FIG 1 Evolved hybrids exhibit changes in copy number and loss of heterozygosity Copy number variants are displayed for selected evolved hybridclones from three nutrient-limited conditions Gh2 glucose Ph4 phosphate and Sh4 sulfate See additional figures in supplementary fig S3Supplementary Material online Hybrid copy number determined by normalized sequencing read depth per open reading frame (ORF) is plottedacross the genome according to S cerevisiae ORF coordinates to account for three reciprocal translocations between S cerevisiae and S uvarumChromosomes are plotted in alternating light and dark purple red indicates a S cerevisiae copy number variant and blue indicates a S uvarumcopy number variant Gh2 has a whole chromosome amplification of S cerevisiae chrIV a small segmental deletion of S uvarum chrIV (non-copyneutral loss of heterozygosity) and an amplification of S uvarum HXT67 Ph4 has a small segmental deletion of S cerevisiae chrIII (non-copyneutral loss of heterozygosity) and an amplification of S cerevisiae chrXIII with corresponding deletion of S uvarum chrXIII (copy neutral loss ofheterozygosity) Sh4 has an amplification of S cerevisiae SUL1 and a whole chromosome amplification of S uvarum chrVIII (note there is areciprocal translocation between chrVIII and chrXV in S uvarum relative to S cerevisiae) Note that Sh4 is plotted on a different scale For specificcoordinates of copy number variants see table 1

Smukowski Heil et al doi101093molbevmsx098 MBE

8

right arm of chromosome 14 an event seen previously inother evolved populations (Dunham et al 2002 Greshamet al 2008 Sunshine et al 2015) All copy number events in

S cerevisiae had break points at repetitive elements knownas Ty elements except those located on chrII which maybe mediated by another mechanism (Brewer et al 2015) In

FIG 2 Repeated loss of heterozygosity at the PHO84 locus in intra- and interspecific hybrids (A) The 25-kb region extending from the left telomereof chromosome XIII to the high-affinity phosphate transporter gene PHO84 (B) Copy number is plotted across part of chromosome XIII in thehybrid ancestor and three evolved hybrid clones in phosphate limitation (clone indicated in upper right corner) Red shows the S cerevisiae alleleblue shows the S uvarum allele and purple shows where both species exhibit the same copy number Note 8 kb of telomere sequence is removeddue to repetitive sequence (C) Alternate allele frequency is plotted for a portion of chromosome XIII in the ancestor and four evolved S cerevisiaeclones in phosphate limitation (clone indicated in upper right corner) All evolved S cerevisiae clones exhibit a loss of heterozygosity at thetelomeric portion of chromosome XIII (loss of S288C amplification of GRF167) as illustrated by an allele frequency of zero compared with theancestor Regions of heterozygosity are interspersed with regions of homozygosity as one of the parents of the diploid was itself the product of across between strains FL100 and S288C and the other parent was S288C Regions of heterozygosity are due to FL100 haplotypes S cerevisiae copynumber for the four evolved clones is shown below the ancestor is diploid across the chromosome (also see table 2 supplementary fig S1Supplementary Material online)

Adaptation in Hybrids doi101093molbevmsx098 MBE

9

contrast copy number variants in the hybrid were rarely facil-itated by repetitive elements perhaps in part because S uva-rum has no full length Ty elements however why S cerevisiaeTy elements and long terminal repeat (LTR) sequences fromeither genome were not utilized remains unknown

Frequently in nutrient-limited evolution experimentscopy number variants involve amplification of the nutrient-specific transporter and indeed we also observed amplifica-tion of these transporters in many of the clones In sulfatelimitation the S cerevisiae allele of the high-affinity sulfatetransporter gene SUL1 is amplified in 77 hybrid clones and 44 S cerevisiae clones (fig 1 tables 1 and 2 supplementary figsS1 and S3 Supplementary Material online) InterestinglySUL2 is the preferred sulfate transporter in S uvarum(Sanchez et al 2017) and was not observed to be amplifiedin the evolved hybrids (supplementary fig S2 and table S2Supplementary Material online) In glucose limitation previ-ous S cerevisiae evolution experiments found frequent am-plification of the high-affinity glucose transporter genesHXT67 (Brown et al 1998 Dunham et al 2002 Greshamet al 2008 Kao and Sherlock 2008 Kvitek and Sherlock2011) In our experiments the S uvarum alleles of theHXT67 transporters are amplified in 33 hybrid clones andboth S uvarum clones but are not amplified in evolved Scerevisiae clones This is suggestive that the S uvarum HXT67alleles confer a greater fitness advantage compared with Scerevisiae alternatively the genomic context could be morepermissive to amplification in S uvarum (fig 1 tables 1 and 2supplementary figs S1ndashS3 Supplementary Material online)Finally in phosphate limitation the S cerevisiae copy of thehigh-affinity phosphate transporter gene PHO84 is amplifiedand the S uvarum allele is lost in 36 hybrid clones in an eventknown as LOH (figs 1 and 2 table 1 supplementary fig 3Supplementary Material online) Intriguingly the evolved Scerevisiae clones also display LOH and accompanied amplifi-cation favoring the allele derived from strain GRF167 over theS288C allele in 44 clones (fig 2 table 2) All hybrid clonescarry the ldquopreferredrdquo GRF167 S cerevisiae allele as this was theallele used to create the de novo hybrid

Loss of Heterozygosity Is a Common Event inHeterozygous Evolving PopulationsSelection on heterozygosity as a LOH event could representis an underappreciated source of adaptation in microbial ex-perimental evolution as typical experiments evolve a haploidor homozygous diploid strain asexually and as a result havelittle variation to select upon LOH is observed in natural andindustrial hybrids (Albertin and Marullo 2012 Louis et al2012 Pryszcz et al 2014 Wolfe 2015 Schroder et al 2016)but here we document its occurrence in both intra- andinterspecific hybrids in the laboratory as a result of short-termevolution (also see (Dunn et al 2013 Burke et al 2014)) LOHis observed across all nutrient conditions with 13 independentLOH events detected in S cerevisiae and 9 independent eventsdocumented in the hybrids (figs 1 and 2 tables 1 and 2 sup-plementary figs S1 and S3 Supplementary Material online) Itthus appears that this type of mutational event is both com-mon and can occur over short evolutionary timescales

The LOH event can result in copy-neutral (where one alleleis lost and the other allele is amplified) or non-neutral chro-mosomal segments (where one allele is lost rendering thestrain hemizygous at that locus) and can favor the retentionof either allele In S cerevisiae there is a bias in resolutionwhere LOH events favor retaining the GRF167 allele over theS288C allele (1013 events Pfrac14 00169 table 2 supplementaryfig S4 Supplementary Material online) One unique case inclone Sc4 has a small approximately 5 kb LOH event onchrXV favoring GRF167 which switches to favoring S288Cfor the rest of the chromosome The retention of S cerevisiaeis slightly more common in the hybrids (59 events Pfrac14 10table 1 supplementary fig S3 Supplementary Material on-line) though not as drastic as the observed genome resolu-tion in the hybrid S pastorianus where LOH favorsS cerevisiae over S eubayanus (Nakao et al 2009) The sizeof the event ranges from approximately 25 kb to the wholechromosome level in the hybrids and from 5 kb to 540 kbin S cerevisiae Where LOH is accompanied by an amplifi-cation event the LOH event always occurs first and doesnot share break points with the amplification event Unlikemany CNV events most LOH events do not appear to bemediated by existing repetitive sequence such as a trans-posable element in the hybrid or S cerevisiae and are mostlikely a product of break-induced replication or mitoticgene conversion with break-induced replication as the fa-vored method as all events extend to the telomere (Hoanget al 2010) The exceptions are in hybrid clones Ph4 Ph5 andSh1 where there is a non-copy neutral loss of S cerevisiaemediated by a Ty element or LTR and S cerevisiae clonesSc1 and Sc4 where there is a 65-kb deletion of the S288Callele flanked by two Ty elements

LOH events in hybrids could signify several ongoing pro-cesses in hybrid genome evolution LOH regions may repre-sent 1) loci with incompatibilities 2) selection on existingvariation or 3) genetic drift eroding genomic segments It isimpossible to definitively rule out any of these hypotheseswithout further experimentation however there are severalarguments disfavoring the incompatibility hypothesis Firstalthough our sample size is modest failing to see repeatedLOH events across nutrient conditions may indicate a lack ofgeneral incompatibility between species (although we cannotpreclude condition-specific incompatibility Hou et al 2015Piatkowska et al 2013) This is consistent with previous stud-ies in yeast which suggest that classic DobzhanksyndashMullerincompatibilities are rare (Liti et al 2006 Greig 2009 Houet al 2014) Furthermore LOH events observed inevolved S cerevisiae suggest that this mutation type isnot unique to interspecific hybrids Instead repeatedevents within a particular condition such as the repeatedLOH at PHO84 in phosphate limitation or the 65 kbsegment on chrXIII in sulfate limitation suggest thatthese events are beneficial and are indeed selection onone allele over the other

LOH Is Driven by Selection on One AlleleTo test the hypothesis that LOH events provide a selectiveadvantage we used allele replacement in which the allele of

Smukowski Heil et al doi101093molbevmsx098 MBE

10

one speciesstrain is replaced with the allele of the otherspeciesstrain in an otherwise isogenic background We testedthis hypothesis using the most commonly seen LOH eventLOH at PHO84 Although the region extends from 25 kb to234 kb in length in the hybrids and from 40 kb to 85 kb in Scerevisiae and thus includes many genes PHO84 was a primecandidate driving this event PHO84 is one of only ten genesencompassed in the region extending from the telomere tothe break point of the shortest LOH event and is included inevery other LOH event on chromosome XIII (fig 2) It is ahigh-affinity phosphate transporter responsible for inorganicphosphate uptake in high and low phosphate conditions(Wykoff and OrsquoShea 2001) and previous work identified apoint mutation in PHO84 (an alanine to valine substitution atthe 50 end of the gene) which increased fitness by 23 inphosphate-limited conditions (Sunshine et al 2015) Finallyprior work with other nutrient transporters has shown am-plification of nutrient transporters to be a key event in adapt-ing to nutrient-limited conditions

We thus selected a region of approximately 25 kb encom-passing the PHO84 ORF its promoter and 30-UTR(Nagalakshmi et al 2008 Yassour et al 2009 Cherry et al2012) We created allele replacement strains using the twoalleles of S cerevisiae in a S cerevisiae diploid background thetwo alleles are 991 identical in this region (supplementaryfig S5 Supplementary Material online) and each strain isidentical to the ancestral strain used in our evolution exper-iments except at the PHO84 locus The S cerevisiae ancestorcarries one copy of GRF167 (preferred) and one copy ofS288C (ldquoun-preferredrdquo) so named due to which allele wasretained and amplified in the evolved clones To measureany resultant changes in fitness we competed each strainindividually against a fluorescent ancestral strain and mea-sured which strain overtook the culture Relative fitness isthus defined as the growth advantage per generation Twocopies of the un-preferred allele decreased relative fitness by531 (6186) whereas two copies of the preferred alleleincreased relative fitness by 993 (6027) This displays anoverall difference in relative fitness of 1524 between the un-preferred and preferred alleles By comparing the fitness ofthese allele replacement strains with the evolved clones (table 2)the allele replacement does not fully recapitulate the fit-ness gain observed in the evolved clone One explanation isthat the additional mutations present in the evolvedstrains also contribute to their total fitness Another expla-nation could be the increased copy number of the PHO84region that we see in these evolved clones To further ex-plore this fitness difference we cloned the GRF167 alleleonto a low copy number plasmid and transformed theallele replacement strain carrying two preferred S cerevi-siae alleles to simulate increased copy number of PHO84and saw only a minimal fitness increase of 176 (6106note all fitness competitions involving plasmids were donein conjunction with empty plasmids to take into accountany fitness effects from the plasmid itself) This supportsthe conclusion that relative fitness gains in the evolvedclone are largely due to the loss of the S288C allele andselection and amplification of the GRF167 allele with little

additional benefit from further amplification It could also bethe case that co-amplification of other genes in the segmentis required to attain the full benefit as previously observedby the contribution of BSD2 to the SUL1 amplicon in sulfatelimitation (Sunshine et al 2015 Payen et al 2016)

To understand the fitness effects of LOH and amplificationin the hybrids we generated hybrid strains with varying num-bers of S cerevisiae GRF167 PHO84 alleles Unfortunately wewere unable to obtain a successful strain carrying the pre-ferred S cerevisiae allele in a S uvarum background howeverwe were able to generate a S uvarum PHO84 knockout strainthus creating a hybrid with one copy of S cerevisiae PHO84When combined with a low copy plasmid carrying the sameallele this strain effectively has two or more copies ofS cerevisiae PHO84 in a hybrid background This hybrid straincan serve as a proxy for the LOH event observed in the evo-lution experiments and has a relative fitness gain of 2557(6288) The ancestral hybrid with the same plasmid (effec-tively 1 S uvarum allele and 2 or more S cerevisiae alleles) hasa relative fitness gain of 1253 (6131) The difference be-tween these two hybrids suggests that while amplification isbeneficial the highest fitness is achieved with the loss of theS uvarum allele (Pfrac14 00061)

Together these results support the conclusion that theS cerevisiae GRF167 allele is preferred over the S288C alleleand that S cerevisiae alleles are preferred over the S uvarumallele in the hybrid and hence that the LOH events seen inboth intra- and interspecific hybrids are the product ofselection

DiscussionIn summary we sought to understand forces underlying ge-nome stabilization and evolution in interspecific and intra-specific hybrids as they adapt to novel environments Weevolved and sequenced clones from 16 hybrid populationsand 16 parental populations to reveal a variety of mutationalevents conferring adaptation to 3 nutrient-limited conditionsOf particular note we find LOH in both evolved intraspecificand interspecific hybrid clones in all nutrient environmentspotentially signifying areas where selection has acted on pre-existing variation present in the ancestral clone We used anallele replacement strategy to test this hypothesis for a com-monly repeated LOH event and show that selection is indeeddriving the homogenization of the genome at this locusAlthough other studies in natural industrial and lab-evolved isolates have observed LOH we present the first em-pirical test of the causal evolutionary forces influencing theseevents This work can begin to help us understand past hy-bridization events and subsequent genome resolution in hy-brids in natural and artificial systems

Similarities and Differences between Intra- andInterspecific HybridsAlthough our sample size is modest we observe several in-teresting trends when comparing S uvarum clones (homo-zygous) S cerevisiae clones (intraspecific hybrid andpreviously published homozygous) and interspecific hybrid

Adaptation in Hybrids doi101093molbevmsx098 MBE

11

clones First theory and previous research predict the inter-specific hybrid may experience more genome instability in theform of chromosomal rearrangements and CNV events(Xiong et al 2011 Lloyd et al 2014 Chester et al 2015Mason and Batley 2015) Instead in our work the interspecifichybrid and S cerevisiae clones experience the same numberof CNV events (both 15 CNVsclone) However the mech-anism of CNV formation seems to differ between the hybridand S cerevisiae clones Whereas S cerevisiae CNV breakpoints typically occur at transposable elements in our studyand previous studies (Dunham et al 2002 Gresham et al2008 Fedoroff 2012) the interspecific hybrid CNVs do notutilize transposable elements although they obviously sharethe same sequence background as the S cerevisiae clones(albeit in one copy) Whether this difference is due to theabsence of full-length transposable elements in S uvarum isunknown but this could potentially explain the lower num-ber of CNV events in S uvarum clones (1 CNVclone) andpresents an intriguing direction for future study

The Predictability of EvolutionWe now have many examples of predictable evolution innatural systems (Losos et al 1998 Rundle et al 2000 Elmerand Meyer 2011 Conte et al 2012 Jones et al 2012 Martinand Orgogozo 2013 Wessinger and Rausher 2014) and inlaboratory experimental evolution in which there often ap-pears to be a limited number of high fitness pathways thatstrains follow when adapting to a particular condition (Fereaet al 1999 Woods et al 2006 Gresham et al 2008 Burke et al2010 Salverda et al 2011 Kawecki et al 2012 Kvitek andSherlock 2013 Lang and Desai 2014) For example it is wellestablished that amplifications of nutrient transporters aredrivers of adaptation in evolution in nutrient-limited condi-tions Previous work in our group has particularly focused onthe amplification of the high-affinity sulfate transporter geneSUL1 in sulfate-limited conditions which occurs in almostevery sulfate-limited evolution experiment and confers a fit-ness advantage of as much as 40 compared with the un-evolved ancestor strain The amplification of phosphatetransporters has been markedly less common and thus driv-ers of adaptation in this condition have been less clearGresham et al (2008) identified a whole chromosome ampli-fication of chrXIII in one population In a follow-up studySunshine et al (2015) found whole or partial amplification ofchrXIII in 38 populations A genome-wide screen for segmen-tal amplifications found a slight increase in fitness for a smalltelomeric segment of chromosome XIII and a A49V pointmutation in PHO84 was observed to increase fitness by 23However screens by Payen et al (2016) showed that althoughPHO84 is recurrently mutated in various experiments itshowed no benefit when amplified or deleted in phos-phate-limited conditions Finally additional evolution exper-iments recapitulated the point mutation seen in Sunshineet al (2015) in 2432 populations and saw amplification ofPHO84 in 832 populations (Miller A Dunham MJ unpub-lished data) It is important to note that all these experimentsused a strain background derived from S288C or CENPKboth of which carry the same (un-preferred) PHO84 allele

In our work we observed amplification of the S cerevisiaeGRF167 allele of PHO84 in 44 S cerevisiae clones from 4populations and 36 hybrid clones from 6 populations Thisamplification was always preceded by the loss of the S288Callele in S cerevisiae clones and the LOH break points arenever shared with the amplification break points There is a15 fitness difference between carrying two copies of theS288C allele of PHO84 compared with carrying two copiesof the GRF167 allele of PHO84 and additional copies of theGRF167 allele do not provide substantial further fitness gainsThe two alleles differ by several noncoding changes and threenonsynonymous substitutions a mutation from glutamicacid to aspartic acid (E229D) leucine to proline (L259P)and leucine to glutamine (L556Q) the latter two of whichare considered ldquononconservativerdquo protein mutations due tochanges in hydrophobicity and structure (supplementary figS5 Supplementary Material online) Intriguingly the L259Pmutation has actually been previously identified as being re-sponsible for resistance to tetrachloroisophthalonitrile andpartial resistance to pentachlorophenol in a QTL study ofsmall-molecule drugs (Perlstein et al 2007) IndeedPerlstein et al found proline at residue 259 to be conservedacross fungal species and even in orthologous human xeno-biotic transporters likely because proline-induced kinks intransmembrane spans have been shown to be essential toprotein function (Cordes et al 2002 Perlstein et al 2007) Itthus appears that amplification of PHO84 has been less pre-dictable as the S288C allele does not confer a fitness advan-tage unless mutated per Sunshine et al (2015) Togetherthese results imply that strain background can constrainadaptive pathways

In hybrids the amplification of the S cerevisiae segmentoccurred in conjunction with the loss of the S uvarum alleleHybrid strains with the LOH had a 2557 relative fitness gainwhereas hybrid strains with amplification of the S cerevisiaePHO84 allele without the LOH had a 1253 fitness gain ThusLOH confers an additional 1304 fitness gain showing thatselection for LOH has a larger impact on fitness than ampli-fication alone Note that S uvarum does have proline at res-idue 259 like the preferred GRF167 allele and differs fromboth S cerevisiae alleles at the other two coding substitutions(229N and 556K) but the amino acid and noncoding diver-gence is too high to speculate what substitutions are respon-sible for the selection of the GRF167 allele (supplementary figS6 Supplementary Material online) Why the loss of one alleleis more beneficial remains unclear as PHO84 is thought tofunction as a monomer (Bun-Ya et al 1991) but it may bedue to competition for cell wall space or negative interactionswith other genes in the PHO pathway (Mouillon and Persson2006)

The infusion of variation created by hybridization providesnew templates for selection to act upon which can be moreimportant than either point mutations or copy number var-iants alone Our work shows that outcrossing need not becommon to have long-lasting effects on adaptation This im-plication is particularly relevant in yeast where outcrossingmay occur quite rarely followed by thousands of asexual gen-erations (Ruderfer et al 2006 Greig and Leu 2009 Liti 2015)

Smukowski Heil et al doi101093molbevmsx098 MBE

12

Applications to Other Hybrids and CancerThe observation that LOH occurs in hybrid genomes is in-creasingly documented (Louis et al 2012 Borneman et al2014 Soltis et al 2014 Marcet-Houben and Gabaldon 2015Pryszcz et al 2014 Schroder et al 2016) although the rea-son(s) for this type of mutation has been unresolved As mostexamples stem from allopolyploid events that occurred mil-lions of years ago understanding why LOH is important inhybrid genome evolution is difficult Cancer cells are alsoknown to experience LOH sometimes involved in the inac-tivation of tumor suppressor genes leaving only one copy ofthe gene that may be mutated or silenced (Thiagalingam et al2001 Tuna et al 2009 Lapunzina and Monk 2011) Datasupport the conclusion that LOH events are selected for dur-ing tumor development as many LOH events involve specificchromosomal segments (Thiagalingam et al 2001) althoughthe underlying molecular and genetic reasons for selection isan open debate (Ryland et al 2015)

Here we experimentally demonstrate that LOH can occurin homoploid interspecific hybrids as well as in intraspecifichybrids These events occur within a few hundred generationsand are common mutations more common on average inthe intraspecific hybrid (13 eventsclone) than the interspe-cific hybrid (056 eventsclone) Interestingly the LOH eventsdo not share break points with the CNV events in S cerevisiaeinstead they appear to occur independently and to precedeany subsequent amplification (amplification occurs following913 LOH events) Competition assays with the PHO84 locusprovide support that LOH itself may be more beneficial thanamplification or at least increase the selective benefit of am-plification events The observation that LOH in intraspecifichybrids occurs independently from copy number change pro-vides different opportunities for adaptation to novelconditions

Other cases of LOH like the copy number neutral eventsobserved in Ph4 Ph5 and Sh4 all of which favor the retentionof the S uvarum allele (supplementary fig S3 SupplementaryMaterial online) may be due to hybrid incompatibility withina particular protein complex other epistatic interactions(Piatkowska et al 2013) or neutral processes We furthermorediscover examples where one species allele appears to bepreferred over the other without LOH such as the repeatedamplification of the S uvarum high-affinity glucose transpor-ters HXT67 When one species allele is amplified and theother is not amplified one explanation is that the local se-quence context can permit or deny amplification In the caseof HXT67 previous experiments in S cerevisiae have shownthat amplification of HXT67 is quite common (Brown et al1998 Dunham et al 2002 Gresham et al 2008 Kao andSherlock 2008 Kvitek and Sherlock 2011) thus suggestingthat when given a choice between this locus in S cerevisiaeor S uvarum in the hybrid the preferred allele is indeed Suvarum though more subtle differences in rate cannot yet beruled out A similar scenario is observed with S cerevisiaeSUL1 (Sanchez et al 2017) Together our results show thatthe heterozygosity supplied by hybridization is an importantcontributor to adaptive routes explored by populations asthey adapt to novel conditions

Although we cannot generalize our results from thePHO84 locus across the many other LOH events discoveredin our hybrids and S cerevisiae in the future we can usesimilar methodology to explore whether positive selectionalways drives LOH or whether other explanations such asincompatibility resolution contribute as well Future experi-ments might also utilize a high throughput method to ex-plore segmental LOH in hybrids at a genome-wide scalesimilar to ongoing experiments at the gene level (LancasterS Dunham MJ unpublished data) Although our sample sizeis moderate this is a novel and necessary step in understand-ing forces underlying hybrid genome stabilization and high-lighting an underappreciated mechanism of hybridadaptation

ConclusionsThe mutation events we observe in our experimentallyevolved hybrids are in many ways quite representative ofmutations observed in ancient hybrid genomes suggestingthat hybrid genome stabilization and adaptation can occurquite rapidly (within several hundred generations)Furthermore our results illustrate that the infusion of varia-tion introduced by hybridization at both the intra- and inter-species level can increase fitness by providing choices of allelesfor selection to act upon even when sexual reproduction israre This may be particularly important for leveraging existingvariation for agricultural and industrial processes and as cli-mate change potentially increases natural hybridization (Kellyet al 2010 Hoffmann and Sgro 2011 Muhlfeld et al 2014)

Materials and Methods

StrainsA list of strains used in this study is included in supplementarytable S1 Supplementary Material online All interspecific hy-brids were created by crossing a ura3 LYS2 haploid parent to aURA3 lys2 haploid parent of the other mating type plating onmedia lacking both uracil and lysine and selecting for proto-trophs The S cerevisiae strain background known asldquoGRF167rdquo is itself a cross between FL100 and the genomictype strain S288C (data not shown) GRF167 was chosen as astrain background for simultaneous work investigating trans-posable elements during experimental evolution which willbe addressed in a future study

Evolution ExperimentsContinuous cultures were established using media and con-ditions previously described (Gresham et al 2008 Sanchezet al 2017) Detailed protocols and media recipes are availableat httpdunhamgswashingtoneduprotocolsshtml (lastaccessed March 6 2017) Samples were taken daily and mea-sured for optical density at 600 nm and cell count micros-copy was performed to check for contamination andarchival glycerol stocks were made daily An experimentwas terminated when contamination growth in tubing orclumping appeared (number of generations at the endpoint for each population are presented in tables 1 and

Adaptation in Hybrids doi101093molbevmsx098 MBE

13

2) Samples from each end point population were colonypurified to yield two clones for further study

Array Comparative Genomic HybridizationPopulations from the end point of each evolution were ana-lyzed for copy number changes using array comparative ge-nomic hybridization following the protocol used in Sanchezet al (2017)

SequencingDNA was extracted from overnight cultures using theHoffmanndashWinston protocol (Hoffman and Winston 1987)and cleaned using the Clean amp Concentrator kit (ZymoResearch) Nextera libraries were prepared following theNextera library kit protocol and sequenced using paired end150 bp reads on the Illumina NextSeq 500 machine (sequenc-ing coverage in supplementary table S2 SupplementaryMaterial online) The reference genomes used were S cerevi-siae v3 (Engel et al 2014) S uvarum (Scannell et al 2011) anda hybrid reference genome created by concatenating the twogenomes Sequence was aligned to the appropriate referencegenome using bwa v062 (Li and Durbin 2009) and mutationswere called using GATK (McKenna et al 2010) and samtools0119 (Li et al 2009) Mutations in evolved clones were filteredin comparison with the ancestor to obtain de novo mutationsAll mutations were first visually inspected using IntegrativeGenomics Viewer (Robinson et al 2011) Subsequently pointmutations in the hybrids were confirmed with Sanger se-quencing (supplementary table S3 Supplementary Materialonline) Copy number variants were visualized usingDNAcopy for S cerevisiae and S uvarum (Seshan andOlshen 2016) LOH events were called based on sequencingcoverage in the hybrids and by identifying homozygous variantcalls in S cerevisiae All break points were called by visual in-spection of sequencing reads and are thus approximate

Fitness AssaysThe pairwise competition experiments were performed in 20ml chemostats (Miller and Dunham 2013) Each competitorstrain was cultured individually until steady state was reachedand then was mixed 5050 with a GFP-tagged ancestor Eachcompetition was conducted in at least two biological repli-cates for approximately 15 generations after mixing Sampleswere collected and analyzed twice daily The proportion ofGFPthorn cells in the population was detected using a BD AccuriC6 flow cytometer (BD Biosciences) The data were plottedwith ln [dark cellsGFPthorn cells] versus generations The rela-tive fitness coefficient was determined from the slope of thelinear region

Strain ConstructionAllele replacements for the PHO84 locus were done followingthe protocol of the Caudy lab with further modifications de-scribed here The native locus was replaced withKluyveromyces lactis URA3 The pho84DURA3 strain wasgrown overnight in 5 ml of C-URA media then inoculatedin a flask of 100 ml yeast extract peptone dextrose (YPD) andgrown to an optical density of 06ndash08 Cells were washed then

aliquoted 275 ml of transformation mix (35 ml 1 M lithiumacetate 240 ml of 50 3500 polyethylene glycol) 10 ml ofsalmon sperm and approximately 3 mg of polymerase chainreaction product were added to the cell pellet It was incu-bated at 37 C (S uvarum) or 42 C (S cerevisiae) for 45 minthen plated to YPD It was replica plated to 5-fluorooroticacid the following day and colonies were tested for the gain ofthe appropriate species allele The GRF167 allele was clonedinto the pIL37 plasmid using Gibson assembly (Gibson et al2009) Correct assembly was verified by Sanger sequencing Allprimers used can be found in supplementary table S3Supplementary Material online

Supplementary MaterialSupplementary data are available at Molecular Biology andEvolution online

AcknowledgmentsWe thank Noah Hanson and Erica Alcantara for technicalassistance We thank Monica Sanchez and three anonymousreviewers for helpful comments on this manuscript JosephSchacherer kindly confirmed the contribution of FL100 to theGRF167 strain background Thanks to Yixian Zheng and DougKoshland for contributing to the initial experimental designcreating yeast strains and purchasing the oligonucleotidesused for the microarrays This work was supported by theNational Science Foundation (grant number 1516330) andthe National Institutes of Health (grant number R01GM094306) MD is a Senior Fellow in the GeneticNetworks program at the Canadian Institute for AdvancedResearch and a Rita Allen Foundation Scholar CSH wassupported in part by National Institutes of Health (grantnumber T32 HG00035) This work was also supported bythe National Institutes of Health (grant number P50GM071508) to the Lewis-Sigler Institute and from theHoward Hughes Medical Institute to Doug Koshland andYixian Zheng Sequencing read data have been deposited atthe NCBI under BioProject accession PRJNA374049Microarray data are deposited in the Gene ExpressionOmnibus (GEO) repository under accession numbersGSE95086 and GSE87401 and in the Princeton MicroarrayDatabase

ReferencesAbbott R Albach D Ansell S Arntzen JW Baird SJ Bierne N Boughman J

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Antunovics Z Nguyen HV Gaillardin C Sipiczki M 2005 Gradual ge-nome stabilisation by progressive reduction of the Saccharomyces

Smukowski Heil et al doi101093molbevmsx098 MBE

14

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Baker E Wang B Bellora N Peris D Hulfachor AB Koshalek JA AdamsM Libkind D Hittinger CT 2015 The Genome sequence ofSaccharomyces eubayanus and the domestication of lager-brewingyeasts Mol Biol Evol 322818ndash2831

Barbosa R Almeida P Safar SVB Santos RO Morais PB Nielly-Thibault LLeducq JB Landry CR Goncalves P Rosa CA Sampaio JP 2016Evidence of natural hybridization in Brazilian wild lineages ofSaccharomyces cerevisiae Genome Biol Evol 8317ndash329

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Burke MK Dunham JP Shahrestani P Thornton KR Rose MR Long AD2010 Genome-wide analysis of a long-term evolution experimentwith Drosophila Nature 467587ndash590

Burke MK Liti G Long AD 2014 Standing genetic variation drives re-peatable experimental evolution in outcrossing populations ofSaccharomyces cerevisiae Mol Biol Evol 313228ndash3239

Cherry JM Hong EL Amundsen C Balakrishnan R Binkley G Chan ETChristie KR Costanzo MC Dwight SS Engel SR et al 2012Saccharomyces genome database the genomics resource of buddingyeast Nucleic Acids Res 40D700ndashD705

Chester M Riley RK Soltis PS Soltis DE 2015 Patterns of chromosomalvariation in natural populations of the neoallotetraploid Tragopogonmirus (Asteraceae) Heredity (Edinb) 114309ndash317

Chou JY Leu JY 2010 Speciation through cytonuclear incompatibilityinsights from yeast and implications for higher eukaryotes Bioessays32401ndash411

Cliften PF Fulton RS Wilson RK Johnston M 2006 After the duplicationgene loss and adaptation in Saccharomyces genomes Genetics172863ndash872

Conte GL Arnegard ME Peichel CL Schluter D 2012 The probability ofgenetic parallelism and convergence in natural populations Proc BiolSci 2795039ndash5047

Cordes FS Bright JN Sansom MS 2002 Proline-induced distortions oftransmembrane helices J Mol Biol 323951ndash960

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Dasmahapatra KK Walters JR Briscoe AD Davey JW Whibley A NadeauNJ Zimin AV Hughes DST Ferguson LC Martin SH et al 2012Butterfly genome reveals promiscuous exchange of mimicry adap-tations among species Nature 48794ndash98

Dowling TE Smith GR Brown WM 1989 Reproductive isolation andintrogression between Notropis-Cornutus and Notropis-Chrysocephalus (Family Cyprinidae)mdashcomparison of morphologyallozymes and mitochondrial-DNA Evolution 43620ndash634

Doyle JJ Flagel LE Paterson AH Rapp RA Soltis DE Soltis PS Wendel JF2008 Evolutionary genetics of genome merger and doubling inplants Annu Rev Genet 42443ndash461

Dunham MJ Badrane H Ferea T Adams J Brown PO Rosenzweig FBotstein D 2002 Characteristic genome rearrangements in experi-mental evolution of Saccharomyces cerevisiae Proc Natl Acad Sci U SA 9916144ndash16149

Dunn B Paulish T Stanbery A Piotrowski J Koniges G Kroll E LouisEJ Liti G Sherlock G Rosenzweig F 2013 Recurrent rearrange-ment during adaptive evolution in an interspecific yeast hybridsuggests a model for rapid introgression PLoS Genet9e1003366

Dunn B Richter C Kvitek DJ Pugh T Sherlock G 2012 Analysis of theSaccharomyces cerevisiae pan-genome reveals a pool of copy num-ber variants distributed in diverse yeast strains from differing indus-trial environments Genome Res 22908ndash924

Dunn B Sherlock G 2008 Reconstruction of the genome origins andevolution of the hybrid lager yeast Saccharomyces pastorianusGenome Res 181610ndash1623

Elmer KR Meyer A 2011 Adaptation in the age of ecological genomicsinsights from parallelism and convergence Trends Ecol Evol26298ndash306

Engel SR Dietrich FS Fisk DG Binkley G Balakrishnan R Costanzo MCDwight SS Hitz BC Karra K Nash RS et al 2014 The referencegenome sequence of Saccharomyces cerevisiae then and now G3(Bethesda) 4389ndash398

Fedoroff NV 2012 Presidential address Transposable elements epige-netics and genome evolution Science 338758ndash767

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Gibson B Liti G 2015 Saccharomyces pastorianus genomic insightsinspiring innovation for industry Yeast 3217ndash27

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Gonzalez SS Barrio E Gafner J Querol A 2006 Natural hybrids fromSaccharomyces cerevisiae Saccharomyces bayanus andSaccharomyces kudriavzevii in wine fermentations FEMS Yeast Res61221ndash1234

Gonzalez SS Barrio E Querol A 2008 Molecular characterization of newnatural hybrids of Saccharomyces cerevisiae and S kudriavzevii inbrewing Appl Environ Microbiol 742314ndash2320

Grant PR Grant BR 1994 Phenotypic and genetic consequences ofhybridization in Darwinrsquos finches Evolution 48297ndash316

Grant PR Grant BR 2002 Unpredictable evolution in a 30-year study ofDarwinrsquos finches Science 296707ndash711

Grant PR Grant BR 2010 Natural selection speciation and Darwinrsquosfinches Proc Calif Acad Sci 61245ndash260

Grant PR Grant BR Petren K 2005 Hybridization in the recent past AmNat 16656ndash67

Greig D 2009 Reproductive isolation in Saccharomyces Heredity (Edinb)10239ndash44

Greig D Leu JY 2009 Natural history of budding yeast Current Biol19R886ndashR890

Greig D Louis EJ Borts RH Travisano M 2002 Hybrid speciation inexperimental populations of yeast Science 2981773ndash1775

Gresham D Desai MM Tucker CM Jenq HT Pai DA Ward A DeSevoCG Botstein D Dunham MJ 2008 The repertoire and dynamics ofevolutionary adaptations to controlled nutrient-limited environ-ments in yeast PLoS Genet 4e1000303

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Heiser CB 1954 Variation and subspeciation in the common sunflowerHelianthus annuus Am Midl Nat 51287ndash305

Hittinger CT 2013 Saccharomyces diversity and evolution a buddingmodel genus Trends Genet 29309ndash317

Hoang ML Tan FJ Lai DC Celniker SE Hoskins RA Dunham MJ ZhengY Koshland D 2010 Competitive repair by naturally dispersed

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repetitive DNA during non-allelic homologous recombination PLoSGenet 6e1001228

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Hoffman CS Winston F 1987 A 10-minute DNA preparation from yeastefficiently releases autonomous plasmids for transformation ofEscherichia coli Gene 57267ndash272

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Landry CR Hartl DL Ranz JM 2007 Genome clashes in hybrids insightsfrom gene expression Heredity (Edinb) 99483ndash493

Lang GI Desai MM 2014 The spectrum of adaptive mutations in ex-perimental evolution Genomics 104412ndash416

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Lee HY Chou JY Cheong L Chang NH Yang SY Leu JY 2008Incompatibility of nuclear and mitochondrial genomes causes hy-brid sterility between two yeast species Cell 1351065ndash1073

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Liti G Barton DB Louis EJ 2006 Sequence diversity reproductiveisolation and species concepts in Saccharomyces Genetics174839ndash850

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plication phylogenetic evidence for an ancient interspecies hybrid-ization in the bakerrsquos yeast lineage PLoS Biol 13e1002220

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Payen C Di Rienzi SC Ong GT Pogachar JL Sanchez JC SunshineAB Raghuraman MK Brewer BJ Dunham MJ 2014 The dynam-ics of diverse segmental amplifications in populations ofSaccharomyces cerevisiae adapting to strong selection G3(Bethesda) 4399ndash409

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Peris D Langdon QK Moriarty RV Sylvester K Bontrager M Charron GLeducq JB Landry CR Libkind D Hittinger CT 2016 Complex ances-tries of lager-brewing hybrids were shaped by standing variation inthe wild yeast Saccharomyces eubayanus PLoS Genet 12e1006155

Perlstein EO Ruderfer DM Roberts DC Schreiber SL Kruglyak L 2007Genetic basis of individual differences in the response to small-molecule drugs in yeast Nat Genet 39496ndash502

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Pryszcz LP Nemeth T Gacser A Gabaldon T 2014 Genome comparisonof Candida orthopsilosis clinical strains reveals the existence of hy-brids between two distinct subspecies Genome Biol Evol61069ndash1078

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Scannell DR Zill OA Rokas A Payen C Dunham MJ Eisen MB Rine JJohnston M Hittinger CT 2011 The awesome power of yeast evo-lutionary genetics new genome sequences and strain resources forthe Saccharomyces sensu stricto genus G3 (Bethesda) 111ndash25

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Schumer M Rosenthal GG Andolfatto P 2014 How common is homo-ploid hybrid speciation Evolution 681553ndash1560

Schwarzbach AE Donovan LA Rieseberg LH 2001 Transgressivecharacter expression in a hybrid sunflower species Am J Bot88270ndash277

Selmecki AM Maruvka YE Richmond PA Guillet M Shoresh NSorenson AL De S Kishony R Michor F Dowell R et al 2015Polyploidy can drive rapid adaptation in yeast Nature 519349ndash52

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Soltis PS 2013 Hybridization speciation and novelty J Evol Biol26291ndash293

Soltis PS Soltis DE 2009 The role of hybridization in plant speciationAnnu Rev Plant Biol 60561ndash588

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Taylor DJ Hebert PDN 1993 Habitat-dependent hybrid parentage anddifferential introgression between neighboringly sympatric Daphniaspecies Proc Natl Acad Sci U S A 907079ndash7083

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Tuna M Knuutila S Mills GB 2009 Uniparental disomy in cancer TrendsMol Med 15120ndash128

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Wayne RK 1993 Molecular evolution of the dog family Trends Genet9218ndash224

Wenger JW Schwartz K Sherlock G 2010 Bulk segregant analysis byhigh-throughput sequencing reveals a novel xylose utilization genefrom Saccharomyces cerevisiae PLoS Genet 6e1000942

Wessinger CA Rausher MD 2014 Predictability and irreversibility ofgenetic changes associated with flower color evolution inPenstemon barbatus Evolution 681058ndash1070

Wolfe KH 2015 Origin of the yeast whole-genome duplication PLoSBiol 13e1002221

Woods R Schneider D Winkworth CL Riley MA Lenski RE 2006 Testsof parallel molecular evolution in a long-term experiment withEscherichia coli Proc Natl Acad Sci U S A 1039107ndash9112

Wykoff DD OrsquoShea EK 2001 Phosphate transport and sensing inSaccharomyces cerevisiae Genetics 1591491ndash1499

Xiong Z Gaeta RT Pires JC 2011 Homoeologous shuffling andchromosome compensation maintain genome balance inresynthesized allopolyploid Brassica napus Proc Natl Acad SciU S A 1087908ndash7913

Yassour M Kapian T Fraser HB Levin JZ Pfiffner J Adiconis X Schroth GLuo SJ Khrebtukova I Gnirke A et al 2009 Ab initio construction ofa eukaryotic transcriptome by massively parallel mRNA sequencingProc Natl Acad Sci U S A 1063264ndash3269

Zhu YO Sherlock G Petrov DA 2016 Whole genome analysis of 132clinical Saccharomyces cerevisiae strains reveals extensive ploidy var-iation G3 (Bethesda) doi101534g3116029397

Adaptation in Hybrids doi101093molbevmsx098 MBE

17

  • msx098-TF1
  • msx098-TF2
Page 6: Loss of Heterozygosity Drives Adaptation in Hybrid Yeastdunham.gs.washington.edu/SmukowskiHeil.pdf · 2017-05-20 · Loss of Heterozygosity Drives Adaptation in Hybrid Yeast Caiti

Tab

le2

Mu

tati

on

san

dFi

tnes

so

fEv

olv

edPa

ren

talC

lon

es

Clo

ne

Loca

tio

nG

ene(

s)M

uta

tio

nSp

ecie

sG

ener

atio

ns

Rel

ativ

eFi

tnes

s6

SE

Gc1

chrX

IV0

561

000

6322

507

8433

329

8ge

nes

79

gen

esC

NV

(am

plifi

cati

on

of

chr

14L

favo

rin

gG

RF1

67

del

etio

no

fch

r14R

)ce

r16

316

42

63

42

chrV

160

000

576

874

220

gen

esLO

H(f

avo

rsG

RF1

67)

Gc2

chrV

431

750

576

874

71ge

nes

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

167

103

66

058

chrX

V7

1000

01

0912

9119

6ge

nes

LOH

CN

V(m

on

oso

my

favo

rin

gS2

88C

)G

u1

chrX

V59

7ge

nes

CN

V(w

ho

lech

rom

oso

me

amp

lifica

tio

n)

uva

468

180

36

212

chrI

I911

925

917

281

HX

T6

7C

NV

(am

plifi

cati

on

)ch

rXV

385

930

NEL

1N

on

syn

on

ymo

us

N12

9Ich

rII9

1190

9In

terg

enic

par

to

fth

eH

XT

67

amp

lifica

tio

nG

u2

chrX

V59

7ge

nes

CN

V(w

ho

lech

rom

oso

me

amp

lifica

tio

n)

uva

486

131

2ch

rII9

1192

59

1728

1H

XT

67

CN

V(a

mp

lifica

tio

n)

chrI

V1

0029

3R

GT

2N

on

syn

on

ymo

us

G10

7Vch

rV4

2093

FRD

1N

on

syn

on

ymo

us

G12

8Ach

rII9

1719

1H

XT

7Sy

no

nym

ou

sH

53H

chrX

I155

787

Inte

rgen

icP

c1ch

rXII

I03

9000

(LO

H)

01

9662

8(C

NV

3co

pie

s)

1966

283

7300

0(C

NV

2co

pie

s)LO

H1

5ge

nes

incl

ud

ing

PH

O84

C

NV

201

gen

esLO

HC

NV

(am

plifi

cati

on

fav

ori

ng

GR

F167

)ce

r15

221

22

60

81

Pc2

chrX

III0

411

00(L

OH

)0

196

628

(CN

V3

cop

ies)

19

6628

373

000

(CN

V2

cop

ies)

LOH

16

gen

esin

clu

din

gP

HO

84

CN

V2

01ge

nes

LOH

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

149

181

36

103

chrV

III5

2034

9In

terg

enic

Pc3

chrX

III0

390

00(L

OH

)0

196

628

(CN

V3

cop

ies)

19

6628

373

000

(CN

V2

cop

ies)

LOH

15

gen

esin

clu

din

gP

HO

84

CN

V2

01ge

nes

LOH

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

127

194

9

Pc4

chrX

III0

855

00(L

OH

)0

196

628

(CN

V3

cop

ies)

19

6628

373

000

(CN

V2

cop

ies)

LOH

40

gen

esin

clu

din

gP

HO

84

CN

V2

01ge

nes

LOH

CN

V(a

mp

lifica

tio

nf

avo

rin

gG

RF1

67)

cer

132

209

66

141

chrX

II2

6400

01

0781

7743

7ge

nes

LOH

(fav

ori

ng

S288

C)

chrX

V1

0231

97P

IP2

No

nsy

no

nym

ou

sE6

QP

u1

uva

240

1

686

110

Pu

2ch

rIX

144

80Y

PS6

5rsquo-u

pst

ream

uva

234

213

06

073

chrI

X2

2531

4SE

C6

No

nsy

no

nym

ou

sI1

84L

chrX

III

1295

67T

CB

3N

on

syn

on

ymo

us

E625

GSc

1ch

rXIV

01

0200

0(C

NV

3co

pie

s)6

3200

07

8433

3(C

NV

1co

py)

LO

H1

0000

07

8433

348

gen

es7

9ge

nes

367

gen

esLO

HC

NV

(am

plifi

cati

on

of

chr

14L

del

etio

no

fch

r14R

LO

Hfa

vori

ng

S288

C)

cer

182

380

66

175

chrV

III2

0796

7SM

F2N

on

syn

on

ymo

us

W10

5Sch

rXII

I190

000

196

500

RR

N11

CA

T2

VP

S71

LOH

CN

V(d

elet

ion

fav

ori

ng

GR

F167

)ch

rII7

8718

07

9735

0V

BA

1SU

L1P

CA

1C

NV

(am

plifi

cati

on

)Sc

2ch

rXII

578

gen

esC

NV

(wh

ole

chro

mo

som

eam

plifi

cati

on

fa

vori

ng

GR

F167

)ce

r17

640

21

61

33

chrX

II6

9200

01

0781

7719

3ge

nes

LOH

(fav

ori

ng

GR

F167

)ch

rII7

7322

08

1318

418

gen

esin

clu

din

gSU

L1C

NV

(am

plifi

cati

on

)Sc

3ch

rVI9

4104

FRS2

No

nsy

no

nym

ou

sV

303I

cer

201

413

46

677

chrV

III3

0890

3T

RA

1N

on

syn

on

ymo

us

V20

48A

chrX

IV2

3226

6P

OP

1N

on

syn

on

ymo

us

S477

ch

rXV

291

219

TLG

2N

on

syn

on

ymo

us

D28

6Ych

rXV

309

86H

PF1

Syn

on

ymo

us

T20

7Tch

rII7

8180

07

9223

05

gen

esin

clu

din

gB

SD2

and

SUL1

CN

V(a

mp

lifica

tio

n)

(co

nti

nu

ed)

Smukowski Heil et al doi101093molbevmsx098 MBE

6

Tab

le2

Co

nti

nu

ed

Clo

ne

Loca

tio

nG

ene(

s)M

uta

tio

nSp

ecie

sG

ener

atio

ns

Rel

ativ

eFi

tnes

s6

SE

Sc4

chrI

I275

000

813

184

289

gen

esLO

H(f

avo

rin

gG

RF1

67)

cer

190

312

56

613

chrI

I788

608

795

833

SUL1

PC

A1

CN

V(a

mp

lifica

tio

n)

chrX

I517

650

666

816

68ge

nes

CN

V(a

mp

lifica

tio

n)

chrX

III1

9000

01

9650

0R

RN

11C

AT

2V

PS7

1LO

HC

NV

(del

etio

nf

avo

rin

gG

RF1

67)

chrX

IV6

3200

07

8433

379

gen

esLO

HC

NV

(del

etio

n)

chrX

V3

3670

03

4200

034

2000

109

1291

2ge

nes

384

gen

esLO

H(f

avo

rin

gG

RF1

67f

avo

rin

gS2

88C

)ch

rIX

233

67C

SS1

No

nsy

no

nym

ou

sD

914N

Su1

chrX

177

350

345

680

96ge

nes

incl

ud

ing

SUL2

CN

V(a

mp

lifica

tio

n)

uva

557

218

62

37(S

anch

eze

tal

201

7)ch

rXV

I466

649

DIG

1N

on

syn

on

ymo

us

E49Q

chrV

188

548

Inte

rgen

icSu

2ch

rX1

7735

03

4568

096

gen

esin

clu

din

gSU

L2C

NV

(am

plifi

cati

on

)ch

rIV

803

704

KT

R3

5rsquo-u

pst

ream

chrI

I121

779

PIN

4N

on

syn

on

ymo

us

N26

3Sch

rVII

165

902

MP

T5

No

nsy

no

nym

ou

sQ

618K

chrI

I836

169

RSC

3Sy

no

nym

ou

sR

4Rch

rIV

107

948

UFD

2Sy

no

nym

ou

sG

691G

chrI

II2

8761

8In

terg

enic

Poin

tm

uta

tio

ns

cop

yn

um

ber

vari

ants

(CN

V)

and

loss

ofh

eter

ozy

gosi

tyev

ents

(LO

H)

are

reco

rded

for

each

evo

lved

par

enta

lclo

ne

Clo

nes

are

iden

tifi

edb

yn

utr

ien

t(G

glu

cose

limit

atio

nP

ph

osp

hat

elim

itat

ion

an

dS

sulfa

telim

itat

ion

)b

ysp

ecie

s(ldquo

crdquod

eno

tes

Sce

revi

siae

ldquou

rdquod

eno

tes

Suv

arum

)an

dth

en

um

ber

ind

icat

esit

sd

eriv

atio

nfr

om

ind

epen

den

tp

op

ula

tio

ns

No

teth

atm

uta

tio

ns

inth

eS

uvar

umge

no

me

use

Suv

arum

chro

mo

som

esan

dco

ord

inat

esA

llb

reak

po

ints

wer

eca

lled

by

visu

alin

spec

tio

no

fse

qu

enci

ng

read

san

dar

eth

us

app

roxi

mat

eR

elat

ive

fitn

ess

isre

po

rted

wit

hst

and

ard

erro

r(S

E)s

ever

alcl

on

esar

ere

po

rted

wit

ho

ut

SEd

ue

tote

chn

ical

diffi

cult

ies

wit

hre

plic

ates

Adaptation in Hybrids doi101093molbevmsx098 MBE

7

had an average of 15 CNV per clone and the evolvedS uvarum clones had an average of 1 CNV per clone (table 2supplementary figs S1 and S2 Supplementary Material online)The most common event across nutrient limitations in theinterspecific hybrids was an amplification of the S cerevisiaecopy of chromosome IV which occurred in four

independent hybrid clones (three in phosphate limitationone in glucose limitation supplementary fig S3Supplementary Material online) Several other characteris-tic rearrangements occurred in the evolved S cerevisiaeclones including the amplification of the left arm of chro-mosome 14 accompanied by segmental monosomy of the

FIG 1 Evolved hybrids exhibit changes in copy number and loss of heterozygosity Copy number variants are displayed for selected evolved hybridclones from three nutrient-limited conditions Gh2 glucose Ph4 phosphate and Sh4 sulfate See additional figures in supplementary fig S3Supplementary Material online Hybrid copy number determined by normalized sequencing read depth per open reading frame (ORF) is plottedacross the genome according to S cerevisiae ORF coordinates to account for three reciprocal translocations between S cerevisiae and S uvarumChromosomes are plotted in alternating light and dark purple red indicates a S cerevisiae copy number variant and blue indicates a S uvarumcopy number variant Gh2 has a whole chromosome amplification of S cerevisiae chrIV a small segmental deletion of S uvarum chrIV (non-copyneutral loss of heterozygosity) and an amplification of S uvarum HXT67 Ph4 has a small segmental deletion of S cerevisiae chrIII (non-copyneutral loss of heterozygosity) and an amplification of S cerevisiae chrXIII with corresponding deletion of S uvarum chrXIII (copy neutral loss ofheterozygosity) Sh4 has an amplification of S cerevisiae SUL1 and a whole chromosome amplification of S uvarum chrVIII (note there is areciprocal translocation between chrVIII and chrXV in S uvarum relative to S cerevisiae) Note that Sh4 is plotted on a different scale For specificcoordinates of copy number variants see table 1

Smukowski Heil et al doi101093molbevmsx098 MBE

8

right arm of chromosome 14 an event seen previously inother evolved populations (Dunham et al 2002 Greshamet al 2008 Sunshine et al 2015) All copy number events in

S cerevisiae had break points at repetitive elements knownas Ty elements except those located on chrII which maybe mediated by another mechanism (Brewer et al 2015) In

FIG 2 Repeated loss of heterozygosity at the PHO84 locus in intra- and interspecific hybrids (A) The 25-kb region extending from the left telomereof chromosome XIII to the high-affinity phosphate transporter gene PHO84 (B) Copy number is plotted across part of chromosome XIII in thehybrid ancestor and three evolved hybrid clones in phosphate limitation (clone indicated in upper right corner) Red shows the S cerevisiae alleleblue shows the S uvarum allele and purple shows where both species exhibit the same copy number Note 8 kb of telomere sequence is removeddue to repetitive sequence (C) Alternate allele frequency is plotted for a portion of chromosome XIII in the ancestor and four evolved S cerevisiaeclones in phosphate limitation (clone indicated in upper right corner) All evolved S cerevisiae clones exhibit a loss of heterozygosity at thetelomeric portion of chromosome XIII (loss of S288C amplification of GRF167) as illustrated by an allele frequency of zero compared with theancestor Regions of heterozygosity are interspersed with regions of homozygosity as one of the parents of the diploid was itself the product of across between strains FL100 and S288C and the other parent was S288C Regions of heterozygosity are due to FL100 haplotypes S cerevisiae copynumber for the four evolved clones is shown below the ancestor is diploid across the chromosome (also see table 2 supplementary fig S1Supplementary Material online)

Adaptation in Hybrids doi101093molbevmsx098 MBE

9

contrast copy number variants in the hybrid were rarely facil-itated by repetitive elements perhaps in part because S uva-rum has no full length Ty elements however why S cerevisiaeTy elements and long terminal repeat (LTR) sequences fromeither genome were not utilized remains unknown

Frequently in nutrient-limited evolution experimentscopy number variants involve amplification of the nutrient-specific transporter and indeed we also observed amplifica-tion of these transporters in many of the clones In sulfatelimitation the S cerevisiae allele of the high-affinity sulfatetransporter gene SUL1 is amplified in 77 hybrid clones and 44 S cerevisiae clones (fig 1 tables 1 and 2 supplementary figsS1 and S3 Supplementary Material online) InterestinglySUL2 is the preferred sulfate transporter in S uvarum(Sanchez et al 2017) and was not observed to be amplifiedin the evolved hybrids (supplementary fig S2 and table S2Supplementary Material online) In glucose limitation previ-ous S cerevisiae evolution experiments found frequent am-plification of the high-affinity glucose transporter genesHXT67 (Brown et al 1998 Dunham et al 2002 Greshamet al 2008 Kao and Sherlock 2008 Kvitek and Sherlock2011) In our experiments the S uvarum alleles of theHXT67 transporters are amplified in 33 hybrid clones andboth S uvarum clones but are not amplified in evolved Scerevisiae clones This is suggestive that the S uvarum HXT67alleles confer a greater fitness advantage compared with Scerevisiae alternatively the genomic context could be morepermissive to amplification in S uvarum (fig 1 tables 1 and 2supplementary figs S1ndashS3 Supplementary Material online)Finally in phosphate limitation the S cerevisiae copy of thehigh-affinity phosphate transporter gene PHO84 is amplifiedand the S uvarum allele is lost in 36 hybrid clones in an eventknown as LOH (figs 1 and 2 table 1 supplementary fig 3Supplementary Material online) Intriguingly the evolved Scerevisiae clones also display LOH and accompanied amplifi-cation favoring the allele derived from strain GRF167 over theS288C allele in 44 clones (fig 2 table 2) All hybrid clonescarry the ldquopreferredrdquo GRF167 S cerevisiae allele as this was theallele used to create the de novo hybrid

Loss of Heterozygosity Is a Common Event inHeterozygous Evolving PopulationsSelection on heterozygosity as a LOH event could representis an underappreciated source of adaptation in microbial ex-perimental evolution as typical experiments evolve a haploidor homozygous diploid strain asexually and as a result havelittle variation to select upon LOH is observed in natural andindustrial hybrids (Albertin and Marullo 2012 Louis et al2012 Pryszcz et al 2014 Wolfe 2015 Schroder et al 2016)but here we document its occurrence in both intra- andinterspecific hybrids in the laboratory as a result of short-termevolution (also see (Dunn et al 2013 Burke et al 2014)) LOHis observed across all nutrient conditions with 13 independentLOH events detected in S cerevisiae and 9 independent eventsdocumented in the hybrids (figs 1 and 2 tables 1 and 2 sup-plementary figs S1 and S3 Supplementary Material online) Itthus appears that this type of mutational event is both com-mon and can occur over short evolutionary timescales

The LOH event can result in copy-neutral (where one alleleis lost and the other allele is amplified) or non-neutral chro-mosomal segments (where one allele is lost rendering thestrain hemizygous at that locus) and can favor the retentionof either allele In S cerevisiae there is a bias in resolutionwhere LOH events favor retaining the GRF167 allele over theS288C allele (1013 events Pfrac14 00169 table 2 supplementaryfig S4 Supplementary Material online) One unique case inclone Sc4 has a small approximately 5 kb LOH event onchrXV favoring GRF167 which switches to favoring S288Cfor the rest of the chromosome The retention of S cerevisiaeis slightly more common in the hybrids (59 events Pfrac14 10table 1 supplementary fig S3 Supplementary Material on-line) though not as drastic as the observed genome resolu-tion in the hybrid S pastorianus where LOH favorsS cerevisiae over S eubayanus (Nakao et al 2009) The sizeof the event ranges from approximately 25 kb to the wholechromosome level in the hybrids and from 5 kb to 540 kbin S cerevisiae Where LOH is accompanied by an amplifi-cation event the LOH event always occurs first and doesnot share break points with the amplification event Unlikemany CNV events most LOH events do not appear to bemediated by existing repetitive sequence such as a trans-posable element in the hybrid or S cerevisiae and are mostlikely a product of break-induced replication or mitoticgene conversion with break-induced replication as the fa-vored method as all events extend to the telomere (Hoanget al 2010) The exceptions are in hybrid clones Ph4 Ph5 andSh1 where there is a non-copy neutral loss of S cerevisiaemediated by a Ty element or LTR and S cerevisiae clonesSc1 and Sc4 where there is a 65-kb deletion of the S288Callele flanked by two Ty elements

LOH events in hybrids could signify several ongoing pro-cesses in hybrid genome evolution LOH regions may repre-sent 1) loci with incompatibilities 2) selection on existingvariation or 3) genetic drift eroding genomic segments It isimpossible to definitively rule out any of these hypotheseswithout further experimentation however there are severalarguments disfavoring the incompatibility hypothesis Firstalthough our sample size is modest failing to see repeatedLOH events across nutrient conditions may indicate a lack ofgeneral incompatibility between species (although we cannotpreclude condition-specific incompatibility Hou et al 2015Piatkowska et al 2013) This is consistent with previous stud-ies in yeast which suggest that classic DobzhanksyndashMullerincompatibilities are rare (Liti et al 2006 Greig 2009 Houet al 2014) Furthermore LOH events observed inevolved S cerevisiae suggest that this mutation type isnot unique to interspecific hybrids Instead repeatedevents within a particular condition such as the repeatedLOH at PHO84 in phosphate limitation or the 65 kbsegment on chrXIII in sulfate limitation suggest thatthese events are beneficial and are indeed selection onone allele over the other

LOH Is Driven by Selection on One AlleleTo test the hypothesis that LOH events provide a selectiveadvantage we used allele replacement in which the allele of

Smukowski Heil et al doi101093molbevmsx098 MBE

10

one speciesstrain is replaced with the allele of the otherspeciesstrain in an otherwise isogenic background We testedthis hypothesis using the most commonly seen LOH eventLOH at PHO84 Although the region extends from 25 kb to234 kb in length in the hybrids and from 40 kb to 85 kb in Scerevisiae and thus includes many genes PHO84 was a primecandidate driving this event PHO84 is one of only ten genesencompassed in the region extending from the telomere tothe break point of the shortest LOH event and is included inevery other LOH event on chromosome XIII (fig 2) It is ahigh-affinity phosphate transporter responsible for inorganicphosphate uptake in high and low phosphate conditions(Wykoff and OrsquoShea 2001) and previous work identified apoint mutation in PHO84 (an alanine to valine substitution atthe 50 end of the gene) which increased fitness by 23 inphosphate-limited conditions (Sunshine et al 2015) Finallyprior work with other nutrient transporters has shown am-plification of nutrient transporters to be a key event in adapt-ing to nutrient-limited conditions

We thus selected a region of approximately 25 kb encom-passing the PHO84 ORF its promoter and 30-UTR(Nagalakshmi et al 2008 Yassour et al 2009 Cherry et al2012) We created allele replacement strains using the twoalleles of S cerevisiae in a S cerevisiae diploid background thetwo alleles are 991 identical in this region (supplementaryfig S5 Supplementary Material online) and each strain isidentical to the ancestral strain used in our evolution exper-iments except at the PHO84 locus The S cerevisiae ancestorcarries one copy of GRF167 (preferred) and one copy ofS288C (ldquoun-preferredrdquo) so named due to which allele wasretained and amplified in the evolved clones To measureany resultant changes in fitness we competed each strainindividually against a fluorescent ancestral strain and mea-sured which strain overtook the culture Relative fitness isthus defined as the growth advantage per generation Twocopies of the un-preferred allele decreased relative fitness by531 (6186) whereas two copies of the preferred alleleincreased relative fitness by 993 (6027) This displays anoverall difference in relative fitness of 1524 between the un-preferred and preferred alleles By comparing the fitness ofthese allele replacement strains with the evolved clones (table 2)the allele replacement does not fully recapitulate the fit-ness gain observed in the evolved clone One explanation isthat the additional mutations present in the evolvedstrains also contribute to their total fitness Another expla-nation could be the increased copy number of the PHO84region that we see in these evolved clones To further ex-plore this fitness difference we cloned the GRF167 alleleonto a low copy number plasmid and transformed theallele replacement strain carrying two preferred S cerevi-siae alleles to simulate increased copy number of PHO84and saw only a minimal fitness increase of 176 (6106note all fitness competitions involving plasmids were donein conjunction with empty plasmids to take into accountany fitness effects from the plasmid itself) This supportsthe conclusion that relative fitness gains in the evolvedclone are largely due to the loss of the S288C allele andselection and amplification of the GRF167 allele with little

additional benefit from further amplification It could also bethe case that co-amplification of other genes in the segmentis required to attain the full benefit as previously observedby the contribution of BSD2 to the SUL1 amplicon in sulfatelimitation (Sunshine et al 2015 Payen et al 2016)

To understand the fitness effects of LOH and amplificationin the hybrids we generated hybrid strains with varying num-bers of S cerevisiae GRF167 PHO84 alleles Unfortunately wewere unable to obtain a successful strain carrying the pre-ferred S cerevisiae allele in a S uvarum background howeverwe were able to generate a S uvarum PHO84 knockout strainthus creating a hybrid with one copy of S cerevisiae PHO84When combined with a low copy plasmid carrying the sameallele this strain effectively has two or more copies ofS cerevisiae PHO84 in a hybrid background This hybrid straincan serve as a proxy for the LOH event observed in the evo-lution experiments and has a relative fitness gain of 2557(6288) The ancestral hybrid with the same plasmid (effec-tively 1 S uvarum allele and 2 or more S cerevisiae alleles) hasa relative fitness gain of 1253 (6131) The difference be-tween these two hybrids suggests that while amplification isbeneficial the highest fitness is achieved with the loss of theS uvarum allele (Pfrac14 00061)

Together these results support the conclusion that theS cerevisiae GRF167 allele is preferred over the S288C alleleand that S cerevisiae alleles are preferred over the S uvarumallele in the hybrid and hence that the LOH events seen inboth intra- and interspecific hybrids are the product ofselection

DiscussionIn summary we sought to understand forces underlying ge-nome stabilization and evolution in interspecific and intra-specific hybrids as they adapt to novel environments Weevolved and sequenced clones from 16 hybrid populationsand 16 parental populations to reveal a variety of mutationalevents conferring adaptation to 3 nutrient-limited conditionsOf particular note we find LOH in both evolved intraspecificand interspecific hybrid clones in all nutrient environmentspotentially signifying areas where selection has acted on pre-existing variation present in the ancestral clone We used anallele replacement strategy to test this hypothesis for a com-monly repeated LOH event and show that selection is indeeddriving the homogenization of the genome at this locusAlthough other studies in natural industrial and lab-evolved isolates have observed LOH we present the first em-pirical test of the causal evolutionary forces influencing theseevents This work can begin to help us understand past hy-bridization events and subsequent genome resolution in hy-brids in natural and artificial systems

Similarities and Differences between Intra- andInterspecific HybridsAlthough our sample size is modest we observe several in-teresting trends when comparing S uvarum clones (homo-zygous) S cerevisiae clones (intraspecific hybrid andpreviously published homozygous) and interspecific hybrid

Adaptation in Hybrids doi101093molbevmsx098 MBE

11

clones First theory and previous research predict the inter-specific hybrid may experience more genome instability in theform of chromosomal rearrangements and CNV events(Xiong et al 2011 Lloyd et al 2014 Chester et al 2015Mason and Batley 2015) Instead in our work the interspecifichybrid and S cerevisiae clones experience the same numberof CNV events (both 15 CNVsclone) However the mech-anism of CNV formation seems to differ between the hybridand S cerevisiae clones Whereas S cerevisiae CNV breakpoints typically occur at transposable elements in our studyand previous studies (Dunham et al 2002 Gresham et al2008 Fedoroff 2012) the interspecific hybrid CNVs do notutilize transposable elements although they obviously sharethe same sequence background as the S cerevisiae clones(albeit in one copy) Whether this difference is due to theabsence of full-length transposable elements in S uvarum isunknown but this could potentially explain the lower num-ber of CNV events in S uvarum clones (1 CNVclone) andpresents an intriguing direction for future study

The Predictability of EvolutionWe now have many examples of predictable evolution innatural systems (Losos et al 1998 Rundle et al 2000 Elmerand Meyer 2011 Conte et al 2012 Jones et al 2012 Martinand Orgogozo 2013 Wessinger and Rausher 2014) and inlaboratory experimental evolution in which there often ap-pears to be a limited number of high fitness pathways thatstrains follow when adapting to a particular condition (Fereaet al 1999 Woods et al 2006 Gresham et al 2008 Burke et al2010 Salverda et al 2011 Kawecki et al 2012 Kvitek andSherlock 2013 Lang and Desai 2014) For example it is wellestablished that amplifications of nutrient transporters aredrivers of adaptation in evolution in nutrient-limited condi-tions Previous work in our group has particularly focused onthe amplification of the high-affinity sulfate transporter geneSUL1 in sulfate-limited conditions which occurs in almostevery sulfate-limited evolution experiment and confers a fit-ness advantage of as much as 40 compared with the un-evolved ancestor strain The amplification of phosphatetransporters has been markedly less common and thus driv-ers of adaptation in this condition have been less clearGresham et al (2008) identified a whole chromosome ampli-fication of chrXIII in one population In a follow-up studySunshine et al (2015) found whole or partial amplification ofchrXIII in 38 populations A genome-wide screen for segmen-tal amplifications found a slight increase in fitness for a smalltelomeric segment of chromosome XIII and a A49V pointmutation in PHO84 was observed to increase fitness by 23However screens by Payen et al (2016) showed that althoughPHO84 is recurrently mutated in various experiments itshowed no benefit when amplified or deleted in phos-phate-limited conditions Finally additional evolution exper-iments recapitulated the point mutation seen in Sunshineet al (2015) in 2432 populations and saw amplification ofPHO84 in 832 populations (Miller A Dunham MJ unpub-lished data) It is important to note that all these experimentsused a strain background derived from S288C or CENPKboth of which carry the same (un-preferred) PHO84 allele

In our work we observed amplification of the S cerevisiaeGRF167 allele of PHO84 in 44 S cerevisiae clones from 4populations and 36 hybrid clones from 6 populations Thisamplification was always preceded by the loss of the S288Callele in S cerevisiae clones and the LOH break points arenever shared with the amplification break points There is a15 fitness difference between carrying two copies of theS288C allele of PHO84 compared with carrying two copiesof the GRF167 allele of PHO84 and additional copies of theGRF167 allele do not provide substantial further fitness gainsThe two alleles differ by several noncoding changes and threenonsynonymous substitutions a mutation from glutamicacid to aspartic acid (E229D) leucine to proline (L259P)and leucine to glutamine (L556Q) the latter two of whichare considered ldquononconservativerdquo protein mutations due tochanges in hydrophobicity and structure (supplementary figS5 Supplementary Material online) Intriguingly the L259Pmutation has actually been previously identified as being re-sponsible for resistance to tetrachloroisophthalonitrile andpartial resistance to pentachlorophenol in a QTL study ofsmall-molecule drugs (Perlstein et al 2007) IndeedPerlstein et al found proline at residue 259 to be conservedacross fungal species and even in orthologous human xeno-biotic transporters likely because proline-induced kinks intransmembrane spans have been shown to be essential toprotein function (Cordes et al 2002 Perlstein et al 2007) Itthus appears that amplification of PHO84 has been less pre-dictable as the S288C allele does not confer a fitness advan-tage unless mutated per Sunshine et al (2015) Togetherthese results imply that strain background can constrainadaptive pathways

In hybrids the amplification of the S cerevisiae segmentoccurred in conjunction with the loss of the S uvarum alleleHybrid strains with the LOH had a 2557 relative fitness gainwhereas hybrid strains with amplification of the S cerevisiaePHO84 allele without the LOH had a 1253 fitness gain ThusLOH confers an additional 1304 fitness gain showing thatselection for LOH has a larger impact on fitness than ampli-fication alone Note that S uvarum does have proline at res-idue 259 like the preferred GRF167 allele and differs fromboth S cerevisiae alleles at the other two coding substitutions(229N and 556K) but the amino acid and noncoding diver-gence is too high to speculate what substitutions are respon-sible for the selection of the GRF167 allele (supplementary figS6 Supplementary Material online) Why the loss of one alleleis more beneficial remains unclear as PHO84 is thought tofunction as a monomer (Bun-Ya et al 1991) but it may bedue to competition for cell wall space or negative interactionswith other genes in the PHO pathway (Mouillon and Persson2006)

The infusion of variation created by hybridization providesnew templates for selection to act upon which can be moreimportant than either point mutations or copy number var-iants alone Our work shows that outcrossing need not becommon to have long-lasting effects on adaptation This im-plication is particularly relevant in yeast where outcrossingmay occur quite rarely followed by thousands of asexual gen-erations (Ruderfer et al 2006 Greig and Leu 2009 Liti 2015)

Smukowski Heil et al doi101093molbevmsx098 MBE

12

Applications to Other Hybrids and CancerThe observation that LOH occurs in hybrid genomes is in-creasingly documented (Louis et al 2012 Borneman et al2014 Soltis et al 2014 Marcet-Houben and Gabaldon 2015Pryszcz et al 2014 Schroder et al 2016) although the rea-son(s) for this type of mutation has been unresolved As mostexamples stem from allopolyploid events that occurred mil-lions of years ago understanding why LOH is important inhybrid genome evolution is difficult Cancer cells are alsoknown to experience LOH sometimes involved in the inac-tivation of tumor suppressor genes leaving only one copy ofthe gene that may be mutated or silenced (Thiagalingam et al2001 Tuna et al 2009 Lapunzina and Monk 2011) Datasupport the conclusion that LOH events are selected for dur-ing tumor development as many LOH events involve specificchromosomal segments (Thiagalingam et al 2001) althoughthe underlying molecular and genetic reasons for selection isan open debate (Ryland et al 2015)

Here we experimentally demonstrate that LOH can occurin homoploid interspecific hybrids as well as in intraspecifichybrids These events occur within a few hundred generationsand are common mutations more common on average inthe intraspecific hybrid (13 eventsclone) than the interspe-cific hybrid (056 eventsclone) Interestingly the LOH eventsdo not share break points with the CNV events in S cerevisiaeinstead they appear to occur independently and to precedeany subsequent amplification (amplification occurs following913 LOH events) Competition assays with the PHO84 locusprovide support that LOH itself may be more beneficial thanamplification or at least increase the selective benefit of am-plification events The observation that LOH in intraspecifichybrids occurs independently from copy number change pro-vides different opportunities for adaptation to novelconditions

Other cases of LOH like the copy number neutral eventsobserved in Ph4 Ph5 and Sh4 all of which favor the retentionof the S uvarum allele (supplementary fig S3 SupplementaryMaterial online) may be due to hybrid incompatibility withina particular protein complex other epistatic interactions(Piatkowska et al 2013) or neutral processes We furthermorediscover examples where one species allele appears to bepreferred over the other without LOH such as the repeatedamplification of the S uvarum high-affinity glucose transpor-ters HXT67 When one species allele is amplified and theother is not amplified one explanation is that the local se-quence context can permit or deny amplification In the caseof HXT67 previous experiments in S cerevisiae have shownthat amplification of HXT67 is quite common (Brown et al1998 Dunham et al 2002 Gresham et al 2008 Kao andSherlock 2008 Kvitek and Sherlock 2011) thus suggestingthat when given a choice between this locus in S cerevisiaeor S uvarum in the hybrid the preferred allele is indeed Suvarum though more subtle differences in rate cannot yet beruled out A similar scenario is observed with S cerevisiaeSUL1 (Sanchez et al 2017) Together our results show thatthe heterozygosity supplied by hybridization is an importantcontributor to adaptive routes explored by populations asthey adapt to novel conditions

Although we cannot generalize our results from thePHO84 locus across the many other LOH events discoveredin our hybrids and S cerevisiae in the future we can usesimilar methodology to explore whether positive selectionalways drives LOH or whether other explanations such asincompatibility resolution contribute as well Future experi-ments might also utilize a high throughput method to ex-plore segmental LOH in hybrids at a genome-wide scalesimilar to ongoing experiments at the gene level (LancasterS Dunham MJ unpublished data) Although our sample sizeis moderate this is a novel and necessary step in understand-ing forces underlying hybrid genome stabilization and high-lighting an underappreciated mechanism of hybridadaptation

ConclusionsThe mutation events we observe in our experimentallyevolved hybrids are in many ways quite representative ofmutations observed in ancient hybrid genomes suggestingthat hybrid genome stabilization and adaptation can occurquite rapidly (within several hundred generations)Furthermore our results illustrate that the infusion of varia-tion introduced by hybridization at both the intra- and inter-species level can increase fitness by providing choices of allelesfor selection to act upon even when sexual reproduction israre This may be particularly important for leveraging existingvariation for agricultural and industrial processes and as cli-mate change potentially increases natural hybridization (Kellyet al 2010 Hoffmann and Sgro 2011 Muhlfeld et al 2014)

Materials and Methods

StrainsA list of strains used in this study is included in supplementarytable S1 Supplementary Material online All interspecific hy-brids were created by crossing a ura3 LYS2 haploid parent to aURA3 lys2 haploid parent of the other mating type plating onmedia lacking both uracil and lysine and selecting for proto-trophs The S cerevisiae strain background known asldquoGRF167rdquo is itself a cross between FL100 and the genomictype strain S288C (data not shown) GRF167 was chosen as astrain background for simultaneous work investigating trans-posable elements during experimental evolution which willbe addressed in a future study

Evolution ExperimentsContinuous cultures were established using media and con-ditions previously described (Gresham et al 2008 Sanchezet al 2017) Detailed protocols and media recipes are availableat httpdunhamgswashingtoneduprotocolsshtml (lastaccessed March 6 2017) Samples were taken daily and mea-sured for optical density at 600 nm and cell count micros-copy was performed to check for contamination andarchival glycerol stocks were made daily An experimentwas terminated when contamination growth in tubing orclumping appeared (number of generations at the endpoint for each population are presented in tables 1 and

Adaptation in Hybrids doi101093molbevmsx098 MBE

13

2) Samples from each end point population were colonypurified to yield two clones for further study

Array Comparative Genomic HybridizationPopulations from the end point of each evolution were ana-lyzed for copy number changes using array comparative ge-nomic hybridization following the protocol used in Sanchezet al (2017)

SequencingDNA was extracted from overnight cultures using theHoffmanndashWinston protocol (Hoffman and Winston 1987)and cleaned using the Clean amp Concentrator kit (ZymoResearch) Nextera libraries were prepared following theNextera library kit protocol and sequenced using paired end150 bp reads on the Illumina NextSeq 500 machine (sequenc-ing coverage in supplementary table S2 SupplementaryMaterial online) The reference genomes used were S cerevi-siae v3 (Engel et al 2014) S uvarum (Scannell et al 2011) anda hybrid reference genome created by concatenating the twogenomes Sequence was aligned to the appropriate referencegenome using bwa v062 (Li and Durbin 2009) and mutationswere called using GATK (McKenna et al 2010) and samtools0119 (Li et al 2009) Mutations in evolved clones were filteredin comparison with the ancestor to obtain de novo mutationsAll mutations were first visually inspected using IntegrativeGenomics Viewer (Robinson et al 2011) Subsequently pointmutations in the hybrids were confirmed with Sanger se-quencing (supplementary table S3 Supplementary Materialonline) Copy number variants were visualized usingDNAcopy for S cerevisiae and S uvarum (Seshan andOlshen 2016) LOH events were called based on sequencingcoverage in the hybrids and by identifying homozygous variantcalls in S cerevisiae All break points were called by visual in-spection of sequencing reads and are thus approximate

Fitness AssaysThe pairwise competition experiments were performed in 20ml chemostats (Miller and Dunham 2013) Each competitorstrain was cultured individually until steady state was reachedand then was mixed 5050 with a GFP-tagged ancestor Eachcompetition was conducted in at least two biological repli-cates for approximately 15 generations after mixing Sampleswere collected and analyzed twice daily The proportion ofGFPthorn cells in the population was detected using a BD AccuriC6 flow cytometer (BD Biosciences) The data were plottedwith ln [dark cellsGFPthorn cells] versus generations The rela-tive fitness coefficient was determined from the slope of thelinear region

Strain ConstructionAllele replacements for the PHO84 locus were done followingthe protocol of the Caudy lab with further modifications de-scribed here The native locus was replaced withKluyveromyces lactis URA3 The pho84DURA3 strain wasgrown overnight in 5 ml of C-URA media then inoculatedin a flask of 100 ml yeast extract peptone dextrose (YPD) andgrown to an optical density of 06ndash08 Cells were washed then

aliquoted 275 ml of transformation mix (35 ml 1 M lithiumacetate 240 ml of 50 3500 polyethylene glycol) 10 ml ofsalmon sperm and approximately 3 mg of polymerase chainreaction product were added to the cell pellet It was incu-bated at 37 C (S uvarum) or 42 C (S cerevisiae) for 45 minthen plated to YPD It was replica plated to 5-fluorooroticacid the following day and colonies were tested for the gain ofthe appropriate species allele The GRF167 allele was clonedinto the pIL37 plasmid using Gibson assembly (Gibson et al2009) Correct assembly was verified by Sanger sequencing Allprimers used can be found in supplementary table S3Supplementary Material online

Supplementary MaterialSupplementary data are available at Molecular Biology andEvolution online

AcknowledgmentsWe thank Noah Hanson and Erica Alcantara for technicalassistance We thank Monica Sanchez and three anonymousreviewers for helpful comments on this manuscript JosephSchacherer kindly confirmed the contribution of FL100 to theGRF167 strain background Thanks to Yixian Zheng and DougKoshland for contributing to the initial experimental designcreating yeast strains and purchasing the oligonucleotidesused for the microarrays This work was supported by theNational Science Foundation (grant number 1516330) andthe National Institutes of Health (grant number R01GM094306) MD is a Senior Fellow in the GeneticNetworks program at the Canadian Institute for AdvancedResearch and a Rita Allen Foundation Scholar CSH wassupported in part by National Institutes of Health (grantnumber T32 HG00035) This work was also supported bythe National Institutes of Health (grant number P50GM071508) to the Lewis-Sigler Institute and from theHoward Hughes Medical Institute to Doug Koshland andYixian Zheng Sequencing read data have been deposited atthe NCBI under BioProject accession PRJNA374049Microarray data are deposited in the Gene ExpressionOmnibus (GEO) repository under accession numbersGSE95086 and GSE87401 and in the Princeton MicroarrayDatabase

ReferencesAbbott R Albach D Ansell S Arntzen JW Baird SJ Bierne N Boughman J

Brelsford A Buerkle CA Buggs R et al 2013 Hybridization andspeciation J Evol Biol 26229ndash246

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Albertin W Marullo P 2012 Polyploidy in fungi evolution after whole-genome duplication Proc Biol Sci 2792497ndash2509

Almeida P Goncalves C Teixeira S Libkind D Bontrager M Masneuf-Pomarede I Albertin W Durrens P Sherman DJ Marullo P et al2014 A Gondwanan imprint on global diversity and domesticationof wine and cider yeast Saccharomyces uvarum Nat Commun54044

Antunovics Z Nguyen HV Gaillardin C Sipiczki M 2005 Gradual ge-nome stabilisation by progressive reduction of the Saccharomyces

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uvarum genome in an interspecific hybrid with Saccharomyces cer-evisiae FEMS Yeast Res 51141ndash1150

Baker E Wang B Bellora N Peris D Hulfachor AB Koshalek JA AdamsM Libkind D Hittinger CT 2015 The Genome sequence ofSaccharomyces eubayanus and the domestication of lager-brewingyeasts Mol Biol Evol 322818ndash2831

Barbosa R Almeida P Safar SVB Santos RO Morais PB Nielly-Thibault LLeducq JB Landry CR Goncalves P Rosa CA Sampaio JP 2016Evidence of natural hybridization in Brazilian wild lineages ofSaccharomyces cerevisiae Genome Biol Evol 8317ndash329

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Belloch C Orlic S Barrio E Querol A 2008 Fermentative stress adapta-tion of hybrids within the Saccharomyces sensu stricto complex Int JFood Microbiol 122188ndash195

Bellon JR Yang F Day MP Inglis DL Chambers PJ 2015 Designingand creating Saccharomyces interspecific hybrids for improvedindustry relevant phenotypes Appl Microbiol Biotechnol998597ndash8609

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Brewer BJ Payen C Di Rienzi SC Higgins MM Ong G Dunham MJRaghuraman MK 2015 Origin-dependent inverted-repeat amplifi-cation tests of a model for inverted DNA amplification PLoS Genet11e1005699

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Bun-Ya M Nishimura M Harashima S Oshima Y 1991 The PHO84 geneof Saccharomyces cerevisiae encodes an inorganic phosphate trans-porter Mol Cell Biol 113229ndash3238

Burke MK Dunham JP Shahrestani P Thornton KR Rose MR Long AD2010 Genome-wide analysis of a long-term evolution experimentwith Drosophila Nature 467587ndash590

Burke MK Liti G Long AD 2014 Standing genetic variation drives re-peatable experimental evolution in outcrossing populations ofSaccharomyces cerevisiae Mol Biol Evol 313228ndash3239

Cherry JM Hong EL Amundsen C Balakrishnan R Binkley G Chan ETChristie KR Costanzo MC Dwight SS Engel SR et al 2012Saccharomyces genome database the genomics resource of buddingyeast Nucleic Acids Res 40D700ndashD705

Chester M Riley RK Soltis PS Soltis DE 2015 Patterns of chromosomalvariation in natural populations of the neoallotetraploid Tragopogonmirus (Asteraceae) Heredity (Edinb) 114309ndash317

Chou JY Leu JY 2010 Speciation through cytonuclear incompatibilityinsights from yeast and implications for higher eukaryotes Bioessays32401ndash411

Cliften PF Fulton RS Wilson RK Johnston M 2006 After the duplicationgene loss and adaptation in Saccharomyces genomes Genetics172863ndash872

Conte GL Arnegard ME Peichel CL Schluter D 2012 The probability ofgenetic parallelism and convergence in natural populations Proc BiolSci 2795039ndash5047

Cordes FS Bright JN Sansom MS 2002 Proline-induced distortions oftransmembrane helices J Mol Biol 323951ndash960

Crow JF 1998 90 years ago the beginning of hybrid maize Genetics148923ndash928

Dasmahapatra KK Walters JR Briscoe AD Davey JW Whibley A NadeauNJ Zimin AV Hughes DST Ferguson LC Martin SH et al 2012Butterfly genome reveals promiscuous exchange of mimicry adap-tations among species Nature 48794ndash98

Dowling TE Smith GR Brown WM 1989 Reproductive isolation andintrogression between Notropis-Cornutus and Notropis-Chrysocephalus (Family Cyprinidae)mdashcomparison of morphologyallozymes and mitochondrial-DNA Evolution 43620ndash634

Doyle JJ Flagel LE Paterson AH Rapp RA Soltis DE Soltis PS Wendel JF2008 Evolutionary genetics of genome merger and doubling inplants Annu Rev Genet 42443ndash461

Dunham MJ Badrane H Ferea T Adams J Brown PO Rosenzweig FBotstein D 2002 Characteristic genome rearrangements in experi-mental evolution of Saccharomyces cerevisiae Proc Natl Acad Sci U SA 9916144ndash16149

Dunn B Paulish T Stanbery A Piotrowski J Koniges G Kroll E LouisEJ Liti G Sherlock G Rosenzweig F 2013 Recurrent rearrange-ment during adaptive evolution in an interspecific yeast hybridsuggests a model for rapid introgression PLoS Genet9e1003366

Dunn B Richter C Kvitek DJ Pugh T Sherlock G 2012 Analysis of theSaccharomyces cerevisiae pan-genome reveals a pool of copy num-ber variants distributed in diverse yeast strains from differing indus-trial environments Genome Res 22908ndash924

Dunn B Sherlock G 2008 Reconstruction of the genome origins andevolution of the hybrid lager yeast Saccharomyces pastorianusGenome Res 181610ndash1623

Elmer KR Meyer A 2011 Adaptation in the age of ecological genomicsinsights from parallelism and convergence Trends Ecol Evol26298ndash306

Engel SR Dietrich FS Fisk DG Binkley G Balakrishnan R Costanzo MCDwight SS Hitz BC Karra K Nash RS et al 2014 The referencegenome sequence of Saccharomyces cerevisiae then and now G3(Bethesda) 4389ndash398

Fedoroff NV 2012 Presidential address Transposable elements epige-netics and genome evolution Science 338758ndash767

Ferea TL Botstein D Brown PO Rosenzweig RF 1999 Systematicchanges in gene expression patterns following adaptive evolutionin yeast Proc Natl Acad Sci U S A 969721ndash9726

Gibson B Liti G 2015 Saccharomyces pastorianus genomic insightsinspiring innovation for industry Yeast 3217ndash27

Gibson DG Young L Chuang RY Venter JC Hutchison CA Smith HO2009 Enzymatic assembly of DNA molecules up to several hundredkilobases Nat Methods 6343ndash5

Gonzalez SS Barrio E Gafner J Querol A 2006 Natural hybrids fromSaccharomyces cerevisiae Saccharomyces bayanus andSaccharomyces kudriavzevii in wine fermentations FEMS Yeast Res61221ndash1234

Gonzalez SS Barrio E Querol A 2008 Molecular characterization of newnatural hybrids of Saccharomyces cerevisiae and S kudriavzevii inbrewing Appl Environ Microbiol 742314ndash2320

Grant PR Grant BR 1994 Phenotypic and genetic consequences ofhybridization in Darwinrsquos finches Evolution 48297ndash316

Grant PR Grant BR 2002 Unpredictable evolution in a 30-year study ofDarwinrsquos finches Science 296707ndash711

Grant PR Grant BR 2010 Natural selection speciation and Darwinrsquosfinches Proc Calif Acad Sci 61245ndash260

Grant PR Grant BR Petren K 2005 Hybridization in the recent past AmNat 16656ndash67

Greig D 2009 Reproductive isolation in Saccharomyces Heredity (Edinb)10239ndash44

Greig D Leu JY 2009 Natural history of budding yeast Current Biol19R886ndashR890

Greig D Louis EJ Borts RH Travisano M 2002 Hybrid speciation inexperimental populations of yeast Science 2981773ndash1775

Gresham D Desai MM Tucker CM Jenq HT Pai DA Ward A DeSevoCG Botstein D Dunham MJ 2008 The repertoire and dynamics ofevolutionary adaptations to controlled nutrient-limited environ-ments in yeast PLoS Genet 4e1000303

Hajjar R Hodgkin T 2007 The use of wild relatives in crop improvementa survey of developments over the last 20 years Euphytica 1561ndash13

Heiser CB 1954 Variation and subspeciation in the common sunflowerHelianthus annuus Am Midl Nat 51287ndash305

Hittinger CT 2013 Saccharomyces diversity and evolution a buddingmodel genus Trends Genet 29309ndash317

Hoang ML Tan FJ Lai DC Celniker SE Hoskins RA Dunham MJ ZhengY Koshland D 2010 Competitive repair by naturally dispersed

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15

repetitive DNA during non-allelic homologous recombination PLoSGenet 6e1001228

Hoffmann AA Sgro CM 2011 Climate change and evolutionary adap-tation Nature 470479ndash485

Hoffman CS Winston F 1987 A 10-minute DNA preparation from yeastefficiently releases autonomous plasmids for transformation ofEscherichia coli Gene 57267ndash272

Hou J Friedrich A de Montigny J Schacherer J 2014 Chromosomalrearrangements as a major mechanism in the onset of reproductiveisolation in Saccharomyces cerevisiae Curr Biol 241153ndash1159

Hou J Friedrich A Gounot JS Schacherer J 2015 Comprehensive surveyof condition-specific reproductive isolation reveals genetic incom-patibility in yeast Nat Commun 67214

Huerta-Sanchez E Casey FP 2015 Archaic inheritance supporting high-altitude life in Tibet J Appl Physiol (1985) 1191129ndash1134

Jones FC Grabherr MG Chan YF Russell P Mauceli E Johnson JSwofford R Pirun M Zody MC White S et al 2012 The genomicbasis of adaptive evolution in threespine sticklebacks Nature48455ndash61

Kao KC Sherlock G 2008 Molecular characterization of clonal interfer-ence during adaptive evolution in asexual populations ofSaccharomyces cerevisiae Nat Genet 401499ndash1504

Kawecki TJ Lenski RE Ebert D Hollis B Olivieri I Whitlock MC 2012Experimental evolution Trends Ecol Evol 27547ndash560

Kellis M Patterson N Endrizzi M Birren B Lander ES 2003 Sequencingand comparison of yeast species to identify genes and regulatoryelements Nature 423241ndash254

Kelly B Whiteley A Tallmon D 2010 The Arctic melting pot Nature468891ndash891

Kvitek DJ Sherlock G 2011 Reciprocal sign epistasis between frequentlyexperimentally evolved adaptive mutations causes a rugged fitnesslandscape PLoS Genet 7e1002056

Kvitek DJ Sherlock G 2013 Whole genome whole population sequenc-ing reveals that loss of signaling networks is the major adaptivestrategy in a constant environment PLoS Genet 9e1003972

Landry CR Hartl DL Ranz JM 2007 Genome clashes in hybrids insightsfrom gene expression Heredity (Edinb) 99483ndash493

Lang GI Desai MM 2014 The spectrum of adaptive mutations in ex-perimental evolution Genomics 104412ndash416

Lapunzina P Monk D 2011 The consequences of uniparental disomyand copy number neutral loss-of-heterozygosity during human de-velopment and cancer Biol Cell 103303ndash317

Leducq JB Nielly-Thibault L Charron G Eberlein C Verta JP Samani PSylvester K Hittinger CT Bell G Landry CR 2016 Speciation drivenby hybridization and chromosomal plasticity in a wild yeast NatMicrobiol 115003

Lee HY Chou JY Cheong L Chang NH Yang SY Leu JY 2008Incompatibility of nuclear and mitochondrial genomes causes hy-brid sterility between two yeast species Cell 1351065ndash1073

Li H Durbin R 2009 Fast and accurate short read alignment withBurrows-Wheeler transform Bioinformatics 251754ndash1760

Li H Handsaker B Wysoker A Fennell T Ruan J Homer N Marth GAbecasis G Durbin R 2009 The Sequence AlignmentMap formatand SAMtools Bioinformatics 252078ndash2079

Liti G 2015 The fascinating and secret wild life of the budding yeast Scerevisiae Elife 4e05835

Liti G Barton DB Louis EJ 2006 Sequence diversity reproductiveisolation and species concepts in Saccharomyces Genetics174839ndash850

Lloyd AH Ranoux M Vautrin S Glover N Fourment J Charif D ChouletF Lassalle G Marande W Tran J et al 2014 Meiotic gene evolutioncan you teach a new dog new tricks Mol Biol Evol 311724ndash1727

Losos JB Jackman TR Larson A de Queiroz K Rodriguez-Schettino L1998 Contingency and determinism in replicated adaptive radia-tions of island lizards Science 2792115ndash2118

Louis VL Despons L Friedrich A Martin T Durrens P Casaregola SNeuveglise C Fairhead C Marck C Cruz JA et al 2012 Pichia sorbi-tophila an interspecies yeast hybrid reveals early steps of genomeresolution after polyploidization G3 (Bethesda) 2299ndash311

Mallet J 2007 Hybrid speciation Nature 446279ndash283Marcet-Houben M Gabaldon T 2015 Beyond the whole-genome du-

plication phylogenetic evidence for an ancient interspecies hybrid-ization in the bakerrsquos yeast lineage PLoS Biol 13e1002220

Marinoni G Manuel M Petersen RF Hvidtfeldt J Sulo P Piskur J 1999Horizontal transfer of genetic material among Saccharomyces yeastsJ Bacteriol 1816488ndash6496

Martin A Orgogozo V 2013 The loci of repeated evolution a catalog ofgenetic hotspots of phenotypic variation Evolution 671235ndash1250

Masly JP Jones CD Noor MAF Locke J Orr HA 2006 Gene transpositionas a cause of hybrid sterility in Drosophila Science 3131448ndash1450

Mason AS Batley J 2015 Creating new interspecific hybrid and polyploidcrops Trends Biotechnol 33436ndash441

Mavarez J Salazar CA Bermingham E Salcedo C Jiggins CD Linares M2006 Speciation by hybridization in Heliconius butterflies Nature441868ndash871

McKenna A Hanna M Banks E Sivachenko A Cibulskis K Kernytsky AGarimella K Altshuler D Gabriel S Daly M et al 2010 The genomeanalysis toolkit a MapReduce framework for analyzing next-generation DNA sequencing data Genome Res 201297ndash1303

Meiklejohn CD Holmbeck MA Siddiq MA Abt DN Rand DMMontooth KL 2013 An Incompatibility between a mitochondrialtRNA and its nuclear-encoded tRNA synthetase compromises de-velopment and fitness in Drosophila PLoS Genet 9e1003238

Meyer A Salzburger W Schartl M 2006 Hybrid origin of a swordtailspecies (Teleostei Xiphophorus clemenciae) driven by sexual selec-tion Mol Ecol 15721ndash730

Michalak P 2009 Epigenetic transposon and small RNA determinants ofhybrid dysfunctions Heredity (Edinb) 10245ndash50

Miller AW Dunham MJ 2013 Design and use of multiplexed chemostatarrays J Vis Exp 72e50262

Monod J 1949 The growth of bacterial cultures Annu Rev Microbiol3371ndash394

Mouillon JM Persson BL 2006 New aspects on phosphate sensing andsignalling in Saccharomyces cerevisiae FEMS Yeast Res 6171ndash176

Muhlfeld CC Kovach RP Jones LA Al-Chokhachy R Boyer MC Leary RFLowe WH Luikart G Allendorf FW 2014 Invasive hybridization in athreatened species is accelerated by climate change Nat ClimChange 4620ndash624

Muller LAH McCusker JH 2009 A multispecies-based taxonomic mi-croarray reveals interspecies hybridization and introgression inSaccharomyces cerevisiae FEMS Yeast Res 9143ndash152

Nagalakshmi U Wang Z Waern K Shou C Raha D Gerstein M SnyderM 2008 The transcriptional landscape of the yeast genome definedby RNA sequencing Science 3201344ndash1349

Nakao Y Kanamori T Itoh T Kodama Y Rainieri S Nakamura NShimonaga T Hattori M Ashikari T 2009 Genome sequence ofthe lager brewing yeast an interspecies hybrid DNA Res 16115ndash129

Nolte AW Freyhof J Stemshorn KC Tautz D 2005 An invasive lineage ofsculpins Cottus sp (Pisces Teleostei) in the Rhine with new habitatadaptations has originated from hybridization between old phylo-geographic groups Proc Biol Sci 2722379ndash2387

Novick A Szilard L 1950a Description of the chemostat Science112715ndash716

Novick A Szilard L 1950b Experiments with the Chemostat on spon-taneous mutations of bacteria Proc Natl Acad Sci U S A 36708ndash719

Ouyang YD Liu YG Zhang QF 2010 Hybrid sterility in plant storiesfrom rice Curr Opin Plant Biol 13186ndash192

Payen C Di Rienzi SC Ong GT Pogachar JL Sanchez JC SunshineAB Raghuraman MK Brewer BJ Dunham MJ 2014 The dynam-ics of diverse segmental amplifications in populations ofSaccharomyces cerevisiae adapting to strong selection G3(Bethesda) 4399ndash409

Payen C Sunshine AB Ong GT Pogachar JL Zhao W Dunham MJ 2016High-throughput identification of adaptive mutations in experimen-tally evolved yeast populations PLoS Genet 12e1006339

Perez-Prat E van Lookeren Campagne MM 2002 Hybrid seed produc-tion and the challenge of propagating male-sterile plants TrendsPlant Sci 7199ndash203

Smukowski Heil et al doi101093molbevmsx098 MBE

16

Peris D Langdon QK Moriarty RV Sylvester K Bontrager M Charron GLeducq JB Landry CR Libkind D Hittinger CT 2016 Complex ances-tries of lager-brewing hybrids were shaped by standing variation inthe wild yeast Saccharomyces eubayanus PLoS Genet 12e1006155

Perlstein EO Ruderfer DM Roberts DC Schreiber SL Kruglyak L 2007Genetic basis of individual differences in the response to small-molecule drugs in yeast Nat Genet 39496ndash502

Piatkowska EM Naseeb S Knight D Delneri D 2013 Chimeric proteincomplexes in hybrid species generate novel phenotypes PLoS Genet9e1003836

Piotrowski JS Nagarajan S Kroll E Stanbery A Chiotti KE Kruckeberg ALDunn B Sherlock G Rosenzweig F 2012 Different selective pressureslead to different genomic outcomes as newly-formed hybrid yeastsevolve BMC Evol Biol 1246

Pryszcz LP Nemeth T Gacser A Gabaldon T 2014 Genome comparisonof Candida orthopsilosis clinical strains reveals the existence of hy-brids between two distinct subspecies Genome Biol Evol61069ndash1078

Racimo F Sankararaman S Nielsen R Huerta-Sanchez E 2015 Evidencefor archaic adaptive introgression in humans Nat Rev Genet16359ndash371

Rieseberg LH 1991 Homoploid reticulate evolution in Helianthus(Asteraceae)mdashevidence from ribosomal genes Am J Bot781218ndash1237

Rieseberg LH 1997 Hybrid origins of plant species Annu Rev EcolSystemat 28359ndash389

Robinson JT Thorvaldsdottir H Winckler W Guttman M Lander ESGetz G Mesirov JP 2011 Integrative genomics viewer NatBiotechnol 2924ndash26

Rosenthal DM Schwarzbach AE Donovan LA Raymond O RiesebergLH 2002 Phenotypic differentiation between three ancient hybridtaxa and their parental species Int J Plant Sci 163387ndash398

Ruderfer DM Pratt SC Seidel HS Kruglyak L 2006 Population genomicanalysis of outcrossing and recombination in yeast Nat Genet381077ndash1081

Rundle HD Nagel L Boughman JW Schluter D 2000 Natural selectionand parallel speciation in sympatric sticklebacks Science287306ndash308

Ryland GL Doyle MA Goode D Boyle SE Choong DY Rowley SM Li JBowtell DD Tothill RW Campbell IG et al 2015 Australian OvarianCancer Study Group Loss of heterozygosity what is it good for BMCMed Genomics 845

Salverda MLM Dellus E Gorter FA Debets AJM van der Oost J HoekstraRF Tawfik DS de Visser JAGM 2011 Initial mutations direct alter-native pathways of protein evolution PLoS Genet 7e1001321

Sanchez MR Miller AW Liachko I Sunshine AB Lynch B Huang MAlcantara E DeSevo CG Pai DA Tucker CM et al 2017 Differentialparalog divergence modulates genome evolution across yeast spe-cies PLoS Genet 13(2)e1006585

Scannell DR Zill OA Rokas A Payen C Dunham MJ Eisen MB Rine JJohnston M Hittinger CT 2011 The awesome power of yeast evo-lutionary genetics new genome sequences and strain resources forthe Saccharomyces sensu stricto genus G3 (Bethesda) 111ndash25

Schroder MS Martinez de San Vicente K Prandini TH Hammel SHiggins DG Bagagli E Wolfe KH Butler G 2016 Multiple originsof the pathogenic yeast Candida orthopsilosis by separate hybridiza-tions between two parental species PLoS Genet 12e1006404

Schumer M Rosenthal GG Andolfatto P 2014 How common is homo-ploid hybrid speciation Evolution 681553ndash1560

Schwarzbach AE Donovan LA Rieseberg LH 2001 Transgressivecharacter expression in a hybrid sunflower species Am J Bot88270ndash277

Selmecki AM Maruvka YE Richmond PA Guillet M Shoresh NSorenson AL De S Kishony R Michor F Dowell R et al 2015Polyploidy can drive rapid adaptation in yeast Nature 519349ndash52

Seshan VE Olshen A 2016 DNAcopy DNA copy number data analysisVersion R package version 1460

Soltis DE Visger CJ Soltis PS 2014 The polyploidy revolution then andnow Stebbins revisited Am J Bot 1011057ndash1078

Soltis PS 2013 Hybridization speciation and novelty J Evol Biol26291ndash293

Soltis PS Soltis DE 2009 The role of hybridization in plant speciationAnnu Rev Plant Biol 60561ndash588

Sunshine AB Payen C Ong GT Liachko I Tan KM Dunham MJ 2015The fitness consequences of aneuploidy are driven by condition-dependent gene effects PLoS Biol 13e1002155

Tamai Y Momma T Yoshimoto H Kaneko Y 1998 Co-existence of twotypes of chromosome in the bottom fermenting yeastSaccharomyces pastorianus Yeast 14923ndash933

Taylor DJ Hebert PDN 1993 Habitat-dependent hybrid parentage anddifferential introgression between neighboringly sympatric Daphniaspecies Proc Natl Acad Sci U S A 907079ndash7083

Thiagalingam S Laken S Willson JK Markowitz SD Kinzler KWVogelstein B Lengauer C 2001 Mechanisms underlying losses ofheterozygosity in human colorectal cancers Proc Natl Acad Sci US A 982698ndash2702

Tuna M Knuutila S Mills GB 2009 Uniparental disomy in cancer TrendsMol Med 15120ndash128

Walther A Hesselbart A Wendland J 2014 Genome sequence ofSaccharomyces carlsbergensis the worldrsquos first pure culture lageryeast G3 (Bethesda) 4783ndash793

Wayne RK 1993 Molecular evolution of the dog family Trends Genet9218ndash224

Wenger JW Schwartz K Sherlock G 2010 Bulk segregant analysis byhigh-throughput sequencing reveals a novel xylose utilization genefrom Saccharomyces cerevisiae PLoS Genet 6e1000942

Wessinger CA Rausher MD 2014 Predictability and irreversibility ofgenetic changes associated with flower color evolution inPenstemon barbatus Evolution 681058ndash1070

Wolfe KH 2015 Origin of the yeast whole-genome duplication PLoSBiol 13e1002221

Woods R Schneider D Winkworth CL Riley MA Lenski RE 2006 Testsof parallel molecular evolution in a long-term experiment withEscherichia coli Proc Natl Acad Sci U S A 1039107ndash9112

Wykoff DD OrsquoShea EK 2001 Phosphate transport and sensing inSaccharomyces cerevisiae Genetics 1591491ndash1499

Xiong Z Gaeta RT Pires JC 2011 Homoeologous shuffling andchromosome compensation maintain genome balance inresynthesized allopolyploid Brassica napus Proc Natl Acad SciU S A 1087908ndash7913

Yassour M Kapian T Fraser HB Levin JZ Pfiffner J Adiconis X Schroth GLuo SJ Khrebtukova I Gnirke A et al 2009 Ab initio construction ofa eukaryotic transcriptome by massively parallel mRNA sequencingProc Natl Acad Sci U S A 1063264ndash3269

Zhu YO Sherlock G Petrov DA 2016 Whole genome analysis of 132clinical Saccharomyces cerevisiae strains reveals extensive ploidy var-iation G3 (Bethesda) doi101534g3116029397

Adaptation in Hybrids doi101093molbevmsx098 MBE

17

  • msx098-TF1
  • msx098-TF2
Page 7: Loss of Heterozygosity Drives Adaptation in Hybrid Yeastdunham.gs.washington.edu/SmukowskiHeil.pdf · 2017-05-20 · Loss of Heterozygosity Drives Adaptation in Hybrid Yeast Caiti

Tab

le2

Co

nti

nu

ed

Clo

ne

Loca

tio

nG

ene(

s)M

uta

tio

nSp

ecie

sG

ener

atio

ns

Rel

ativ

eFi

tnes

s6

SE

Sc4

chrI

I275

000

813

184

289

gen

esLO

H(f

avo

rin

gG

RF1

67)

cer

190

312

56

613

chrI

I788

608

795

833

SUL1

PC

A1

CN

V(a

mp

lifica

tio

n)

chrX

I517

650

666

816

68ge

nes

CN

V(a

mp

lifica

tio

n)

chrX

III1

9000

01

9650

0R

RN

11C

AT

2V

PS7

1LO

HC

NV

(del

etio

nf

avo

rin

gG

RF1

67)

chrX

IV6

3200

07

8433

379

gen

esLO

HC

NV

(del

etio

n)

chrX

V3

3670

03

4200

034

2000

109

1291

2ge

nes

384

gen

esLO

H(f

avo

rin

gG

RF1

67f

avo

rin

gS2

88C

)ch

rIX

233

67C

SS1

No

nsy

no

nym

ou

sD

914N

Su1

chrX

177

350

345

680

96ge

nes

incl

ud

ing

SUL2

CN

V(a

mp

lifica

tio

n)

uva

557

218

62

37(S

anch

eze

tal

201

7)ch

rXV

I466

649

DIG

1N

on

syn

on

ymo

us

E49Q

chrV

188

548

Inte

rgen

icSu

2ch

rX1

7735

03

4568

096

gen

esin

clu

din

gSU

L2C

NV

(am

plifi

cati

on

)ch

rIV

803

704

KT

R3

5rsquo-u

pst

ream

chrI

I121

779

PIN

4N

on

syn

on

ymo

us

N26

3Sch

rVII

165

902

MP

T5

No

nsy

no

nym

ou

sQ

618K

chrI

I836

169

RSC

3Sy

no

nym

ou

sR

4Rch

rIV

107

948

UFD

2Sy

no

nym

ou

sG

691G

chrI

II2

8761

8In

terg

enic

Poin

tm

uta

tio

ns

cop

yn

um

ber

vari

ants

(CN

V)

and

loss

ofh

eter

ozy

gosi

tyev

ents

(LO

H)

are

reco

rded

for

each

evo

lved

par

enta

lclo

ne

Clo

nes

are

iden

tifi

edb

yn

utr

ien

t(G

glu

cose

limit

atio

nP

ph

osp

hat

elim

itat

ion

an

dS

sulfa

telim

itat

ion

)b

ysp

ecie

s(ldquo

crdquod

eno

tes

Sce

revi

siae

ldquou

rdquod

eno

tes

Suv

arum

)an

dth

en

um

ber

ind

icat

esit

sd

eriv

atio

nfr

om

ind

epen

den

tp

op

ula

tio

ns

No

teth

atm

uta

tio

ns

inth

eS

uvar

umge

no

me

use

Suv

arum

chro

mo

som

esan

dco

ord

inat

esA

llb

reak

po

ints

wer

eca

lled

by

visu

alin

spec

tio

no

fse

qu

enci

ng

read

san

dar

eth

us

app

roxi

mat

eR

elat

ive

fitn

ess

isre

po

rted

wit

hst

and

ard

erro

r(S

E)s

ever

alcl

on

esar

ere

po

rted

wit

ho

ut

SEd

ue

tote

chn

ical

diffi

cult

ies

wit

hre

plic

ates

Adaptation in Hybrids doi101093molbevmsx098 MBE

7

had an average of 15 CNV per clone and the evolvedS uvarum clones had an average of 1 CNV per clone (table 2supplementary figs S1 and S2 Supplementary Material online)The most common event across nutrient limitations in theinterspecific hybrids was an amplification of the S cerevisiaecopy of chromosome IV which occurred in four

independent hybrid clones (three in phosphate limitationone in glucose limitation supplementary fig S3Supplementary Material online) Several other characteris-tic rearrangements occurred in the evolved S cerevisiaeclones including the amplification of the left arm of chro-mosome 14 accompanied by segmental monosomy of the

FIG 1 Evolved hybrids exhibit changes in copy number and loss of heterozygosity Copy number variants are displayed for selected evolved hybridclones from three nutrient-limited conditions Gh2 glucose Ph4 phosphate and Sh4 sulfate See additional figures in supplementary fig S3Supplementary Material online Hybrid copy number determined by normalized sequencing read depth per open reading frame (ORF) is plottedacross the genome according to S cerevisiae ORF coordinates to account for three reciprocal translocations between S cerevisiae and S uvarumChromosomes are plotted in alternating light and dark purple red indicates a S cerevisiae copy number variant and blue indicates a S uvarumcopy number variant Gh2 has a whole chromosome amplification of S cerevisiae chrIV a small segmental deletion of S uvarum chrIV (non-copyneutral loss of heterozygosity) and an amplification of S uvarum HXT67 Ph4 has a small segmental deletion of S cerevisiae chrIII (non-copyneutral loss of heterozygosity) and an amplification of S cerevisiae chrXIII with corresponding deletion of S uvarum chrXIII (copy neutral loss ofheterozygosity) Sh4 has an amplification of S cerevisiae SUL1 and a whole chromosome amplification of S uvarum chrVIII (note there is areciprocal translocation between chrVIII and chrXV in S uvarum relative to S cerevisiae) Note that Sh4 is plotted on a different scale For specificcoordinates of copy number variants see table 1

Smukowski Heil et al doi101093molbevmsx098 MBE

8

right arm of chromosome 14 an event seen previously inother evolved populations (Dunham et al 2002 Greshamet al 2008 Sunshine et al 2015) All copy number events in

S cerevisiae had break points at repetitive elements knownas Ty elements except those located on chrII which maybe mediated by another mechanism (Brewer et al 2015) In

FIG 2 Repeated loss of heterozygosity at the PHO84 locus in intra- and interspecific hybrids (A) The 25-kb region extending from the left telomereof chromosome XIII to the high-affinity phosphate transporter gene PHO84 (B) Copy number is plotted across part of chromosome XIII in thehybrid ancestor and three evolved hybrid clones in phosphate limitation (clone indicated in upper right corner) Red shows the S cerevisiae alleleblue shows the S uvarum allele and purple shows where both species exhibit the same copy number Note 8 kb of telomere sequence is removeddue to repetitive sequence (C) Alternate allele frequency is plotted for a portion of chromosome XIII in the ancestor and four evolved S cerevisiaeclones in phosphate limitation (clone indicated in upper right corner) All evolved S cerevisiae clones exhibit a loss of heterozygosity at thetelomeric portion of chromosome XIII (loss of S288C amplification of GRF167) as illustrated by an allele frequency of zero compared with theancestor Regions of heterozygosity are interspersed with regions of homozygosity as one of the parents of the diploid was itself the product of across between strains FL100 and S288C and the other parent was S288C Regions of heterozygosity are due to FL100 haplotypes S cerevisiae copynumber for the four evolved clones is shown below the ancestor is diploid across the chromosome (also see table 2 supplementary fig S1Supplementary Material online)

Adaptation in Hybrids doi101093molbevmsx098 MBE

9

contrast copy number variants in the hybrid were rarely facil-itated by repetitive elements perhaps in part because S uva-rum has no full length Ty elements however why S cerevisiaeTy elements and long terminal repeat (LTR) sequences fromeither genome were not utilized remains unknown

Frequently in nutrient-limited evolution experimentscopy number variants involve amplification of the nutrient-specific transporter and indeed we also observed amplifica-tion of these transporters in many of the clones In sulfatelimitation the S cerevisiae allele of the high-affinity sulfatetransporter gene SUL1 is amplified in 77 hybrid clones and 44 S cerevisiae clones (fig 1 tables 1 and 2 supplementary figsS1 and S3 Supplementary Material online) InterestinglySUL2 is the preferred sulfate transporter in S uvarum(Sanchez et al 2017) and was not observed to be amplifiedin the evolved hybrids (supplementary fig S2 and table S2Supplementary Material online) In glucose limitation previ-ous S cerevisiae evolution experiments found frequent am-plification of the high-affinity glucose transporter genesHXT67 (Brown et al 1998 Dunham et al 2002 Greshamet al 2008 Kao and Sherlock 2008 Kvitek and Sherlock2011) In our experiments the S uvarum alleles of theHXT67 transporters are amplified in 33 hybrid clones andboth S uvarum clones but are not amplified in evolved Scerevisiae clones This is suggestive that the S uvarum HXT67alleles confer a greater fitness advantage compared with Scerevisiae alternatively the genomic context could be morepermissive to amplification in S uvarum (fig 1 tables 1 and 2supplementary figs S1ndashS3 Supplementary Material online)Finally in phosphate limitation the S cerevisiae copy of thehigh-affinity phosphate transporter gene PHO84 is amplifiedand the S uvarum allele is lost in 36 hybrid clones in an eventknown as LOH (figs 1 and 2 table 1 supplementary fig 3Supplementary Material online) Intriguingly the evolved Scerevisiae clones also display LOH and accompanied amplifi-cation favoring the allele derived from strain GRF167 over theS288C allele in 44 clones (fig 2 table 2) All hybrid clonescarry the ldquopreferredrdquo GRF167 S cerevisiae allele as this was theallele used to create the de novo hybrid

Loss of Heterozygosity Is a Common Event inHeterozygous Evolving PopulationsSelection on heterozygosity as a LOH event could representis an underappreciated source of adaptation in microbial ex-perimental evolution as typical experiments evolve a haploidor homozygous diploid strain asexually and as a result havelittle variation to select upon LOH is observed in natural andindustrial hybrids (Albertin and Marullo 2012 Louis et al2012 Pryszcz et al 2014 Wolfe 2015 Schroder et al 2016)but here we document its occurrence in both intra- andinterspecific hybrids in the laboratory as a result of short-termevolution (also see (Dunn et al 2013 Burke et al 2014)) LOHis observed across all nutrient conditions with 13 independentLOH events detected in S cerevisiae and 9 independent eventsdocumented in the hybrids (figs 1 and 2 tables 1 and 2 sup-plementary figs S1 and S3 Supplementary Material online) Itthus appears that this type of mutational event is both com-mon and can occur over short evolutionary timescales

The LOH event can result in copy-neutral (where one alleleis lost and the other allele is amplified) or non-neutral chro-mosomal segments (where one allele is lost rendering thestrain hemizygous at that locus) and can favor the retentionof either allele In S cerevisiae there is a bias in resolutionwhere LOH events favor retaining the GRF167 allele over theS288C allele (1013 events Pfrac14 00169 table 2 supplementaryfig S4 Supplementary Material online) One unique case inclone Sc4 has a small approximately 5 kb LOH event onchrXV favoring GRF167 which switches to favoring S288Cfor the rest of the chromosome The retention of S cerevisiaeis slightly more common in the hybrids (59 events Pfrac14 10table 1 supplementary fig S3 Supplementary Material on-line) though not as drastic as the observed genome resolu-tion in the hybrid S pastorianus where LOH favorsS cerevisiae over S eubayanus (Nakao et al 2009) The sizeof the event ranges from approximately 25 kb to the wholechromosome level in the hybrids and from 5 kb to 540 kbin S cerevisiae Where LOH is accompanied by an amplifi-cation event the LOH event always occurs first and doesnot share break points with the amplification event Unlikemany CNV events most LOH events do not appear to bemediated by existing repetitive sequence such as a trans-posable element in the hybrid or S cerevisiae and are mostlikely a product of break-induced replication or mitoticgene conversion with break-induced replication as the fa-vored method as all events extend to the telomere (Hoanget al 2010) The exceptions are in hybrid clones Ph4 Ph5 andSh1 where there is a non-copy neutral loss of S cerevisiaemediated by a Ty element or LTR and S cerevisiae clonesSc1 and Sc4 where there is a 65-kb deletion of the S288Callele flanked by two Ty elements

LOH events in hybrids could signify several ongoing pro-cesses in hybrid genome evolution LOH regions may repre-sent 1) loci with incompatibilities 2) selection on existingvariation or 3) genetic drift eroding genomic segments It isimpossible to definitively rule out any of these hypotheseswithout further experimentation however there are severalarguments disfavoring the incompatibility hypothesis Firstalthough our sample size is modest failing to see repeatedLOH events across nutrient conditions may indicate a lack ofgeneral incompatibility between species (although we cannotpreclude condition-specific incompatibility Hou et al 2015Piatkowska et al 2013) This is consistent with previous stud-ies in yeast which suggest that classic DobzhanksyndashMullerincompatibilities are rare (Liti et al 2006 Greig 2009 Houet al 2014) Furthermore LOH events observed inevolved S cerevisiae suggest that this mutation type isnot unique to interspecific hybrids Instead repeatedevents within a particular condition such as the repeatedLOH at PHO84 in phosphate limitation or the 65 kbsegment on chrXIII in sulfate limitation suggest thatthese events are beneficial and are indeed selection onone allele over the other

LOH Is Driven by Selection on One AlleleTo test the hypothesis that LOH events provide a selectiveadvantage we used allele replacement in which the allele of

Smukowski Heil et al doi101093molbevmsx098 MBE

10

one speciesstrain is replaced with the allele of the otherspeciesstrain in an otherwise isogenic background We testedthis hypothesis using the most commonly seen LOH eventLOH at PHO84 Although the region extends from 25 kb to234 kb in length in the hybrids and from 40 kb to 85 kb in Scerevisiae and thus includes many genes PHO84 was a primecandidate driving this event PHO84 is one of only ten genesencompassed in the region extending from the telomere tothe break point of the shortest LOH event and is included inevery other LOH event on chromosome XIII (fig 2) It is ahigh-affinity phosphate transporter responsible for inorganicphosphate uptake in high and low phosphate conditions(Wykoff and OrsquoShea 2001) and previous work identified apoint mutation in PHO84 (an alanine to valine substitution atthe 50 end of the gene) which increased fitness by 23 inphosphate-limited conditions (Sunshine et al 2015) Finallyprior work with other nutrient transporters has shown am-plification of nutrient transporters to be a key event in adapt-ing to nutrient-limited conditions

We thus selected a region of approximately 25 kb encom-passing the PHO84 ORF its promoter and 30-UTR(Nagalakshmi et al 2008 Yassour et al 2009 Cherry et al2012) We created allele replacement strains using the twoalleles of S cerevisiae in a S cerevisiae diploid background thetwo alleles are 991 identical in this region (supplementaryfig S5 Supplementary Material online) and each strain isidentical to the ancestral strain used in our evolution exper-iments except at the PHO84 locus The S cerevisiae ancestorcarries one copy of GRF167 (preferred) and one copy ofS288C (ldquoun-preferredrdquo) so named due to which allele wasretained and amplified in the evolved clones To measureany resultant changes in fitness we competed each strainindividually against a fluorescent ancestral strain and mea-sured which strain overtook the culture Relative fitness isthus defined as the growth advantage per generation Twocopies of the un-preferred allele decreased relative fitness by531 (6186) whereas two copies of the preferred alleleincreased relative fitness by 993 (6027) This displays anoverall difference in relative fitness of 1524 between the un-preferred and preferred alleles By comparing the fitness ofthese allele replacement strains with the evolved clones (table 2)the allele replacement does not fully recapitulate the fit-ness gain observed in the evolved clone One explanation isthat the additional mutations present in the evolvedstrains also contribute to their total fitness Another expla-nation could be the increased copy number of the PHO84region that we see in these evolved clones To further ex-plore this fitness difference we cloned the GRF167 alleleonto a low copy number plasmid and transformed theallele replacement strain carrying two preferred S cerevi-siae alleles to simulate increased copy number of PHO84and saw only a minimal fitness increase of 176 (6106note all fitness competitions involving plasmids were donein conjunction with empty plasmids to take into accountany fitness effects from the plasmid itself) This supportsthe conclusion that relative fitness gains in the evolvedclone are largely due to the loss of the S288C allele andselection and amplification of the GRF167 allele with little

additional benefit from further amplification It could also bethe case that co-amplification of other genes in the segmentis required to attain the full benefit as previously observedby the contribution of BSD2 to the SUL1 amplicon in sulfatelimitation (Sunshine et al 2015 Payen et al 2016)

To understand the fitness effects of LOH and amplificationin the hybrids we generated hybrid strains with varying num-bers of S cerevisiae GRF167 PHO84 alleles Unfortunately wewere unable to obtain a successful strain carrying the pre-ferred S cerevisiae allele in a S uvarum background howeverwe were able to generate a S uvarum PHO84 knockout strainthus creating a hybrid with one copy of S cerevisiae PHO84When combined with a low copy plasmid carrying the sameallele this strain effectively has two or more copies ofS cerevisiae PHO84 in a hybrid background This hybrid straincan serve as a proxy for the LOH event observed in the evo-lution experiments and has a relative fitness gain of 2557(6288) The ancestral hybrid with the same plasmid (effec-tively 1 S uvarum allele and 2 or more S cerevisiae alleles) hasa relative fitness gain of 1253 (6131) The difference be-tween these two hybrids suggests that while amplification isbeneficial the highest fitness is achieved with the loss of theS uvarum allele (Pfrac14 00061)

Together these results support the conclusion that theS cerevisiae GRF167 allele is preferred over the S288C alleleand that S cerevisiae alleles are preferred over the S uvarumallele in the hybrid and hence that the LOH events seen inboth intra- and interspecific hybrids are the product ofselection

DiscussionIn summary we sought to understand forces underlying ge-nome stabilization and evolution in interspecific and intra-specific hybrids as they adapt to novel environments Weevolved and sequenced clones from 16 hybrid populationsand 16 parental populations to reveal a variety of mutationalevents conferring adaptation to 3 nutrient-limited conditionsOf particular note we find LOH in both evolved intraspecificand interspecific hybrid clones in all nutrient environmentspotentially signifying areas where selection has acted on pre-existing variation present in the ancestral clone We used anallele replacement strategy to test this hypothesis for a com-monly repeated LOH event and show that selection is indeeddriving the homogenization of the genome at this locusAlthough other studies in natural industrial and lab-evolved isolates have observed LOH we present the first em-pirical test of the causal evolutionary forces influencing theseevents This work can begin to help us understand past hy-bridization events and subsequent genome resolution in hy-brids in natural and artificial systems

Similarities and Differences between Intra- andInterspecific HybridsAlthough our sample size is modest we observe several in-teresting trends when comparing S uvarum clones (homo-zygous) S cerevisiae clones (intraspecific hybrid andpreviously published homozygous) and interspecific hybrid

Adaptation in Hybrids doi101093molbevmsx098 MBE

11

clones First theory and previous research predict the inter-specific hybrid may experience more genome instability in theform of chromosomal rearrangements and CNV events(Xiong et al 2011 Lloyd et al 2014 Chester et al 2015Mason and Batley 2015) Instead in our work the interspecifichybrid and S cerevisiae clones experience the same numberof CNV events (both 15 CNVsclone) However the mech-anism of CNV formation seems to differ between the hybridand S cerevisiae clones Whereas S cerevisiae CNV breakpoints typically occur at transposable elements in our studyand previous studies (Dunham et al 2002 Gresham et al2008 Fedoroff 2012) the interspecific hybrid CNVs do notutilize transposable elements although they obviously sharethe same sequence background as the S cerevisiae clones(albeit in one copy) Whether this difference is due to theabsence of full-length transposable elements in S uvarum isunknown but this could potentially explain the lower num-ber of CNV events in S uvarum clones (1 CNVclone) andpresents an intriguing direction for future study

The Predictability of EvolutionWe now have many examples of predictable evolution innatural systems (Losos et al 1998 Rundle et al 2000 Elmerand Meyer 2011 Conte et al 2012 Jones et al 2012 Martinand Orgogozo 2013 Wessinger and Rausher 2014) and inlaboratory experimental evolution in which there often ap-pears to be a limited number of high fitness pathways thatstrains follow when adapting to a particular condition (Fereaet al 1999 Woods et al 2006 Gresham et al 2008 Burke et al2010 Salverda et al 2011 Kawecki et al 2012 Kvitek andSherlock 2013 Lang and Desai 2014) For example it is wellestablished that amplifications of nutrient transporters aredrivers of adaptation in evolution in nutrient-limited condi-tions Previous work in our group has particularly focused onthe amplification of the high-affinity sulfate transporter geneSUL1 in sulfate-limited conditions which occurs in almostevery sulfate-limited evolution experiment and confers a fit-ness advantage of as much as 40 compared with the un-evolved ancestor strain The amplification of phosphatetransporters has been markedly less common and thus driv-ers of adaptation in this condition have been less clearGresham et al (2008) identified a whole chromosome ampli-fication of chrXIII in one population In a follow-up studySunshine et al (2015) found whole or partial amplification ofchrXIII in 38 populations A genome-wide screen for segmen-tal amplifications found a slight increase in fitness for a smalltelomeric segment of chromosome XIII and a A49V pointmutation in PHO84 was observed to increase fitness by 23However screens by Payen et al (2016) showed that althoughPHO84 is recurrently mutated in various experiments itshowed no benefit when amplified or deleted in phos-phate-limited conditions Finally additional evolution exper-iments recapitulated the point mutation seen in Sunshineet al (2015) in 2432 populations and saw amplification ofPHO84 in 832 populations (Miller A Dunham MJ unpub-lished data) It is important to note that all these experimentsused a strain background derived from S288C or CENPKboth of which carry the same (un-preferred) PHO84 allele

In our work we observed amplification of the S cerevisiaeGRF167 allele of PHO84 in 44 S cerevisiae clones from 4populations and 36 hybrid clones from 6 populations Thisamplification was always preceded by the loss of the S288Callele in S cerevisiae clones and the LOH break points arenever shared with the amplification break points There is a15 fitness difference between carrying two copies of theS288C allele of PHO84 compared with carrying two copiesof the GRF167 allele of PHO84 and additional copies of theGRF167 allele do not provide substantial further fitness gainsThe two alleles differ by several noncoding changes and threenonsynonymous substitutions a mutation from glutamicacid to aspartic acid (E229D) leucine to proline (L259P)and leucine to glutamine (L556Q) the latter two of whichare considered ldquononconservativerdquo protein mutations due tochanges in hydrophobicity and structure (supplementary figS5 Supplementary Material online) Intriguingly the L259Pmutation has actually been previously identified as being re-sponsible for resistance to tetrachloroisophthalonitrile andpartial resistance to pentachlorophenol in a QTL study ofsmall-molecule drugs (Perlstein et al 2007) IndeedPerlstein et al found proline at residue 259 to be conservedacross fungal species and even in orthologous human xeno-biotic transporters likely because proline-induced kinks intransmembrane spans have been shown to be essential toprotein function (Cordes et al 2002 Perlstein et al 2007) Itthus appears that amplification of PHO84 has been less pre-dictable as the S288C allele does not confer a fitness advan-tage unless mutated per Sunshine et al (2015) Togetherthese results imply that strain background can constrainadaptive pathways

In hybrids the amplification of the S cerevisiae segmentoccurred in conjunction with the loss of the S uvarum alleleHybrid strains with the LOH had a 2557 relative fitness gainwhereas hybrid strains with amplification of the S cerevisiaePHO84 allele without the LOH had a 1253 fitness gain ThusLOH confers an additional 1304 fitness gain showing thatselection for LOH has a larger impact on fitness than ampli-fication alone Note that S uvarum does have proline at res-idue 259 like the preferred GRF167 allele and differs fromboth S cerevisiae alleles at the other two coding substitutions(229N and 556K) but the amino acid and noncoding diver-gence is too high to speculate what substitutions are respon-sible for the selection of the GRF167 allele (supplementary figS6 Supplementary Material online) Why the loss of one alleleis more beneficial remains unclear as PHO84 is thought tofunction as a monomer (Bun-Ya et al 1991) but it may bedue to competition for cell wall space or negative interactionswith other genes in the PHO pathway (Mouillon and Persson2006)

The infusion of variation created by hybridization providesnew templates for selection to act upon which can be moreimportant than either point mutations or copy number var-iants alone Our work shows that outcrossing need not becommon to have long-lasting effects on adaptation This im-plication is particularly relevant in yeast where outcrossingmay occur quite rarely followed by thousands of asexual gen-erations (Ruderfer et al 2006 Greig and Leu 2009 Liti 2015)

Smukowski Heil et al doi101093molbevmsx098 MBE

12

Applications to Other Hybrids and CancerThe observation that LOH occurs in hybrid genomes is in-creasingly documented (Louis et al 2012 Borneman et al2014 Soltis et al 2014 Marcet-Houben and Gabaldon 2015Pryszcz et al 2014 Schroder et al 2016) although the rea-son(s) for this type of mutation has been unresolved As mostexamples stem from allopolyploid events that occurred mil-lions of years ago understanding why LOH is important inhybrid genome evolution is difficult Cancer cells are alsoknown to experience LOH sometimes involved in the inac-tivation of tumor suppressor genes leaving only one copy ofthe gene that may be mutated or silenced (Thiagalingam et al2001 Tuna et al 2009 Lapunzina and Monk 2011) Datasupport the conclusion that LOH events are selected for dur-ing tumor development as many LOH events involve specificchromosomal segments (Thiagalingam et al 2001) althoughthe underlying molecular and genetic reasons for selection isan open debate (Ryland et al 2015)

Here we experimentally demonstrate that LOH can occurin homoploid interspecific hybrids as well as in intraspecifichybrids These events occur within a few hundred generationsand are common mutations more common on average inthe intraspecific hybrid (13 eventsclone) than the interspe-cific hybrid (056 eventsclone) Interestingly the LOH eventsdo not share break points with the CNV events in S cerevisiaeinstead they appear to occur independently and to precedeany subsequent amplification (amplification occurs following913 LOH events) Competition assays with the PHO84 locusprovide support that LOH itself may be more beneficial thanamplification or at least increase the selective benefit of am-plification events The observation that LOH in intraspecifichybrids occurs independently from copy number change pro-vides different opportunities for adaptation to novelconditions

Other cases of LOH like the copy number neutral eventsobserved in Ph4 Ph5 and Sh4 all of which favor the retentionof the S uvarum allele (supplementary fig S3 SupplementaryMaterial online) may be due to hybrid incompatibility withina particular protein complex other epistatic interactions(Piatkowska et al 2013) or neutral processes We furthermorediscover examples where one species allele appears to bepreferred over the other without LOH such as the repeatedamplification of the S uvarum high-affinity glucose transpor-ters HXT67 When one species allele is amplified and theother is not amplified one explanation is that the local se-quence context can permit or deny amplification In the caseof HXT67 previous experiments in S cerevisiae have shownthat amplification of HXT67 is quite common (Brown et al1998 Dunham et al 2002 Gresham et al 2008 Kao andSherlock 2008 Kvitek and Sherlock 2011) thus suggestingthat when given a choice between this locus in S cerevisiaeor S uvarum in the hybrid the preferred allele is indeed Suvarum though more subtle differences in rate cannot yet beruled out A similar scenario is observed with S cerevisiaeSUL1 (Sanchez et al 2017) Together our results show thatthe heterozygosity supplied by hybridization is an importantcontributor to adaptive routes explored by populations asthey adapt to novel conditions

Although we cannot generalize our results from thePHO84 locus across the many other LOH events discoveredin our hybrids and S cerevisiae in the future we can usesimilar methodology to explore whether positive selectionalways drives LOH or whether other explanations such asincompatibility resolution contribute as well Future experi-ments might also utilize a high throughput method to ex-plore segmental LOH in hybrids at a genome-wide scalesimilar to ongoing experiments at the gene level (LancasterS Dunham MJ unpublished data) Although our sample sizeis moderate this is a novel and necessary step in understand-ing forces underlying hybrid genome stabilization and high-lighting an underappreciated mechanism of hybridadaptation

ConclusionsThe mutation events we observe in our experimentallyevolved hybrids are in many ways quite representative ofmutations observed in ancient hybrid genomes suggestingthat hybrid genome stabilization and adaptation can occurquite rapidly (within several hundred generations)Furthermore our results illustrate that the infusion of varia-tion introduced by hybridization at both the intra- and inter-species level can increase fitness by providing choices of allelesfor selection to act upon even when sexual reproduction israre This may be particularly important for leveraging existingvariation for agricultural and industrial processes and as cli-mate change potentially increases natural hybridization (Kellyet al 2010 Hoffmann and Sgro 2011 Muhlfeld et al 2014)

Materials and Methods

StrainsA list of strains used in this study is included in supplementarytable S1 Supplementary Material online All interspecific hy-brids were created by crossing a ura3 LYS2 haploid parent to aURA3 lys2 haploid parent of the other mating type plating onmedia lacking both uracil and lysine and selecting for proto-trophs The S cerevisiae strain background known asldquoGRF167rdquo is itself a cross between FL100 and the genomictype strain S288C (data not shown) GRF167 was chosen as astrain background for simultaneous work investigating trans-posable elements during experimental evolution which willbe addressed in a future study

Evolution ExperimentsContinuous cultures were established using media and con-ditions previously described (Gresham et al 2008 Sanchezet al 2017) Detailed protocols and media recipes are availableat httpdunhamgswashingtoneduprotocolsshtml (lastaccessed March 6 2017) Samples were taken daily and mea-sured for optical density at 600 nm and cell count micros-copy was performed to check for contamination andarchival glycerol stocks were made daily An experimentwas terminated when contamination growth in tubing orclumping appeared (number of generations at the endpoint for each population are presented in tables 1 and

Adaptation in Hybrids doi101093molbevmsx098 MBE

13

2) Samples from each end point population were colonypurified to yield two clones for further study

Array Comparative Genomic HybridizationPopulations from the end point of each evolution were ana-lyzed for copy number changes using array comparative ge-nomic hybridization following the protocol used in Sanchezet al (2017)

SequencingDNA was extracted from overnight cultures using theHoffmanndashWinston protocol (Hoffman and Winston 1987)and cleaned using the Clean amp Concentrator kit (ZymoResearch) Nextera libraries were prepared following theNextera library kit protocol and sequenced using paired end150 bp reads on the Illumina NextSeq 500 machine (sequenc-ing coverage in supplementary table S2 SupplementaryMaterial online) The reference genomes used were S cerevi-siae v3 (Engel et al 2014) S uvarum (Scannell et al 2011) anda hybrid reference genome created by concatenating the twogenomes Sequence was aligned to the appropriate referencegenome using bwa v062 (Li and Durbin 2009) and mutationswere called using GATK (McKenna et al 2010) and samtools0119 (Li et al 2009) Mutations in evolved clones were filteredin comparison with the ancestor to obtain de novo mutationsAll mutations were first visually inspected using IntegrativeGenomics Viewer (Robinson et al 2011) Subsequently pointmutations in the hybrids were confirmed with Sanger se-quencing (supplementary table S3 Supplementary Materialonline) Copy number variants were visualized usingDNAcopy for S cerevisiae and S uvarum (Seshan andOlshen 2016) LOH events were called based on sequencingcoverage in the hybrids and by identifying homozygous variantcalls in S cerevisiae All break points were called by visual in-spection of sequencing reads and are thus approximate

Fitness AssaysThe pairwise competition experiments were performed in 20ml chemostats (Miller and Dunham 2013) Each competitorstrain was cultured individually until steady state was reachedand then was mixed 5050 with a GFP-tagged ancestor Eachcompetition was conducted in at least two biological repli-cates for approximately 15 generations after mixing Sampleswere collected and analyzed twice daily The proportion ofGFPthorn cells in the population was detected using a BD AccuriC6 flow cytometer (BD Biosciences) The data were plottedwith ln [dark cellsGFPthorn cells] versus generations The rela-tive fitness coefficient was determined from the slope of thelinear region

Strain ConstructionAllele replacements for the PHO84 locus were done followingthe protocol of the Caudy lab with further modifications de-scribed here The native locus was replaced withKluyveromyces lactis URA3 The pho84DURA3 strain wasgrown overnight in 5 ml of C-URA media then inoculatedin a flask of 100 ml yeast extract peptone dextrose (YPD) andgrown to an optical density of 06ndash08 Cells were washed then

aliquoted 275 ml of transformation mix (35 ml 1 M lithiumacetate 240 ml of 50 3500 polyethylene glycol) 10 ml ofsalmon sperm and approximately 3 mg of polymerase chainreaction product were added to the cell pellet It was incu-bated at 37 C (S uvarum) or 42 C (S cerevisiae) for 45 minthen plated to YPD It was replica plated to 5-fluorooroticacid the following day and colonies were tested for the gain ofthe appropriate species allele The GRF167 allele was clonedinto the pIL37 plasmid using Gibson assembly (Gibson et al2009) Correct assembly was verified by Sanger sequencing Allprimers used can be found in supplementary table S3Supplementary Material online

Supplementary MaterialSupplementary data are available at Molecular Biology andEvolution online

AcknowledgmentsWe thank Noah Hanson and Erica Alcantara for technicalassistance We thank Monica Sanchez and three anonymousreviewers for helpful comments on this manuscript JosephSchacherer kindly confirmed the contribution of FL100 to theGRF167 strain background Thanks to Yixian Zheng and DougKoshland for contributing to the initial experimental designcreating yeast strains and purchasing the oligonucleotidesused for the microarrays This work was supported by theNational Science Foundation (grant number 1516330) andthe National Institutes of Health (grant number R01GM094306) MD is a Senior Fellow in the GeneticNetworks program at the Canadian Institute for AdvancedResearch and a Rita Allen Foundation Scholar CSH wassupported in part by National Institutes of Health (grantnumber T32 HG00035) This work was also supported bythe National Institutes of Health (grant number P50GM071508) to the Lewis-Sigler Institute and from theHoward Hughes Medical Institute to Doug Koshland andYixian Zheng Sequencing read data have been deposited atthe NCBI under BioProject accession PRJNA374049Microarray data are deposited in the Gene ExpressionOmnibus (GEO) repository under accession numbersGSE95086 and GSE87401 and in the Princeton MicroarrayDatabase

ReferencesAbbott R Albach D Ansell S Arntzen JW Baird SJ Bierne N Boughman J

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Almeida P Goncalves C Teixeira S Libkind D Bontrager M Masneuf-Pomarede I Albertin W Durrens P Sherman DJ Marullo P et al2014 A Gondwanan imprint on global diversity and domesticationof wine and cider yeast Saccharomyces uvarum Nat Commun54044

Antunovics Z Nguyen HV Gaillardin C Sipiczki M 2005 Gradual ge-nome stabilisation by progressive reduction of the Saccharomyces

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14

uvarum genome in an interspecific hybrid with Saccharomyces cer-evisiae FEMS Yeast Res 51141ndash1150

Baker E Wang B Bellora N Peris D Hulfachor AB Koshalek JA AdamsM Libkind D Hittinger CT 2015 The Genome sequence ofSaccharomyces eubayanus and the domestication of lager-brewingyeasts Mol Biol Evol 322818ndash2831

Barbosa R Almeida P Safar SVB Santos RO Morais PB Nielly-Thibault LLeducq JB Landry CR Goncalves P Rosa CA Sampaio JP 2016Evidence of natural hybridization in Brazilian wild lineages ofSaccharomyces cerevisiae Genome Biol Evol 8317ndash329

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Bellon JR Yang F Day MP Inglis DL Chambers PJ 2015 Designingand creating Saccharomyces interspecific hybrids for improvedindustry relevant phenotypes Appl Microbiol Biotechnol998597ndash8609

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Brewer BJ Payen C Di Rienzi SC Higgins MM Ong G Dunham MJRaghuraman MK 2015 Origin-dependent inverted-repeat amplifi-cation tests of a model for inverted DNA amplification PLoS Genet11e1005699

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Bun-Ya M Nishimura M Harashima S Oshima Y 1991 The PHO84 geneof Saccharomyces cerevisiae encodes an inorganic phosphate trans-porter Mol Cell Biol 113229ndash3238

Burke MK Dunham JP Shahrestani P Thornton KR Rose MR Long AD2010 Genome-wide analysis of a long-term evolution experimentwith Drosophila Nature 467587ndash590

Burke MK Liti G Long AD 2014 Standing genetic variation drives re-peatable experimental evolution in outcrossing populations ofSaccharomyces cerevisiae Mol Biol Evol 313228ndash3239

Cherry JM Hong EL Amundsen C Balakrishnan R Binkley G Chan ETChristie KR Costanzo MC Dwight SS Engel SR et al 2012Saccharomyces genome database the genomics resource of buddingyeast Nucleic Acids Res 40D700ndashD705

Chester M Riley RK Soltis PS Soltis DE 2015 Patterns of chromosomalvariation in natural populations of the neoallotetraploid Tragopogonmirus (Asteraceae) Heredity (Edinb) 114309ndash317

Chou JY Leu JY 2010 Speciation through cytonuclear incompatibilityinsights from yeast and implications for higher eukaryotes Bioessays32401ndash411

Cliften PF Fulton RS Wilson RK Johnston M 2006 After the duplicationgene loss and adaptation in Saccharomyces genomes Genetics172863ndash872

Conte GL Arnegard ME Peichel CL Schluter D 2012 The probability ofgenetic parallelism and convergence in natural populations Proc BiolSci 2795039ndash5047

Cordes FS Bright JN Sansom MS 2002 Proline-induced distortions oftransmembrane helices J Mol Biol 323951ndash960

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Dunham MJ Badrane H Ferea T Adams J Brown PO Rosenzweig FBotstein D 2002 Characteristic genome rearrangements in experi-mental evolution of Saccharomyces cerevisiae Proc Natl Acad Sci U SA 9916144ndash16149

Dunn B Paulish T Stanbery A Piotrowski J Koniges G Kroll E LouisEJ Liti G Sherlock G Rosenzweig F 2013 Recurrent rearrange-ment during adaptive evolution in an interspecific yeast hybridsuggests a model for rapid introgression PLoS Genet9e1003366

Dunn B Richter C Kvitek DJ Pugh T Sherlock G 2012 Analysis of theSaccharomyces cerevisiae pan-genome reveals a pool of copy num-ber variants distributed in diverse yeast strains from differing indus-trial environments Genome Res 22908ndash924

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Elmer KR Meyer A 2011 Adaptation in the age of ecological genomicsinsights from parallelism and convergence Trends Ecol Evol26298ndash306

Engel SR Dietrich FS Fisk DG Binkley G Balakrishnan R Costanzo MCDwight SS Hitz BC Karra K Nash RS et al 2014 The referencegenome sequence of Saccharomyces cerevisiae then and now G3(Bethesda) 4389ndash398

Fedoroff NV 2012 Presidential address Transposable elements epige-netics and genome evolution Science 338758ndash767

Ferea TL Botstein D Brown PO Rosenzweig RF 1999 Systematicchanges in gene expression patterns following adaptive evolutionin yeast Proc Natl Acad Sci U S A 969721ndash9726

Gibson B Liti G 2015 Saccharomyces pastorianus genomic insightsinspiring innovation for industry Yeast 3217ndash27

Gibson DG Young L Chuang RY Venter JC Hutchison CA Smith HO2009 Enzymatic assembly of DNA molecules up to several hundredkilobases Nat Methods 6343ndash5

Gonzalez SS Barrio E Gafner J Querol A 2006 Natural hybrids fromSaccharomyces cerevisiae Saccharomyces bayanus andSaccharomyces kudriavzevii in wine fermentations FEMS Yeast Res61221ndash1234

Gonzalez SS Barrio E Querol A 2008 Molecular characterization of newnatural hybrids of Saccharomyces cerevisiae and S kudriavzevii inbrewing Appl Environ Microbiol 742314ndash2320

Grant PR Grant BR 1994 Phenotypic and genetic consequences ofhybridization in Darwinrsquos finches Evolution 48297ndash316

Grant PR Grant BR 2002 Unpredictable evolution in a 30-year study ofDarwinrsquos finches Science 296707ndash711

Grant PR Grant BR 2010 Natural selection speciation and Darwinrsquosfinches Proc Calif Acad Sci 61245ndash260

Grant PR Grant BR Petren K 2005 Hybridization in the recent past AmNat 16656ndash67

Greig D 2009 Reproductive isolation in Saccharomyces Heredity (Edinb)10239ndash44

Greig D Leu JY 2009 Natural history of budding yeast Current Biol19R886ndashR890

Greig D Louis EJ Borts RH Travisano M 2002 Hybrid speciation inexperimental populations of yeast Science 2981773ndash1775

Gresham D Desai MM Tucker CM Jenq HT Pai DA Ward A DeSevoCG Botstein D Dunham MJ 2008 The repertoire and dynamics ofevolutionary adaptations to controlled nutrient-limited environ-ments in yeast PLoS Genet 4e1000303

Hajjar R Hodgkin T 2007 The use of wild relatives in crop improvementa survey of developments over the last 20 years Euphytica 1561ndash13

Heiser CB 1954 Variation and subspeciation in the common sunflowerHelianthus annuus Am Midl Nat 51287ndash305

Hittinger CT 2013 Saccharomyces diversity and evolution a buddingmodel genus Trends Genet 29309ndash317

Hoang ML Tan FJ Lai DC Celniker SE Hoskins RA Dunham MJ ZhengY Koshland D 2010 Competitive repair by naturally dispersed

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repetitive DNA during non-allelic homologous recombination PLoSGenet 6e1001228

Hoffmann AA Sgro CM 2011 Climate change and evolutionary adap-tation Nature 470479ndash485

Hoffman CS Winston F 1987 A 10-minute DNA preparation from yeastefficiently releases autonomous plasmids for transformation ofEscherichia coli Gene 57267ndash272

Hou J Friedrich A de Montigny J Schacherer J 2014 Chromosomalrearrangements as a major mechanism in the onset of reproductiveisolation in Saccharomyces cerevisiae Curr Biol 241153ndash1159

Hou J Friedrich A Gounot JS Schacherer J 2015 Comprehensive surveyof condition-specific reproductive isolation reveals genetic incom-patibility in yeast Nat Commun 67214

Huerta-Sanchez E Casey FP 2015 Archaic inheritance supporting high-altitude life in Tibet J Appl Physiol (1985) 1191129ndash1134

Jones FC Grabherr MG Chan YF Russell P Mauceli E Johnson JSwofford R Pirun M Zody MC White S et al 2012 The genomicbasis of adaptive evolution in threespine sticklebacks Nature48455ndash61

Kao KC Sherlock G 2008 Molecular characterization of clonal interfer-ence during adaptive evolution in asexual populations ofSaccharomyces cerevisiae Nat Genet 401499ndash1504

Kawecki TJ Lenski RE Ebert D Hollis B Olivieri I Whitlock MC 2012Experimental evolution Trends Ecol Evol 27547ndash560

Kellis M Patterson N Endrizzi M Birren B Lander ES 2003 Sequencingand comparison of yeast species to identify genes and regulatoryelements Nature 423241ndash254

Kelly B Whiteley A Tallmon D 2010 The Arctic melting pot Nature468891ndash891

Kvitek DJ Sherlock G 2011 Reciprocal sign epistasis between frequentlyexperimentally evolved adaptive mutations causes a rugged fitnesslandscape PLoS Genet 7e1002056

Kvitek DJ Sherlock G 2013 Whole genome whole population sequenc-ing reveals that loss of signaling networks is the major adaptivestrategy in a constant environment PLoS Genet 9e1003972

Landry CR Hartl DL Ranz JM 2007 Genome clashes in hybrids insightsfrom gene expression Heredity (Edinb) 99483ndash493

Lang GI Desai MM 2014 The spectrum of adaptive mutations in ex-perimental evolution Genomics 104412ndash416

Lapunzina P Monk D 2011 The consequences of uniparental disomyand copy number neutral loss-of-heterozygosity during human de-velopment and cancer Biol Cell 103303ndash317

Leducq JB Nielly-Thibault L Charron G Eberlein C Verta JP Samani PSylvester K Hittinger CT Bell G Landry CR 2016 Speciation drivenby hybridization and chromosomal plasticity in a wild yeast NatMicrobiol 115003

Lee HY Chou JY Cheong L Chang NH Yang SY Leu JY 2008Incompatibility of nuclear and mitochondrial genomes causes hy-brid sterility between two yeast species Cell 1351065ndash1073

Li H Durbin R 2009 Fast and accurate short read alignment withBurrows-Wheeler transform Bioinformatics 251754ndash1760

Li H Handsaker B Wysoker A Fennell T Ruan J Homer N Marth GAbecasis G Durbin R 2009 The Sequence AlignmentMap formatand SAMtools Bioinformatics 252078ndash2079

Liti G 2015 The fascinating and secret wild life of the budding yeast Scerevisiae Elife 4e05835

Liti G Barton DB Louis EJ 2006 Sequence diversity reproductiveisolation and species concepts in Saccharomyces Genetics174839ndash850

Lloyd AH Ranoux M Vautrin S Glover N Fourment J Charif D ChouletF Lassalle G Marande W Tran J et al 2014 Meiotic gene evolutioncan you teach a new dog new tricks Mol Biol Evol 311724ndash1727

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Mallet J 2007 Hybrid speciation Nature 446279ndash283Marcet-Houben M Gabaldon T 2015 Beyond the whole-genome du-

plication phylogenetic evidence for an ancient interspecies hybrid-ization in the bakerrsquos yeast lineage PLoS Biol 13e1002220

Marinoni G Manuel M Petersen RF Hvidtfeldt J Sulo P Piskur J 1999Horizontal transfer of genetic material among Saccharomyces yeastsJ Bacteriol 1816488ndash6496

Martin A Orgogozo V 2013 The loci of repeated evolution a catalog ofgenetic hotspots of phenotypic variation Evolution 671235ndash1250

Masly JP Jones CD Noor MAF Locke J Orr HA 2006 Gene transpositionas a cause of hybrid sterility in Drosophila Science 3131448ndash1450

Mason AS Batley J 2015 Creating new interspecific hybrid and polyploidcrops Trends Biotechnol 33436ndash441

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Meiklejohn CD Holmbeck MA Siddiq MA Abt DN Rand DMMontooth KL 2013 An Incompatibility between a mitochondrialtRNA and its nuclear-encoded tRNA synthetase compromises de-velopment and fitness in Drosophila PLoS Genet 9e1003238

Meyer A Salzburger W Schartl M 2006 Hybrid origin of a swordtailspecies (Teleostei Xiphophorus clemenciae) driven by sexual selec-tion Mol Ecol 15721ndash730

Michalak P 2009 Epigenetic transposon and small RNA determinants ofhybrid dysfunctions Heredity (Edinb) 10245ndash50

Miller AW Dunham MJ 2013 Design and use of multiplexed chemostatarrays J Vis Exp 72e50262

Monod J 1949 The growth of bacterial cultures Annu Rev Microbiol3371ndash394

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Muhlfeld CC Kovach RP Jones LA Al-Chokhachy R Boyer MC Leary RFLowe WH Luikart G Allendorf FW 2014 Invasive hybridization in athreatened species is accelerated by climate change Nat ClimChange 4620ndash624

Muller LAH McCusker JH 2009 A multispecies-based taxonomic mi-croarray reveals interspecies hybridization and introgression inSaccharomyces cerevisiae FEMS Yeast Res 9143ndash152

Nagalakshmi U Wang Z Waern K Shou C Raha D Gerstein M SnyderM 2008 The transcriptional landscape of the yeast genome definedby RNA sequencing Science 3201344ndash1349

Nakao Y Kanamori T Itoh T Kodama Y Rainieri S Nakamura NShimonaga T Hattori M Ashikari T 2009 Genome sequence ofthe lager brewing yeast an interspecies hybrid DNA Res 16115ndash129

Nolte AW Freyhof J Stemshorn KC Tautz D 2005 An invasive lineage ofsculpins Cottus sp (Pisces Teleostei) in the Rhine with new habitatadaptations has originated from hybridization between old phylo-geographic groups Proc Biol Sci 2722379ndash2387

Novick A Szilard L 1950a Description of the chemostat Science112715ndash716

Novick A Szilard L 1950b Experiments with the Chemostat on spon-taneous mutations of bacteria Proc Natl Acad Sci U S A 36708ndash719

Ouyang YD Liu YG Zhang QF 2010 Hybrid sterility in plant storiesfrom rice Curr Opin Plant Biol 13186ndash192

Payen C Di Rienzi SC Ong GT Pogachar JL Sanchez JC SunshineAB Raghuraman MK Brewer BJ Dunham MJ 2014 The dynam-ics of diverse segmental amplifications in populations ofSaccharomyces cerevisiae adapting to strong selection G3(Bethesda) 4399ndash409

Payen C Sunshine AB Ong GT Pogachar JL Zhao W Dunham MJ 2016High-throughput identification of adaptive mutations in experimen-tally evolved yeast populations PLoS Genet 12e1006339

Perez-Prat E van Lookeren Campagne MM 2002 Hybrid seed produc-tion and the challenge of propagating male-sterile plants TrendsPlant Sci 7199ndash203

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16

Peris D Langdon QK Moriarty RV Sylvester K Bontrager M Charron GLeducq JB Landry CR Libkind D Hittinger CT 2016 Complex ances-tries of lager-brewing hybrids were shaped by standing variation inthe wild yeast Saccharomyces eubayanus PLoS Genet 12e1006155

Perlstein EO Ruderfer DM Roberts DC Schreiber SL Kruglyak L 2007Genetic basis of individual differences in the response to small-molecule drugs in yeast Nat Genet 39496ndash502

Piatkowska EM Naseeb S Knight D Delneri D 2013 Chimeric proteincomplexes in hybrid species generate novel phenotypes PLoS Genet9e1003836

Piotrowski JS Nagarajan S Kroll E Stanbery A Chiotti KE Kruckeberg ALDunn B Sherlock G Rosenzweig F 2012 Different selective pressureslead to different genomic outcomes as newly-formed hybrid yeastsevolve BMC Evol Biol 1246

Pryszcz LP Nemeth T Gacser A Gabaldon T 2014 Genome comparisonof Candida orthopsilosis clinical strains reveals the existence of hy-brids between two distinct subspecies Genome Biol Evol61069ndash1078

Racimo F Sankararaman S Nielsen R Huerta-Sanchez E 2015 Evidencefor archaic adaptive introgression in humans Nat Rev Genet16359ndash371

Rieseberg LH 1991 Homoploid reticulate evolution in Helianthus(Asteraceae)mdashevidence from ribosomal genes Am J Bot781218ndash1237

Rieseberg LH 1997 Hybrid origins of plant species Annu Rev EcolSystemat 28359ndash389

Robinson JT Thorvaldsdottir H Winckler W Guttman M Lander ESGetz G Mesirov JP 2011 Integrative genomics viewer NatBiotechnol 2924ndash26

Rosenthal DM Schwarzbach AE Donovan LA Raymond O RiesebergLH 2002 Phenotypic differentiation between three ancient hybridtaxa and their parental species Int J Plant Sci 163387ndash398

Ruderfer DM Pratt SC Seidel HS Kruglyak L 2006 Population genomicanalysis of outcrossing and recombination in yeast Nat Genet381077ndash1081

Rundle HD Nagel L Boughman JW Schluter D 2000 Natural selectionand parallel speciation in sympatric sticklebacks Science287306ndash308

Ryland GL Doyle MA Goode D Boyle SE Choong DY Rowley SM Li JBowtell DD Tothill RW Campbell IG et al 2015 Australian OvarianCancer Study Group Loss of heterozygosity what is it good for BMCMed Genomics 845

Salverda MLM Dellus E Gorter FA Debets AJM van der Oost J HoekstraRF Tawfik DS de Visser JAGM 2011 Initial mutations direct alter-native pathways of protein evolution PLoS Genet 7e1001321

Sanchez MR Miller AW Liachko I Sunshine AB Lynch B Huang MAlcantara E DeSevo CG Pai DA Tucker CM et al 2017 Differentialparalog divergence modulates genome evolution across yeast spe-cies PLoS Genet 13(2)e1006585

Scannell DR Zill OA Rokas A Payen C Dunham MJ Eisen MB Rine JJohnston M Hittinger CT 2011 The awesome power of yeast evo-lutionary genetics new genome sequences and strain resources forthe Saccharomyces sensu stricto genus G3 (Bethesda) 111ndash25

Schroder MS Martinez de San Vicente K Prandini TH Hammel SHiggins DG Bagagli E Wolfe KH Butler G 2016 Multiple originsof the pathogenic yeast Candida orthopsilosis by separate hybridiza-tions between two parental species PLoS Genet 12e1006404

Schumer M Rosenthal GG Andolfatto P 2014 How common is homo-ploid hybrid speciation Evolution 681553ndash1560

Schwarzbach AE Donovan LA Rieseberg LH 2001 Transgressivecharacter expression in a hybrid sunflower species Am J Bot88270ndash277

Selmecki AM Maruvka YE Richmond PA Guillet M Shoresh NSorenson AL De S Kishony R Michor F Dowell R et al 2015Polyploidy can drive rapid adaptation in yeast Nature 519349ndash52

Seshan VE Olshen A 2016 DNAcopy DNA copy number data analysisVersion R package version 1460

Soltis DE Visger CJ Soltis PS 2014 The polyploidy revolution then andnow Stebbins revisited Am J Bot 1011057ndash1078

Soltis PS 2013 Hybridization speciation and novelty J Evol Biol26291ndash293

Soltis PS Soltis DE 2009 The role of hybridization in plant speciationAnnu Rev Plant Biol 60561ndash588

Sunshine AB Payen C Ong GT Liachko I Tan KM Dunham MJ 2015The fitness consequences of aneuploidy are driven by condition-dependent gene effects PLoS Biol 13e1002155

Tamai Y Momma T Yoshimoto H Kaneko Y 1998 Co-existence of twotypes of chromosome in the bottom fermenting yeastSaccharomyces pastorianus Yeast 14923ndash933

Taylor DJ Hebert PDN 1993 Habitat-dependent hybrid parentage anddifferential introgression between neighboringly sympatric Daphniaspecies Proc Natl Acad Sci U S A 907079ndash7083

Thiagalingam S Laken S Willson JK Markowitz SD Kinzler KWVogelstein B Lengauer C 2001 Mechanisms underlying losses ofheterozygosity in human colorectal cancers Proc Natl Acad Sci US A 982698ndash2702

Tuna M Knuutila S Mills GB 2009 Uniparental disomy in cancer TrendsMol Med 15120ndash128

Walther A Hesselbart A Wendland J 2014 Genome sequence ofSaccharomyces carlsbergensis the worldrsquos first pure culture lageryeast G3 (Bethesda) 4783ndash793

Wayne RK 1993 Molecular evolution of the dog family Trends Genet9218ndash224

Wenger JW Schwartz K Sherlock G 2010 Bulk segregant analysis byhigh-throughput sequencing reveals a novel xylose utilization genefrom Saccharomyces cerevisiae PLoS Genet 6e1000942

Wessinger CA Rausher MD 2014 Predictability and irreversibility ofgenetic changes associated with flower color evolution inPenstemon barbatus Evolution 681058ndash1070

Wolfe KH 2015 Origin of the yeast whole-genome duplication PLoSBiol 13e1002221

Woods R Schneider D Winkworth CL Riley MA Lenski RE 2006 Testsof parallel molecular evolution in a long-term experiment withEscherichia coli Proc Natl Acad Sci U S A 1039107ndash9112

Wykoff DD OrsquoShea EK 2001 Phosphate transport and sensing inSaccharomyces cerevisiae Genetics 1591491ndash1499

Xiong Z Gaeta RT Pires JC 2011 Homoeologous shuffling andchromosome compensation maintain genome balance inresynthesized allopolyploid Brassica napus Proc Natl Acad SciU S A 1087908ndash7913

Yassour M Kapian T Fraser HB Levin JZ Pfiffner J Adiconis X Schroth GLuo SJ Khrebtukova I Gnirke A et al 2009 Ab initio construction ofa eukaryotic transcriptome by massively parallel mRNA sequencingProc Natl Acad Sci U S A 1063264ndash3269

Zhu YO Sherlock G Petrov DA 2016 Whole genome analysis of 132clinical Saccharomyces cerevisiae strains reveals extensive ploidy var-iation G3 (Bethesda) doi101534g3116029397

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had an average of 15 CNV per clone and the evolvedS uvarum clones had an average of 1 CNV per clone (table 2supplementary figs S1 and S2 Supplementary Material online)The most common event across nutrient limitations in theinterspecific hybrids was an amplification of the S cerevisiaecopy of chromosome IV which occurred in four

independent hybrid clones (three in phosphate limitationone in glucose limitation supplementary fig S3Supplementary Material online) Several other characteris-tic rearrangements occurred in the evolved S cerevisiaeclones including the amplification of the left arm of chro-mosome 14 accompanied by segmental monosomy of the

FIG 1 Evolved hybrids exhibit changes in copy number and loss of heterozygosity Copy number variants are displayed for selected evolved hybridclones from three nutrient-limited conditions Gh2 glucose Ph4 phosphate and Sh4 sulfate See additional figures in supplementary fig S3Supplementary Material online Hybrid copy number determined by normalized sequencing read depth per open reading frame (ORF) is plottedacross the genome according to S cerevisiae ORF coordinates to account for three reciprocal translocations between S cerevisiae and S uvarumChromosomes are plotted in alternating light and dark purple red indicates a S cerevisiae copy number variant and blue indicates a S uvarumcopy number variant Gh2 has a whole chromosome amplification of S cerevisiae chrIV a small segmental deletion of S uvarum chrIV (non-copyneutral loss of heterozygosity) and an amplification of S uvarum HXT67 Ph4 has a small segmental deletion of S cerevisiae chrIII (non-copyneutral loss of heterozygosity) and an amplification of S cerevisiae chrXIII with corresponding deletion of S uvarum chrXIII (copy neutral loss ofheterozygosity) Sh4 has an amplification of S cerevisiae SUL1 and a whole chromosome amplification of S uvarum chrVIII (note there is areciprocal translocation between chrVIII and chrXV in S uvarum relative to S cerevisiae) Note that Sh4 is plotted on a different scale For specificcoordinates of copy number variants see table 1

Smukowski Heil et al doi101093molbevmsx098 MBE

8

right arm of chromosome 14 an event seen previously inother evolved populations (Dunham et al 2002 Greshamet al 2008 Sunshine et al 2015) All copy number events in

S cerevisiae had break points at repetitive elements knownas Ty elements except those located on chrII which maybe mediated by another mechanism (Brewer et al 2015) In

FIG 2 Repeated loss of heterozygosity at the PHO84 locus in intra- and interspecific hybrids (A) The 25-kb region extending from the left telomereof chromosome XIII to the high-affinity phosphate transporter gene PHO84 (B) Copy number is plotted across part of chromosome XIII in thehybrid ancestor and three evolved hybrid clones in phosphate limitation (clone indicated in upper right corner) Red shows the S cerevisiae alleleblue shows the S uvarum allele and purple shows where both species exhibit the same copy number Note 8 kb of telomere sequence is removeddue to repetitive sequence (C) Alternate allele frequency is plotted for a portion of chromosome XIII in the ancestor and four evolved S cerevisiaeclones in phosphate limitation (clone indicated in upper right corner) All evolved S cerevisiae clones exhibit a loss of heterozygosity at thetelomeric portion of chromosome XIII (loss of S288C amplification of GRF167) as illustrated by an allele frequency of zero compared with theancestor Regions of heterozygosity are interspersed with regions of homozygosity as one of the parents of the diploid was itself the product of across between strains FL100 and S288C and the other parent was S288C Regions of heterozygosity are due to FL100 haplotypes S cerevisiae copynumber for the four evolved clones is shown below the ancestor is diploid across the chromosome (also see table 2 supplementary fig S1Supplementary Material online)

Adaptation in Hybrids doi101093molbevmsx098 MBE

9

contrast copy number variants in the hybrid were rarely facil-itated by repetitive elements perhaps in part because S uva-rum has no full length Ty elements however why S cerevisiaeTy elements and long terminal repeat (LTR) sequences fromeither genome were not utilized remains unknown

Frequently in nutrient-limited evolution experimentscopy number variants involve amplification of the nutrient-specific transporter and indeed we also observed amplifica-tion of these transporters in many of the clones In sulfatelimitation the S cerevisiae allele of the high-affinity sulfatetransporter gene SUL1 is amplified in 77 hybrid clones and 44 S cerevisiae clones (fig 1 tables 1 and 2 supplementary figsS1 and S3 Supplementary Material online) InterestinglySUL2 is the preferred sulfate transporter in S uvarum(Sanchez et al 2017) and was not observed to be amplifiedin the evolved hybrids (supplementary fig S2 and table S2Supplementary Material online) In glucose limitation previ-ous S cerevisiae evolution experiments found frequent am-plification of the high-affinity glucose transporter genesHXT67 (Brown et al 1998 Dunham et al 2002 Greshamet al 2008 Kao and Sherlock 2008 Kvitek and Sherlock2011) In our experiments the S uvarum alleles of theHXT67 transporters are amplified in 33 hybrid clones andboth S uvarum clones but are not amplified in evolved Scerevisiae clones This is suggestive that the S uvarum HXT67alleles confer a greater fitness advantage compared with Scerevisiae alternatively the genomic context could be morepermissive to amplification in S uvarum (fig 1 tables 1 and 2supplementary figs S1ndashS3 Supplementary Material online)Finally in phosphate limitation the S cerevisiae copy of thehigh-affinity phosphate transporter gene PHO84 is amplifiedand the S uvarum allele is lost in 36 hybrid clones in an eventknown as LOH (figs 1 and 2 table 1 supplementary fig 3Supplementary Material online) Intriguingly the evolved Scerevisiae clones also display LOH and accompanied amplifi-cation favoring the allele derived from strain GRF167 over theS288C allele in 44 clones (fig 2 table 2) All hybrid clonescarry the ldquopreferredrdquo GRF167 S cerevisiae allele as this was theallele used to create the de novo hybrid

Loss of Heterozygosity Is a Common Event inHeterozygous Evolving PopulationsSelection on heterozygosity as a LOH event could representis an underappreciated source of adaptation in microbial ex-perimental evolution as typical experiments evolve a haploidor homozygous diploid strain asexually and as a result havelittle variation to select upon LOH is observed in natural andindustrial hybrids (Albertin and Marullo 2012 Louis et al2012 Pryszcz et al 2014 Wolfe 2015 Schroder et al 2016)but here we document its occurrence in both intra- andinterspecific hybrids in the laboratory as a result of short-termevolution (also see (Dunn et al 2013 Burke et al 2014)) LOHis observed across all nutrient conditions with 13 independentLOH events detected in S cerevisiae and 9 independent eventsdocumented in the hybrids (figs 1 and 2 tables 1 and 2 sup-plementary figs S1 and S3 Supplementary Material online) Itthus appears that this type of mutational event is both com-mon and can occur over short evolutionary timescales

The LOH event can result in copy-neutral (where one alleleis lost and the other allele is amplified) or non-neutral chro-mosomal segments (where one allele is lost rendering thestrain hemizygous at that locus) and can favor the retentionof either allele In S cerevisiae there is a bias in resolutionwhere LOH events favor retaining the GRF167 allele over theS288C allele (1013 events Pfrac14 00169 table 2 supplementaryfig S4 Supplementary Material online) One unique case inclone Sc4 has a small approximately 5 kb LOH event onchrXV favoring GRF167 which switches to favoring S288Cfor the rest of the chromosome The retention of S cerevisiaeis slightly more common in the hybrids (59 events Pfrac14 10table 1 supplementary fig S3 Supplementary Material on-line) though not as drastic as the observed genome resolu-tion in the hybrid S pastorianus where LOH favorsS cerevisiae over S eubayanus (Nakao et al 2009) The sizeof the event ranges from approximately 25 kb to the wholechromosome level in the hybrids and from 5 kb to 540 kbin S cerevisiae Where LOH is accompanied by an amplifi-cation event the LOH event always occurs first and doesnot share break points with the amplification event Unlikemany CNV events most LOH events do not appear to bemediated by existing repetitive sequence such as a trans-posable element in the hybrid or S cerevisiae and are mostlikely a product of break-induced replication or mitoticgene conversion with break-induced replication as the fa-vored method as all events extend to the telomere (Hoanget al 2010) The exceptions are in hybrid clones Ph4 Ph5 andSh1 where there is a non-copy neutral loss of S cerevisiaemediated by a Ty element or LTR and S cerevisiae clonesSc1 and Sc4 where there is a 65-kb deletion of the S288Callele flanked by two Ty elements

LOH events in hybrids could signify several ongoing pro-cesses in hybrid genome evolution LOH regions may repre-sent 1) loci with incompatibilities 2) selection on existingvariation or 3) genetic drift eroding genomic segments It isimpossible to definitively rule out any of these hypotheseswithout further experimentation however there are severalarguments disfavoring the incompatibility hypothesis Firstalthough our sample size is modest failing to see repeatedLOH events across nutrient conditions may indicate a lack ofgeneral incompatibility between species (although we cannotpreclude condition-specific incompatibility Hou et al 2015Piatkowska et al 2013) This is consistent with previous stud-ies in yeast which suggest that classic DobzhanksyndashMullerincompatibilities are rare (Liti et al 2006 Greig 2009 Houet al 2014) Furthermore LOH events observed inevolved S cerevisiae suggest that this mutation type isnot unique to interspecific hybrids Instead repeatedevents within a particular condition such as the repeatedLOH at PHO84 in phosphate limitation or the 65 kbsegment on chrXIII in sulfate limitation suggest thatthese events are beneficial and are indeed selection onone allele over the other

LOH Is Driven by Selection on One AlleleTo test the hypothesis that LOH events provide a selectiveadvantage we used allele replacement in which the allele of

Smukowski Heil et al doi101093molbevmsx098 MBE

10

one speciesstrain is replaced with the allele of the otherspeciesstrain in an otherwise isogenic background We testedthis hypothesis using the most commonly seen LOH eventLOH at PHO84 Although the region extends from 25 kb to234 kb in length in the hybrids and from 40 kb to 85 kb in Scerevisiae and thus includes many genes PHO84 was a primecandidate driving this event PHO84 is one of only ten genesencompassed in the region extending from the telomere tothe break point of the shortest LOH event and is included inevery other LOH event on chromosome XIII (fig 2) It is ahigh-affinity phosphate transporter responsible for inorganicphosphate uptake in high and low phosphate conditions(Wykoff and OrsquoShea 2001) and previous work identified apoint mutation in PHO84 (an alanine to valine substitution atthe 50 end of the gene) which increased fitness by 23 inphosphate-limited conditions (Sunshine et al 2015) Finallyprior work with other nutrient transporters has shown am-plification of nutrient transporters to be a key event in adapt-ing to nutrient-limited conditions

We thus selected a region of approximately 25 kb encom-passing the PHO84 ORF its promoter and 30-UTR(Nagalakshmi et al 2008 Yassour et al 2009 Cherry et al2012) We created allele replacement strains using the twoalleles of S cerevisiae in a S cerevisiae diploid background thetwo alleles are 991 identical in this region (supplementaryfig S5 Supplementary Material online) and each strain isidentical to the ancestral strain used in our evolution exper-iments except at the PHO84 locus The S cerevisiae ancestorcarries one copy of GRF167 (preferred) and one copy ofS288C (ldquoun-preferredrdquo) so named due to which allele wasretained and amplified in the evolved clones To measureany resultant changes in fitness we competed each strainindividually against a fluorescent ancestral strain and mea-sured which strain overtook the culture Relative fitness isthus defined as the growth advantage per generation Twocopies of the un-preferred allele decreased relative fitness by531 (6186) whereas two copies of the preferred alleleincreased relative fitness by 993 (6027) This displays anoverall difference in relative fitness of 1524 between the un-preferred and preferred alleles By comparing the fitness ofthese allele replacement strains with the evolved clones (table 2)the allele replacement does not fully recapitulate the fit-ness gain observed in the evolved clone One explanation isthat the additional mutations present in the evolvedstrains also contribute to their total fitness Another expla-nation could be the increased copy number of the PHO84region that we see in these evolved clones To further ex-plore this fitness difference we cloned the GRF167 alleleonto a low copy number plasmid and transformed theallele replacement strain carrying two preferred S cerevi-siae alleles to simulate increased copy number of PHO84and saw only a minimal fitness increase of 176 (6106note all fitness competitions involving plasmids were donein conjunction with empty plasmids to take into accountany fitness effects from the plasmid itself) This supportsthe conclusion that relative fitness gains in the evolvedclone are largely due to the loss of the S288C allele andselection and amplification of the GRF167 allele with little

additional benefit from further amplification It could also bethe case that co-amplification of other genes in the segmentis required to attain the full benefit as previously observedby the contribution of BSD2 to the SUL1 amplicon in sulfatelimitation (Sunshine et al 2015 Payen et al 2016)

To understand the fitness effects of LOH and amplificationin the hybrids we generated hybrid strains with varying num-bers of S cerevisiae GRF167 PHO84 alleles Unfortunately wewere unable to obtain a successful strain carrying the pre-ferred S cerevisiae allele in a S uvarum background howeverwe were able to generate a S uvarum PHO84 knockout strainthus creating a hybrid with one copy of S cerevisiae PHO84When combined with a low copy plasmid carrying the sameallele this strain effectively has two or more copies ofS cerevisiae PHO84 in a hybrid background This hybrid straincan serve as a proxy for the LOH event observed in the evo-lution experiments and has a relative fitness gain of 2557(6288) The ancestral hybrid with the same plasmid (effec-tively 1 S uvarum allele and 2 or more S cerevisiae alleles) hasa relative fitness gain of 1253 (6131) The difference be-tween these two hybrids suggests that while amplification isbeneficial the highest fitness is achieved with the loss of theS uvarum allele (Pfrac14 00061)

Together these results support the conclusion that theS cerevisiae GRF167 allele is preferred over the S288C alleleand that S cerevisiae alleles are preferred over the S uvarumallele in the hybrid and hence that the LOH events seen inboth intra- and interspecific hybrids are the product ofselection

DiscussionIn summary we sought to understand forces underlying ge-nome stabilization and evolution in interspecific and intra-specific hybrids as they adapt to novel environments Weevolved and sequenced clones from 16 hybrid populationsand 16 parental populations to reveal a variety of mutationalevents conferring adaptation to 3 nutrient-limited conditionsOf particular note we find LOH in both evolved intraspecificand interspecific hybrid clones in all nutrient environmentspotentially signifying areas where selection has acted on pre-existing variation present in the ancestral clone We used anallele replacement strategy to test this hypothesis for a com-monly repeated LOH event and show that selection is indeeddriving the homogenization of the genome at this locusAlthough other studies in natural industrial and lab-evolved isolates have observed LOH we present the first em-pirical test of the causal evolutionary forces influencing theseevents This work can begin to help us understand past hy-bridization events and subsequent genome resolution in hy-brids in natural and artificial systems

Similarities and Differences between Intra- andInterspecific HybridsAlthough our sample size is modest we observe several in-teresting trends when comparing S uvarum clones (homo-zygous) S cerevisiae clones (intraspecific hybrid andpreviously published homozygous) and interspecific hybrid

Adaptation in Hybrids doi101093molbevmsx098 MBE

11

clones First theory and previous research predict the inter-specific hybrid may experience more genome instability in theform of chromosomal rearrangements and CNV events(Xiong et al 2011 Lloyd et al 2014 Chester et al 2015Mason and Batley 2015) Instead in our work the interspecifichybrid and S cerevisiae clones experience the same numberof CNV events (both 15 CNVsclone) However the mech-anism of CNV formation seems to differ between the hybridand S cerevisiae clones Whereas S cerevisiae CNV breakpoints typically occur at transposable elements in our studyand previous studies (Dunham et al 2002 Gresham et al2008 Fedoroff 2012) the interspecific hybrid CNVs do notutilize transposable elements although they obviously sharethe same sequence background as the S cerevisiae clones(albeit in one copy) Whether this difference is due to theabsence of full-length transposable elements in S uvarum isunknown but this could potentially explain the lower num-ber of CNV events in S uvarum clones (1 CNVclone) andpresents an intriguing direction for future study

The Predictability of EvolutionWe now have many examples of predictable evolution innatural systems (Losos et al 1998 Rundle et al 2000 Elmerand Meyer 2011 Conte et al 2012 Jones et al 2012 Martinand Orgogozo 2013 Wessinger and Rausher 2014) and inlaboratory experimental evolution in which there often ap-pears to be a limited number of high fitness pathways thatstrains follow when adapting to a particular condition (Fereaet al 1999 Woods et al 2006 Gresham et al 2008 Burke et al2010 Salverda et al 2011 Kawecki et al 2012 Kvitek andSherlock 2013 Lang and Desai 2014) For example it is wellestablished that amplifications of nutrient transporters aredrivers of adaptation in evolution in nutrient-limited condi-tions Previous work in our group has particularly focused onthe amplification of the high-affinity sulfate transporter geneSUL1 in sulfate-limited conditions which occurs in almostevery sulfate-limited evolution experiment and confers a fit-ness advantage of as much as 40 compared with the un-evolved ancestor strain The amplification of phosphatetransporters has been markedly less common and thus driv-ers of adaptation in this condition have been less clearGresham et al (2008) identified a whole chromosome ampli-fication of chrXIII in one population In a follow-up studySunshine et al (2015) found whole or partial amplification ofchrXIII in 38 populations A genome-wide screen for segmen-tal amplifications found a slight increase in fitness for a smalltelomeric segment of chromosome XIII and a A49V pointmutation in PHO84 was observed to increase fitness by 23However screens by Payen et al (2016) showed that althoughPHO84 is recurrently mutated in various experiments itshowed no benefit when amplified or deleted in phos-phate-limited conditions Finally additional evolution exper-iments recapitulated the point mutation seen in Sunshineet al (2015) in 2432 populations and saw amplification ofPHO84 in 832 populations (Miller A Dunham MJ unpub-lished data) It is important to note that all these experimentsused a strain background derived from S288C or CENPKboth of which carry the same (un-preferred) PHO84 allele

In our work we observed amplification of the S cerevisiaeGRF167 allele of PHO84 in 44 S cerevisiae clones from 4populations and 36 hybrid clones from 6 populations Thisamplification was always preceded by the loss of the S288Callele in S cerevisiae clones and the LOH break points arenever shared with the amplification break points There is a15 fitness difference between carrying two copies of theS288C allele of PHO84 compared with carrying two copiesof the GRF167 allele of PHO84 and additional copies of theGRF167 allele do not provide substantial further fitness gainsThe two alleles differ by several noncoding changes and threenonsynonymous substitutions a mutation from glutamicacid to aspartic acid (E229D) leucine to proline (L259P)and leucine to glutamine (L556Q) the latter two of whichare considered ldquononconservativerdquo protein mutations due tochanges in hydrophobicity and structure (supplementary figS5 Supplementary Material online) Intriguingly the L259Pmutation has actually been previously identified as being re-sponsible for resistance to tetrachloroisophthalonitrile andpartial resistance to pentachlorophenol in a QTL study ofsmall-molecule drugs (Perlstein et al 2007) IndeedPerlstein et al found proline at residue 259 to be conservedacross fungal species and even in orthologous human xeno-biotic transporters likely because proline-induced kinks intransmembrane spans have been shown to be essential toprotein function (Cordes et al 2002 Perlstein et al 2007) Itthus appears that amplification of PHO84 has been less pre-dictable as the S288C allele does not confer a fitness advan-tage unless mutated per Sunshine et al (2015) Togetherthese results imply that strain background can constrainadaptive pathways

In hybrids the amplification of the S cerevisiae segmentoccurred in conjunction with the loss of the S uvarum alleleHybrid strains with the LOH had a 2557 relative fitness gainwhereas hybrid strains with amplification of the S cerevisiaePHO84 allele without the LOH had a 1253 fitness gain ThusLOH confers an additional 1304 fitness gain showing thatselection for LOH has a larger impact on fitness than ampli-fication alone Note that S uvarum does have proline at res-idue 259 like the preferred GRF167 allele and differs fromboth S cerevisiae alleles at the other two coding substitutions(229N and 556K) but the amino acid and noncoding diver-gence is too high to speculate what substitutions are respon-sible for the selection of the GRF167 allele (supplementary figS6 Supplementary Material online) Why the loss of one alleleis more beneficial remains unclear as PHO84 is thought tofunction as a monomer (Bun-Ya et al 1991) but it may bedue to competition for cell wall space or negative interactionswith other genes in the PHO pathway (Mouillon and Persson2006)

The infusion of variation created by hybridization providesnew templates for selection to act upon which can be moreimportant than either point mutations or copy number var-iants alone Our work shows that outcrossing need not becommon to have long-lasting effects on adaptation This im-plication is particularly relevant in yeast where outcrossingmay occur quite rarely followed by thousands of asexual gen-erations (Ruderfer et al 2006 Greig and Leu 2009 Liti 2015)

Smukowski Heil et al doi101093molbevmsx098 MBE

12

Applications to Other Hybrids and CancerThe observation that LOH occurs in hybrid genomes is in-creasingly documented (Louis et al 2012 Borneman et al2014 Soltis et al 2014 Marcet-Houben and Gabaldon 2015Pryszcz et al 2014 Schroder et al 2016) although the rea-son(s) for this type of mutation has been unresolved As mostexamples stem from allopolyploid events that occurred mil-lions of years ago understanding why LOH is important inhybrid genome evolution is difficult Cancer cells are alsoknown to experience LOH sometimes involved in the inac-tivation of tumor suppressor genes leaving only one copy ofthe gene that may be mutated or silenced (Thiagalingam et al2001 Tuna et al 2009 Lapunzina and Monk 2011) Datasupport the conclusion that LOH events are selected for dur-ing tumor development as many LOH events involve specificchromosomal segments (Thiagalingam et al 2001) althoughthe underlying molecular and genetic reasons for selection isan open debate (Ryland et al 2015)

Here we experimentally demonstrate that LOH can occurin homoploid interspecific hybrids as well as in intraspecifichybrids These events occur within a few hundred generationsand are common mutations more common on average inthe intraspecific hybrid (13 eventsclone) than the interspe-cific hybrid (056 eventsclone) Interestingly the LOH eventsdo not share break points with the CNV events in S cerevisiaeinstead they appear to occur independently and to precedeany subsequent amplification (amplification occurs following913 LOH events) Competition assays with the PHO84 locusprovide support that LOH itself may be more beneficial thanamplification or at least increase the selective benefit of am-plification events The observation that LOH in intraspecifichybrids occurs independently from copy number change pro-vides different opportunities for adaptation to novelconditions

Other cases of LOH like the copy number neutral eventsobserved in Ph4 Ph5 and Sh4 all of which favor the retentionof the S uvarum allele (supplementary fig S3 SupplementaryMaterial online) may be due to hybrid incompatibility withina particular protein complex other epistatic interactions(Piatkowska et al 2013) or neutral processes We furthermorediscover examples where one species allele appears to bepreferred over the other without LOH such as the repeatedamplification of the S uvarum high-affinity glucose transpor-ters HXT67 When one species allele is amplified and theother is not amplified one explanation is that the local se-quence context can permit or deny amplification In the caseof HXT67 previous experiments in S cerevisiae have shownthat amplification of HXT67 is quite common (Brown et al1998 Dunham et al 2002 Gresham et al 2008 Kao andSherlock 2008 Kvitek and Sherlock 2011) thus suggestingthat when given a choice between this locus in S cerevisiaeor S uvarum in the hybrid the preferred allele is indeed Suvarum though more subtle differences in rate cannot yet beruled out A similar scenario is observed with S cerevisiaeSUL1 (Sanchez et al 2017) Together our results show thatthe heterozygosity supplied by hybridization is an importantcontributor to adaptive routes explored by populations asthey adapt to novel conditions

Although we cannot generalize our results from thePHO84 locus across the many other LOH events discoveredin our hybrids and S cerevisiae in the future we can usesimilar methodology to explore whether positive selectionalways drives LOH or whether other explanations such asincompatibility resolution contribute as well Future experi-ments might also utilize a high throughput method to ex-plore segmental LOH in hybrids at a genome-wide scalesimilar to ongoing experiments at the gene level (LancasterS Dunham MJ unpublished data) Although our sample sizeis moderate this is a novel and necessary step in understand-ing forces underlying hybrid genome stabilization and high-lighting an underappreciated mechanism of hybridadaptation

ConclusionsThe mutation events we observe in our experimentallyevolved hybrids are in many ways quite representative ofmutations observed in ancient hybrid genomes suggestingthat hybrid genome stabilization and adaptation can occurquite rapidly (within several hundred generations)Furthermore our results illustrate that the infusion of varia-tion introduced by hybridization at both the intra- and inter-species level can increase fitness by providing choices of allelesfor selection to act upon even when sexual reproduction israre This may be particularly important for leveraging existingvariation for agricultural and industrial processes and as cli-mate change potentially increases natural hybridization (Kellyet al 2010 Hoffmann and Sgro 2011 Muhlfeld et al 2014)

Materials and Methods

StrainsA list of strains used in this study is included in supplementarytable S1 Supplementary Material online All interspecific hy-brids were created by crossing a ura3 LYS2 haploid parent to aURA3 lys2 haploid parent of the other mating type plating onmedia lacking both uracil and lysine and selecting for proto-trophs The S cerevisiae strain background known asldquoGRF167rdquo is itself a cross between FL100 and the genomictype strain S288C (data not shown) GRF167 was chosen as astrain background for simultaneous work investigating trans-posable elements during experimental evolution which willbe addressed in a future study

Evolution ExperimentsContinuous cultures were established using media and con-ditions previously described (Gresham et al 2008 Sanchezet al 2017) Detailed protocols and media recipes are availableat httpdunhamgswashingtoneduprotocolsshtml (lastaccessed March 6 2017) Samples were taken daily and mea-sured for optical density at 600 nm and cell count micros-copy was performed to check for contamination andarchival glycerol stocks were made daily An experimentwas terminated when contamination growth in tubing orclumping appeared (number of generations at the endpoint for each population are presented in tables 1 and

Adaptation in Hybrids doi101093molbevmsx098 MBE

13

2) Samples from each end point population were colonypurified to yield two clones for further study

Array Comparative Genomic HybridizationPopulations from the end point of each evolution were ana-lyzed for copy number changes using array comparative ge-nomic hybridization following the protocol used in Sanchezet al (2017)

SequencingDNA was extracted from overnight cultures using theHoffmanndashWinston protocol (Hoffman and Winston 1987)and cleaned using the Clean amp Concentrator kit (ZymoResearch) Nextera libraries were prepared following theNextera library kit protocol and sequenced using paired end150 bp reads on the Illumina NextSeq 500 machine (sequenc-ing coverage in supplementary table S2 SupplementaryMaterial online) The reference genomes used were S cerevi-siae v3 (Engel et al 2014) S uvarum (Scannell et al 2011) anda hybrid reference genome created by concatenating the twogenomes Sequence was aligned to the appropriate referencegenome using bwa v062 (Li and Durbin 2009) and mutationswere called using GATK (McKenna et al 2010) and samtools0119 (Li et al 2009) Mutations in evolved clones were filteredin comparison with the ancestor to obtain de novo mutationsAll mutations were first visually inspected using IntegrativeGenomics Viewer (Robinson et al 2011) Subsequently pointmutations in the hybrids were confirmed with Sanger se-quencing (supplementary table S3 Supplementary Materialonline) Copy number variants were visualized usingDNAcopy for S cerevisiae and S uvarum (Seshan andOlshen 2016) LOH events were called based on sequencingcoverage in the hybrids and by identifying homozygous variantcalls in S cerevisiae All break points were called by visual in-spection of sequencing reads and are thus approximate

Fitness AssaysThe pairwise competition experiments were performed in 20ml chemostats (Miller and Dunham 2013) Each competitorstrain was cultured individually until steady state was reachedand then was mixed 5050 with a GFP-tagged ancestor Eachcompetition was conducted in at least two biological repli-cates for approximately 15 generations after mixing Sampleswere collected and analyzed twice daily The proportion ofGFPthorn cells in the population was detected using a BD AccuriC6 flow cytometer (BD Biosciences) The data were plottedwith ln [dark cellsGFPthorn cells] versus generations The rela-tive fitness coefficient was determined from the slope of thelinear region

Strain ConstructionAllele replacements for the PHO84 locus were done followingthe protocol of the Caudy lab with further modifications de-scribed here The native locus was replaced withKluyveromyces lactis URA3 The pho84DURA3 strain wasgrown overnight in 5 ml of C-URA media then inoculatedin a flask of 100 ml yeast extract peptone dextrose (YPD) andgrown to an optical density of 06ndash08 Cells were washed then

aliquoted 275 ml of transformation mix (35 ml 1 M lithiumacetate 240 ml of 50 3500 polyethylene glycol) 10 ml ofsalmon sperm and approximately 3 mg of polymerase chainreaction product were added to the cell pellet It was incu-bated at 37 C (S uvarum) or 42 C (S cerevisiae) for 45 minthen plated to YPD It was replica plated to 5-fluorooroticacid the following day and colonies were tested for the gain ofthe appropriate species allele The GRF167 allele was clonedinto the pIL37 plasmid using Gibson assembly (Gibson et al2009) Correct assembly was verified by Sanger sequencing Allprimers used can be found in supplementary table S3Supplementary Material online

Supplementary MaterialSupplementary data are available at Molecular Biology andEvolution online

AcknowledgmentsWe thank Noah Hanson and Erica Alcantara for technicalassistance We thank Monica Sanchez and three anonymousreviewers for helpful comments on this manuscript JosephSchacherer kindly confirmed the contribution of FL100 to theGRF167 strain background Thanks to Yixian Zheng and DougKoshland for contributing to the initial experimental designcreating yeast strains and purchasing the oligonucleotidesused for the microarrays This work was supported by theNational Science Foundation (grant number 1516330) andthe National Institutes of Health (grant number R01GM094306) MD is a Senior Fellow in the GeneticNetworks program at the Canadian Institute for AdvancedResearch and a Rita Allen Foundation Scholar CSH wassupported in part by National Institutes of Health (grantnumber T32 HG00035) This work was also supported bythe National Institutes of Health (grant number P50GM071508) to the Lewis-Sigler Institute and from theHoward Hughes Medical Institute to Doug Koshland andYixian Zheng Sequencing read data have been deposited atthe NCBI under BioProject accession PRJNA374049Microarray data are deposited in the Gene ExpressionOmnibus (GEO) repository under accession numbersGSE95086 and GSE87401 and in the Princeton MicroarrayDatabase

ReferencesAbbott R Albach D Ansell S Arntzen JW Baird SJ Bierne N Boughman J

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Almeida P Goncalves C Teixeira S Libkind D Bontrager M Masneuf-Pomarede I Albertin W Durrens P Sherman DJ Marullo P et al2014 A Gondwanan imprint on global diversity and domesticationof wine and cider yeast Saccharomyces uvarum Nat Commun54044

Antunovics Z Nguyen HV Gaillardin C Sipiczki M 2005 Gradual ge-nome stabilisation by progressive reduction of the Saccharomyces

Smukowski Heil et al doi101093molbevmsx098 MBE

14

uvarum genome in an interspecific hybrid with Saccharomyces cer-evisiae FEMS Yeast Res 51141ndash1150

Baker E Wang B Bellora N Peris D Hulfachor AB Koshalek JA AdamsM Libkind D Hittinger CT 2015 The Genome sequence ofSaccharomyces eubayanus and the domestication of lager-brewingyeasts Mol Biol Evol 322818ndash2831

Barbosa R Almeida P Safar SVB Santos RO Morais PB Nielly-Thibault LLeducq JB Landry CR Goncalves P Rosa CA Sampaio JP 2016Evidence of natural hybridization in Brazilian wild lineages ofSaccharomyces cerevisiae Genome Biol Evol 8317ndash329

Barton NH 2001 The role of hybridization in evolution Mol Ecol10551ndash568

Belloch C Orlic S Barrio E Querol A 2008 Fermentative stress adapta-tion of hybrids within the Saccharomyces sensu stricto complex Int JFood Microbiol 122188ndash195

Bellon JR Yang F Day MP Inglis DL Chambers PJ 2015 Designingand creating Saccharomyces interspecific hybrids for improvedindustry relevant phenotypes Appl Microbiol Biotechnol998597ndash8609

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Brewer BJ Payen C Di Rienzi SC Higgins MM Ong G Dunham MJRaghuraman MK 2015 Origin-dependent inverted-repeat amplifi-cation tests of a model for inverted DNA amplification PLoS Genet11e1005699

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Bullini L 1994 Origin and evolution of animal hybrid species Trends EcolEvol 9422ndash426

Bun-Ya M Nishimura M Harashima S Oshima Y 1991 The PHO84 geneof Saccharomyces cerevisiae encodes an inorganic phosphate trans-porter Mol Cell Biol 113229ndash3238

Burke MK Dunham JP Shahrestani P Thornton KR Rose MR Long AD2010 Genome-wide analysis of a long-term evolution experimentwith Drosophila Nature 467587ndash590

Burke MK Liti G Long AD 2014 Standing genetic variation drives re-peatable experimental evolution in outcrossing populations ofSaccharomyces cerevisiae Mol Biol Evol 313228ndash3239

Cherry JM Hong EL Amundsen C Balakrishnan R Binkley G Chan ETChristie KR Costanzo MC Dwight SS Engel SR et al 2012Saccharomyces genome database the genomics resource of buddingyeast Nucleic Acids Res 40D700ndashD705

Chester M Riley RK Soltis PS Soltis DE 2015 Patterns of chromosomalvariation in natural populations of the neoallotetraploid Tragopogonmirus (Asteraceae) Heredity (Edinb) 114309ndash317

Chou JY Leu JY 2010 Speciation through cytonuclear incompatibilityinsights from yeast and implications for higher eukaryotes Bioessays32401ndash411

Cliften PF Fulton RS Wilson RK Johnston M 2006 After the duplicationgene loss and adaptation in Saccharomyces genomes Genetics172863ndash872

Conte GL Arnegard ME Peichel CL Schluter D 2012 The probability ofgenetic parallelism and convergence in natural populations Proc BiolSci 2795039ndash5047

Cordes FS Bright JN Sansom MS 2002 Proline-induced distortions oftransmembrane helices J Mol Biol 323951ndash960

Crow JF 1998 90 years ago the beginning of hybrid maize Genetics148923ndash928

Dasmahapatra KK Walters JR Briscoe AD Davey JW Whibley A NadeauNJ Zimin AV Hughes DST Ferguson LC Martin SH et al 2012Butterfly genome reveals promiscuous exchange of mimicry adap-tations among species Nature 48794ndash98

Dowling TE Smith GR Brown WM 1989 Reproductive isolation andintrogression between Notropis-Cornutus and Notropis-Chrysocephalus (Family Cyprinidae)mdashcomparison of morphologyallozymes and mitochondrial-DNA Evolution 43620ndash634

Doyle JJ Flagel LE Paterson AH Rapp RA Soltis DE Soltis PS Wendel JF2008 Evolutionary genetics of genome merger and doubling inplants Annu Rev Genet 42443ndash461

Dunham MJ Badrane H Ferea T Adams J Brown PO Rosenzweig FBotstein D 2002 Characteristic genome rearrangements in experi-mental evolution of Saccharomyces cerevisiae Proc Natl Acad Sci U SA 9916144ndash16149

Dunn B Paulish T Stanbery A Piotrowski J Koniges G Kroll E LouisEJ Liti G Sherlock G Rosenzweig F 2013 Recurrent rearrange-ment during adaptive evolution in an interspecific yeast hybridsuggests a model for rapid introgression PLoS Genet9e1003366

Dunn B Richter C Kvitek DJ Pugh T Sherlock G 2012 Analysis of theSaccharomyces cerevisiae pan-genome reveals a pool of copy num-ber variants distributed in diverse yeast strains from differing indus-trial environments Genome Res 22908ndash924

Dunn B Sherlock G 2008 Reconstruction of the genome origins andevolution of the hybrid lager yeast Saccharomyces pastorianusGenome Res 181610ndash1623

Elmer KR Meyer A 2011 Adaptation in the age of ecological genomicsinsights from parallelism and convergence Trends Ecol Evol26298ndash306

Engel SR Dietrich FS Fisk DG Binkley G Balakrishnan R Costanzo MCDwight SS Hitz BC Karra K Nash RS et al 2014 The referencegenome sequence of Saccharomyces cerevisiae then and now G3(Bethesda) 4389ndash398

Fedoroff NV 2012 Presidential address Transposable elements epige-netics and genome evolution Science 338758ndash767

Ferea TL Botstein D Brown PO Rosenzweig RF 1999 Systematicchanges in gene expression patterns following adaptive evolutionin yeast Proc Natl Acad Sci U S A 969721ndash9726

Gibson B Liti G 2015 Saccharomyces pastorianus genomic insightsinspiring innovation for industry Yeast 3217ndash27

Gibson DG Young L Chuang RY Venter JC Hutchison CA Smith HO2009 Enzymatic assembly of DNA molecules up to several hundredkilobases Nat Methods 6343ndash5

Gonzalez SS Barrio E Gafner J Querol A 2006 Natural hybrids fromSaccharomyces cerevisiae Saccharomyces bayanus andSaccharomyces kudriavzevii in wine fermentations FEMS Yeast Res61221ndash1234

Gonzalez SS Barrio E Querol A 2008 Molecular characterization of newnatural hybrids of Saccharomyces cerevisiae and S kudriavzevii inbrewing Appl Environ Microbiol 742314ndash2320

Grant PR Grant BR 1994 Phenotypic and genetic consequences ofhybridization in Darwinrsquos finches Evolution 48297ndash316

Grant PR Grant BR 2002 Unpredictable evolution in a 30-year study ofDarwinrsquos finches Science 296707ndash711

Grant PR Grant BR 2010 Natural selection speciation and Darwinrsquosfinches Proc Calif Acad Sci 61245ndash260

Grant PR Grant BR Petren K 2005 Hybridization in the recent past AmNat 16656ndash67

Greig D 2009 Reproductive isolation in Saccharomyces Heredity (Edinb)10239ndash44

Greig D Leu JY 2009 Natural history of budding yeast Current Biol19R886ndashR890

Greig D Louis EJ Borts RH Travisano M 2002 Hybrid speciation inexperimental populations of yeast Science 2981773ndash1775

Gresham D Desai MM Tucker CM Jenq HT Pai DA Ward A DeSevoCG Botstein D Dunham MJ 2008 The repertoire and dynamics ofevolutionary adaptations to controlled nutrient-limited environ-ments in yeast PLoS Genet 4e1000303

Hajjar R Hodgkin T 2007 The use of wild relatives in crop improvementa survey of developments over the last 20 years Euphytica 1561ndash13

Heiser CB 1954 Variation and subspeciation in the common sunflowerHelianthus annuus Am Midl Nat 51287ndash305

Hittinger CT 2013 Saccharomyces diversity and evolution a buddingmodel genus Trends Genet 29309ndash317

Hoang ML Tan FJ Lai DC Celniker SE Hoskins RA Dunham MJ ZhengY Koshland D 2010 Competitive repair by naturally dispersed

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repetitive DNA during non-allelic homologous recombination PLoSGenet 6e1001228

Hoffmann AA Sgro CM 2011 Climate change and evolutionary adap-tation Nature 470479ndash485

Hoffman CS Winston F 1987 A 10-minute DNA preparation from yeastefficiently releases autonomous plasmids for transformation ofEscherichia coli Gene 57267ndash272

Hou J Friedrich A de Montigny J Schacherer J 2014 Chromosomalrearrangements as a major mechanism in the onset of reproductiveisolation in Saccharomyces cerevisiae Curr Biol 241153ndash1159

Hou J Friedrich A Gounot JS Schacherer J 2015 Comprehensive surveyof condition-specific reproductive isolation reveals genetic incom-patibility in yeast Nat Commun 67214

Huerta-Sanchez E Casey FP 2015 Archaic inheritance supporting high-altitude life in Tibet J Appl Physiol (1985) 1191129ndash1134

Jones FC Grabherr MG Chan YF Russell P Mauceli E Johnson JSwofford R Pirun M Zody MC White S et al 2012 The genomicbasis of adaptive evolution in threespine sticklebacks Nature48455ndash61

Kao KC Sherlock G 2008 Molecular characterization of clonal interfer-ence during adaptive evolution in asexual populations ofSaccharomyces cerevisiae Nat Genet 401499ndash1504

Kawecki TJ Lenski RE Ebert D Hollis B Olivieri I Whitlock MC 2012Experimental evolution Trends Ecol Evol 27547ndash560

Kellis M Patterson N Endrizzi M Birren B Lander ES 2003 Sequencingand comparison of yeast species to identify genes and regulatoryelements Nature 423241ndash254

Kelly B Whiteley A Tallmon D 2010 The Arctic melting pot Nature468891ndash891

Kvitek DJ Sherlock G 2011 Reciprocal sign epistasis between frequentlyexperimentally evolved adaptive mutations causes a rugged fitnesslandscape PLoS Genet 7e1002056

Kvitek DJ Sherlock G 2013 Whole genome whole population sequenc-ing reveals that loss of signaling networks is the major adaptivestrategy in a constant environment PLoS Genet 9e1003972

Landry CR Hartl DL Ranz JM 2007 Genome clashes in hybrids insightsfrom gene expression Heredity (Edinb) 99483ndash493

Lang GI Desai MM 2014 The spectrum of adaptive mutations in ex-perimental evolution Genomics 104412ndash416

Lapunzina P Monk D 2011 The consequences of uniparental disomyand copy number neutral loss-of-heterozygosity during human de-velopment and cancer Biol Cell 103303ndash317

Leducq JB Nielly-Thibault L Charron G Eberlein C Verta JP Samani PSylvester K Hittinger CT Bell G Landry CR 2016 Speciation drivenby hybridization and chromosomal plasticity in a wild yeast NatMicrobiol 115003

Lee HY Chou JY Cheong L Chang NH Yang SY Leu JY 2008Incompatibility of nuclear and mitochondrial genomes causes hy-brid sterility between two yeast species Cell 1351065ndash1073

Li H Durbin R 2009 Fast and accurate short read alignment withBurrows-Wheeler transform Bioinformatics 251754ndash1760

Li H Handsaker B Wysoker A Fennell T Ruan J Homer N Marth GAbecasis G Durbin R 2009 The Sequence AlignmentMap formatand SAMtools Bioinformatics 252078ndash2079

Liti G 2015 The fascinating and secret wild life of the budding yeast Scerevisiae Elife 4e05835

Liti G Barton DB Louis EJ 2006 Sequence diversity reproductiveisolation and species concepts in Saccharomyces Genetics174839ndash850

Lloyd AH Ranoux M Vautrin S Glover N Fourment J Charif D ChouletF Lassalle G Marande W Tran J et al 2014 Meiotic gene evolutioncan you teach a new dog new tricks Mol Biol Evol 311724ndash1727

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Mallet J 2007 Hybrid speciation Nature 446279ndash283Marcet-Houben M Gabaldon T 2015 Beyond the whole-genome du-

plication phylogenetic evidence for an ancient interspecies hybrid-ization in the bakerrsquos yeast lineage PLoS Biol 13e1002220

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Masly JP Jones CD Noor MAF Locke J Orr HA 2006 Gene transpositionas a cause of hybrid sterility in Drosophila Science 3131448ndash1450

Mason AS Batley J 2015 Creating new interspecific hybrid and polyploidcrops Trends Biotechnol 33436ndash441

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Meyer A Salzburger W Schartl M 2006 Hybrid origin of a swordtailspecies (Teleostei Xiphophorus clemenciae) driven by sexual selec-tion Mol Ecol 15721ndash730

Michalak P 2009 Epigenetic transposon and small RNA determinants ofhybrid dysfunctions Heredity (Edinb) 10245ndash50

Miller AW Dunham MJ 2013 Design and use of multiplexed chemostatarrays J Vis Exp 72e50262

Monod J 1949 The growth of bacterial cultures Annu Rev Microbiol3371ndash394

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Novick A Szilard L 1950b Experiments with the Chemostat on spon-taneous mutations of bacteria Proc Natl Acad Sci U S A 36708ndash719

Ouyang YD Liu YG Zhang QF 2010 Hybrid sterility in plant storiesfrom rice Curr Opin Plant Biol 13186ndash192

Payen C Di Rienzi SC Ong GT Pogachar JL Sanchez JC SunshineAB Raghuraman MK Brewer BJ Dunham MJ 2014 The dynam-ics of diverse segmental amplifications in populations ofSaccharomyces cerevisiae adapting to strong selection G3(Bethesda) 4399ndash409

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Perez-Prat E van Lookeren Campagne MM 2002 Hybrid seed produc-tion and the challenge of propagating male-sterile plants TrendsPlant Sci 7199ndash203

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Peris D Langdon QK Moriarty RV Sylvester K Bontrager M Charron GLeducq JB Landry CR Libkind D Hittinger CT 2016 Complex ances-tries of lager-brewing hybrids were shaped by standing variation inthe wild yeast Saccharomyces eubayanus PLoS Genet 12e1006155

Perlstein EO Ruderfer DM Roberts DC Schreiber SL Kruglyak L 2007Genetic basis of individual differences in the response to small-molecule drugs in yeast Nat Genet 39496ndash502

Piatkowska EM Naseeb S Knight D Delneri D 2013 Chimeric proteincomplexes in hybrid species generate novel phenotypes PLoS Genet9e1003836

Piotrowski JS Nagarajan S Kroll E Stanbery A Chiotti KE Kruckeberg ALDunn B Sherlock G Rosenzweig F 2012 Different selective pressureslead to different genomic outcomes as newly-formed hybrid yeastsevolve BMC Evol Biol 1246

Pryszcz LP Nemeth T Gacser A Gabaldon T 2014 Genome comparisonof Candida orthopsilosis clinical strains reveals the existence of hy-brids between two distinct subspecies Genome Biol Evol61069ndash1078

Racimo F Sankararaman S Nielsen R Huerta-Sanchez E 2015 Evidencefor archaic adaptive introgression in humans Nat Rev Genet16359ndash371

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Rieseberg LH 1997 Hybrid origins of plant species Annu Rev EcolSystemat 28359ndash389

Robinson JT Thorvaldsdottir H Winckler W Guttman M Lander ESGetz G Mesirov JP 2011 Integrative genomics viewer NatBiotechnol 2924ndash26

Rosenthal DM Schwarzbach AE Donovan LA Raymond O RiesebergLH 2002 Phenotypic differentiation between three ancient hybridtaxa and their parental species Int J Plant Sci 163387ndash398

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Rundle HD Nagel L Boughman JW Schluter D 2000 Natural selectionand parallel speciation in sympatric sticklebacks Science287306ndash308

Ryland GL Doyle MA Goode D Boyle SE Choong DY Rowley SM Li JBowtell DD Tothill RW Campbell IG et al 2015 Australian OvarianCancer Study Group Loss of heterozygosity what is it good for BMCMed Genomics 845

Salverda MLM Dellus E Gorter FA Debets AJM van der Oost J HoekstraRF Tawfik DS de Visser JAGM 2011 Initial mutations direct alter-native pathways of protein evolution PLoS Genet 7e1001321

Sanchez MR Miller AW Liachko I Sunshine AB Lynch B Huang MAlcantara E DeSevo CG Pai DA Tucker CM et al 2017 Differentialparalog divergence modulates genome evolution across yeast spe-cies PLoS Genet 13(2)e1006585

Scannell DR Zill OA Rokas A Payen C Dunham MJ Eisen MB Rine JJohnston M Hittinger CT 2011 The awesome power of yeast evo-lutionary genetics new genome sequences and strain resources forthe Saccharomyces sensu stricto genus G3 (Bethesda) 111ndash25

Schroder MS Martinez de San Vicente K Prandini TH Hammel SHiggins DG Bagagli E Wolfe KH Butler G 2016 Multiple originsof the pathogenic yeast Candida orthopsilosis by separate hybridiza-tions between two parental species PLoS Genet 12e1006404

Schumer M Rosenthal GG Andolfatto P 2014 How common is homo-ploid hybrid speciation Evolution 681553ndash1560

Schwarzbach AE Donovan LA Rieseberg LH 2001 Transgressivecharacter expression in a hybrid sunflower species Am J Bot88270ndash277

Selmecki AM Maruvka YE Richmond PA Guillet M Shoresh NSorenson AL De S Kishony R Michor F Dowell R et al 2015Polyploidy can drive rapid adaptation in yeast Nature 519349ndash52

Seshan VE Olshen A 2016 DNAcopy DNA copy number data analysisVersion R package version 1460

Soltis DE Visger CJ Soltis PS 2014 The polyploidy revolution then andnow Stebbins revisited Am J Bot 1011057ndash1078

Soltis PS 2013 Hybridization speciation and novelty J Evol Biol26291ndash293

Soltis PS Soltis DE 2009 The role of hybridization in plant speciationAnnu Rev Plant Biol 60561ndash588

Sunshine AB Payen C Ong GT Liachko I Tan KM Dunham MJ 2015The fitness consequences of aneuploidy are driven by condition-dependent gene effects PLoS Biol 13e1002155

Tamai Y Momma T Yoshimoto H Kaneko Y 1998 Co-existence of twotypes of chromosome in the bottom fermenting yeastSaccharomyces pastorianus Yeast 14923ndash933

Taylor DJ Hebert PDN 1993 Habitat-dependent hybrid parentage anddifferential introgression between neighboringly sympatric Daphniaspecies Proc Natl Acad Sci U S A 907079ndash7083

Thiagalingam S Laken S Willson JK Markowitz SD Kinzler KWVogelstein B Lengauer C 2001 Mechanisms underlying losses ofheterozygosity in human colorectal cancers Proc Natl Acad Sci US A 982698ndash2702

Tuna M Knuutila S Mills GB 2009 Uniparental disomy in cancer TrendsMol Med 15120ndash128

Walther A Hesselbart A Wendland J 2014 Genome sequence ofSaccharomyces carlsbergensis the worldrsquos first pure culture lageryeast G3 (Bethesda) 4783ndash793

Wayne RK 1993 Molecular evolution of the dog family Trends Genet9218ndash224

Wenger JW Schwartz K Sherlock G 2010 Bulk segregant analysis byhigh-throughput sequencing reveals a novel xylose utilization genefrom Saccharomyces cerevisiae PLoS Genet 6e1000942

Wessinger CA Rausher MD 2014 Predictability and irreversibility ofgenetic changes associated with flower color evolution inPenstemon barbatus Evolution 681058ndash1070

Wolfe KH 2015 Origin of the yeast whole-genome duplication PLoSBiol 13e1002221

Woods R Schneider D Winkworth CL Riley MA Lenski RE 2006 Testsof parallel molecular evolution in a long-term experiment withEscherichia coli Proc Natl Acad Sci U S A 1039107ndash9112

Wykoff DD OrsquoShea EK 2001 Phosphate transport and sensing inSaccharomyces cerevisiae Genetics 1591491ndash1499

Xiong Z Gaeta RT Pires JC 2011 Homoeologous shuffling andchromosome compensation maintain genome balance inresynthesized allopolyploid Brassica napus Proc Natl Acad SciU S A 1087908ndash7913

Yassour M Kapian T Fraser HB Levin JZ Pfiffner J Adiconis X Schroth GLuo SJ Khrebtukova I Gnirke A et al 2009 Ab initio construction ofa eukaryotic transcriptome by massively parallel mRNA sequencingProc Natl Acad Sci U S A 1063264ndash3269

Zhu YO Sherlock G Petrov DA 2016 Whole genome analysis of 132clinical Saccharomyces cerevisiae strains reveals extensive ploidy var-iation G3 (Bethesda) doi101534g3116029397

Adaptation in Hybrids doi101093molbevmsx098 MBE

17

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Page 9: Loss of Heterozygosity Drives Adaptation in Hybrid Yeastdunham.gs.washington.edu/SmukowskiHeil.pdf · 2017-05-20 · Loss of Heterozygosity Drives Adaptation in Hybrid Yeast Caiti

right arm of chromosome 14 an event seen previously inother evolved populations (Dunham et al 2002 Greshamet al 2008 Sunshine et al 2015) All copy number events in

S cerevisiae had break points at repetitive elements knownas Ty elements except those located on chrII which maybe mediated by another mechanism (Brewer et al 2015) In

FIG 2 Repeated loss of heterozygosity at the PHO84 locus in intra- and interspecific hybrids (A) The 25-kb region extending from the left telomereof chromosome XIII to the high-affinity phosphate transporter gene PHO84 (B) Copy number is plotted across part of chromosome XIII in thehybrid ancestor and three evolved hybrid clones in phosphate limitation (clone indicated in upper right corner) Red shows the S cerevisiae alleleblue shows the S uvarum allele and purple shows where both species exhibit the same copy number Note 8 kb of telomere sequence is removeddue to repetitive sequence (C) Alternate allele frequency is plotted for a portion of chromosome XIII in the ancestor and four evolved S cerevisiaeclones in phosphate limitation (clone indicated in upper right corner) All evolved S cerevisiae clones exhibit a loss of heterozygosity at thetelomeric portion of chromosome XIII (loss of S288C amplification of GRF167) as illustrated by an allele frequency of zero compared with theancestor Regions of heterozygosity are interspersed with regions of homozygosity as one of the parents of the diploid was itself the product of across between strains FL100 and S288C and the other parent was S288C Regions of heterozygosity are due to FL100 haplotypes S cerevisiae copynumber for the four evolved clones is shown below the ancestor is diploid across the chromosome (also see table 2 supplementary fig S1Supplementary Material online)

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9

contrast copy number variants in the hybrid were rarely facil-itated by repetitive elements perhaps in part because S uva-rum has no full length Ty elements however why S cerevisiaeTy elements and long terminal repeat (LTR) sequences fromeither genome were not utilized remains unknown

Frequently in nutrient-limited evolution experimentscopy number variants involve amplification of the nutrient-specific transporter and indeed we also observed amplifica-tion of these transporters in many of the clones In sulfatelimitation the S cerevisiae allele of the high-affinity sulfatetransporter gene SUL1 is amplified in 77 hybrid clones and 44 S cerevisiae clones (fig 1 tables 1 and 2 supplementary figsS1 and S3 Supplementary Material online) InterestinglySUL2 is the preferred sulfate transporter in S uvarum(Sanchez et al 2017) and was not observed to be amplifiedin the evolved hybrids (supplementary fig S2 and table S2Supplementary Material online) In glucose limitation previ-ous S cerevisiae evolution experiments found frequent am-plification of the high-affinity glucose transporter genesHXT67 (Brown et al 1998 Dunham et al 2002 Greshamet al 2008 Kao and Sherlock 2008 Kvitek and Sherlock2011) In our experiments the S uvarum alleles of theHXT67 transporters are amplified in 33 hybrid clones andboth S uvarum clones but are not amplified in evolved Scerevisiae clones This is suggestive that the S uvarum HXT67alleles confer a greater fitness advantage compared with Scerevisiae alternatively the genomic context could be morepermissive to amplification in S uvarum (fig 1 tables 1 and 2supplementary figs S1ndashS3 Supplementary Material online)Finally in phosphate limitation the S cerevisiae copy of thehigh-affinity phosphate transporter gene PHO84 is amplifiedand the S uvarum allele is lost in 36 hybrid clones in an eventknown as LOH (figs 1 and 2 table 1 supplementary fig 3Supplementary Material online) Intriguingly the evolved Scerevisiae clones also display LOH and accompanied amplifi-cation favoring the allele derived from strain GRF167 over theS288C allele in 44 clones (fig 2 table 2) All hybrid clonescarry the ldquopreferredrdquo GRF167 S cerevisiae allele as this was theallele used to create the de novo hybrid

Loss of Heterozygosity Is a Common Event inHeterozygous Evolving PopulationsSelection on heterozygosity as a LOH event could representis an underappreciated source of adaptation in microbial ex-perimental evolution as typical experiments evolve a haploidor homozygous diploid strain asexually and as a result havelittle variation to select upon LOH is observed in natural andindustrial hybrids (Albertin and Marullo 2012 Louis et al2012 Pryszcz et al 2014 Wolfe 2015 Schroder et al 2016)but here we document its occurrence in both intra- andinterspecific hybrids in the laboratory as a result of short-termevolution (also see (Dunn et al 2013 Burke et al 2014)) LOHis observed across all nutrient conditions with 13 independentLOH events detected in S cerevisiae and 9 independent eventsdocumented in the hybrids (figs 1 and 2 tables 1 and 2 sup-plementary figs S1 and S3 Supplementary Material online) Itthus appears that this type of mutational event is both com-mon and can occur over short evolutionary timescales

The LOH event can result in copy-neutral (where one alleleis lost and the other allele is amplified) or non-neutral chro-mosomal segments (where one allele is lost rendering thestrain hemizygous at that locus) and can favor the retentionof either allele In S cerevisiae there is a bias in resolutionwhere LOH events favor retaining the GRF167 allele over theS288C allele (1013 events Pfrac14 00169 table 2 supplementaryfig S4 Supplementary Material online) One unique case inclone Sc4 has a small approximately 5 kb LOH event onchrXV favoring GRF167 which switches to favoring S288Cfor the rest of the chromosome The retention of S cerevisiaeis slightly more common in the hybrids (59 events Pfrac14 10table 1 supplementary fig S3 Supplementary Material on-line) though not as drastic as the observed genome resolu-tion in the hybrid S pastorianus where LOH favorsS cerevisiae over S eubayanus (Nakao et al 2009) The sizeof the event ranges from approximately 25 kb to the wholechromosome level in the hybrids and from 5 kb to 540 kbin S cerevisiae Where LOH is accompanied by an amplifi-cation event the LOH event always occurs first and doesnot share break points with the amplification event Unlikemany CNV events most LOH events do not appear to bemediated by existing repetitive sequence such as a trans-posable element in the hybrid or S cerevisiae and are mostlikely a product of break-induced replication or mitoticgene conversion with break-induced replication as the fa-vored method as all events extend to the telomere (Hoanget al 2010) The exceptions are in hybrid clones Ph4 Ph5 andSh1 where there is a non-copy neutral loss of S cerevisiaemediated by a Ty element or LTR and S cerevisiae clonesSc1 and Sc4 where there is a 65-kb deletion of the S288Callele flanked by two Ty elements

LOH events in hybrids could signify several ongoing pro-cesses in hybrid genome evolution LOH regions may repre-sent 1) loci with incompatibilities 2) selection on existingvariation or 3) genetic drift eroding genomic segments It isimpossible to definitively rule out any of these hypotheseswithout further experimentation however there are severalarguments disfavoring the incompatibility hypothesis Firstalthough our sample size is modest failing to see repeatedLOH events across nutrient conditions may indicate a lack ofgeneral incompatibility between species (although we cannotpreclude condition-specific incompatibility Hou et al 2015Piatkowska et al 2013) This is consistent with previous stud-ies in yeast which suggest that classic DobzhanksyndashMullerincompatibilities are rare (Liti et al 2006 Greig 2009 Houet al 2014) Furthermore LOH events observed inevolved S cerevisiae suggest that this mutation type isnot unique to interspecific hybrids Instead repeatedevents within a particular condition such as the repeatedLOH at PHO84 in phosphate limitation or the 65 kbsegment on chrXIII in sulfate limitation suggest thatthese events are beneficial and are indeed selection onone allele over the other

LOH Is Driven by Selection on One AlleleTo test the hypothesis that LOH events provide a selectiveadvantage we used allele replacement in which the allele of

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10

one speciesstrain is replaced with the allele of the otherspeciesstrain in an otherwise isogenic background We testedthis hypothesis using the most commonly seen LOH eventLOH at PHO84 Although the region extends from 25 kb to234 kb in length in the hybrids and from 40 kb to 85 kb in Scerevisiae and thus includes many genes PHO84 was a primecandidate driving this event PHO84 is one of only ten genesencompassed in the region extending from the telomere tothe break point of the shortest LOH event and is included inevery other LOH event on chromosome XIII (fig 2) It is ahigh-affinity phosphate transporter responsible for inorganicphosphate uptake in high and low phosphate conditions(Wykoff and OrsquoShea 2001) and previous work identified apoint mutation in PHO84 (an alanine to valine substitution atthe 50 end of the gene) which increased fitness by 23 inphosphate-limited conditions (Sunshine et al 2015) Finallyprior work with other nutrient transporters has shown am-plification of nutrient transporters to be a key event in adapt-ing to nutrient-limited conditions

We thus selected a region of approximately 25 kb encom-passing the PHO84 ORF its promoter and 30-UTR(Nagalakshmi et al 2008 Yassour et al 2009 Cherry et al2012) We created allele replacement strains using the twoalleles of S cerevisiae in a S cerevisiae diploid background thetwo alleles are 991 identical in this region (supplementaryfig S5 Supplementary Material online) and each strain isidentical to the ancestral strain used in our evolution exper-iments except at the PHO84 locus The S cerevisiae ancestorcarries one copy of GRF167 (preferred) and one copy ofS288C (ldquoun-preferredrdquo) so named due to which allele wasretained and amplified in the evolved clones To measureany resultant changes in fitness we competed each strainindividually against a fluorescent ancestral strain and mea-sured which strain overtook the culture Relative fitness isthus defined as the growth advantage per generation Twocopies of the un-preferred allele decreased relative fitness by531 (6186) whereas two copies of the preferred alleleincreased relative fitness by 993 (6027) This displays anoverall difference in relative fitness of 1524 between the un-preferred and preferred alleles By comparing the fitness ofthese allele replacement strains with the evolved clones (table 2)the allele replacement does not fully recapitulate the fit-ness gain observed in the evolved clone One explanation isthat the additional mutations present in the evolvedstrains also contribute to their total fitness Another expla-nation could be the increased copy number of the PHO84region that we see in these evolved clones To further ex-plore this fitness difference we cloned the GRF167 alleleonto a low copy number plasmid and transformed theallele replacement strain carrying two preferred S cerevi-siae alleles to simulate increased copy number of PHO84and saw only a minimal fitness increase of 176 (6106note all fitness competitions involving plasmids were donein conjunction with empty plasmids to take into accountany fitness effects from the plasmid itself) This supportsthe conclusion that relative fitness gains in the evolvedclone are largely due to the loss of the S288C allele andselection and amplification of the GRF167 allele with little

additional benefit from further amplification It could also bethe case that co-amplification of other genes in the segmentis required to attain the full benefit as previously observedby the contribution of BSD2 to the SUL1 amplicon in sulfatelimitation (Sunshine et al 2015 Payen et al 2016)

To understand the fitness effects of LOH and amplificationin the hybrids we generated hybrid strains with varying num-bers of S cerevisiae GRF167 PHO84 alleles Unfortunately wewere unable to obtain a successful strain carrying the pre-ferred S cerevisiae allele in a S uvarum background howeverwe were able to generate a S uvarum PHO84 knockout strainthus creating a hybrid with one copy of S cerevisiae PHO84When combined with a low copy plasmid carrying the sameallele this strain effectively has two or more copies ofS cerevisiae PHO84 in a hybrid background This hybrid straincan serve as a proxy for the LOH event observed in the evo-lution experiments and has a relative fitness gain of 2557(6288) The ancestral hybrid with the same plasmid (effec-tively 1 S uvarum allele and 2 or more S cerevisiae alleles) hasa relative fitness gain of 1253 (6131) The difference be-tween these two hybrids suggests that while amplification isbeneficial the highest fitness is achieved with the loss of theS uvarum allele (Pfrac14 00061)

Together these results support the conclusion that theS cerevisiae GRF167 allele is preferred over the S288C alleleand that S cerevisiae alleles are preferred over the S uvarumallele in the hybrid and hence that the LOH events seen inboth intra- and interspecific hybrids are the product ofselection

DiscussionIn summary we sought to understand forces underlying ge-nome stabilization and evolution in interspecific and intra-specific hybrids as they adapt to novel environments Weevolved and sequenced clones from 16 hybrid populationsand 16 parental populations to reveal a variety of mutationalevents conferring adaptation to 3 nutrient-limited conditionsOf particular note we find LOH in both evolved intraspecificand interspecific hybrid clones in all nutrient environmentspotentially signifying areas where selection has acted on pre-existing variation present in the ancestral clone We used anallele replacement strategy to test this hypothesis for a com-monly repeated LOH event and show that selection is indeeddriving the homogenization of the genome at this locusAlthough other studies in natural industrial and lab-evolved isolates have observed LOH we present the first em-pirical test of the causal evolutionary forces influencing theseevents This work can begin to help us understand past hy-bridization events and subsequent genome resolution in hy-brids in natural and artificial systems

Similarities and Differences between Intra- andInterspecific HybridsAlthough our sample size is modest we observe several in-teresting trends when comparing S uvarum clones (homo-zygous) S cerevisiae clones (intraspecific hybrid andpreviously published homozygous) and interspecific hybrid

Adaptation in Hybrids doi101093molbevmsx098 MBE

11

clones First theory and previous research predict the inter-specific hybrid may experience more genome instability in theform of chromosomal rearrangements and CNV events(Xiong et al 2011 Lloyd et al 2014 Chester et al 2015Mason and Batley 2015) Instead in our work the interspecifichybrid and S cerevisiae clones experience the same numberof CNV events (both 15 CNVsclone) However the mech-anism of CNV formation seems to differ between the hybridand S cerevisiae clones Whereas S cerevisiae CNV breakpoints typically occur at transposable elements in our studyand previous studies (Dunham et al 2002 Gresham et al2008 Fedoroff 2012) the interspecific hybrid CNVs do notutilize transposable elements although they obviously sharethe same sequence background as the S cerevisiae clones(albeit in one copy) Whether this difference is due to theabsence of full-length transposable elements in S uvarum isunknown but this could potentially explain the lower num-ber of CNV events in S uvarum clones (1 CNVclone) andpresents an intriguing direction for future study

The Predictability of EvolutionWe now have many examples of predictable evolution innatural systems (Losos et al 1998 Rundle et al 2000 Elmerand Meyer 2011 Conte et al 2012 Jones et al 2012 Martinand Orgogozo 2013 Wessinger and Rausher 2014) and inlaboratory experimental evolution in which there often ap-pears to be a limited number of high fitness pathways thatstrains follow when adapting to a particular condition (Fereaet al 1999 Woods et al 2006 Gresham et al 2008 Burke et al2010 Salverda et al 2011 Kawecki et al 2012 Kvitek andSherlock 2013 Lang and Desai 2014) For example it is wellestablished that amplifications of nutrient transporters aredrivers of adaptation in evolution in nutrient-limited condi-tions Previous work in our group has particularly focused onthe amplification of the high-affinity sulfate transporter geneSUL1 in sulfate-limited conditions which occurs in almostevery sulfate-limited evolution experiment and confers a fit-ness advantage of as much as 40 compared with the un-evolved ancestor strain The amplification of phosphatetransporters has been markedly less common and thus driv-ers of adaptation in this condition have been less clearGresham et al (2008) identified a whole chromosome ampli-fication of chrXIII in one population In a follow-up studySunshine et al (2015) found whole or partial amplification ofchrXIII in 38 populations A genome-wide screen for segmen-tal amplifications found a slight increase in fitness for a smalltelomeric segment of chromosome XIII and a A49V pointmutation in PHO84 was observed to increase fitness by 23However screens by Payen et al (2016) showed that althoughPHO84 is recurrently mutated in various experiments itshowed no benefit when amplified or deleted in phos-phate-limited conditions Finally additional evolution exper-iments recapitulated the point mutation seen in Sunshineet al (2015) in 2432 populations and saw amplification ofPHO84 in 832 populations (Miller A Dunham MJ unpub-lished data) It is important to note that all these experimentsused a strain background derived from S288C or CENPKboth of which carry the same (un-preferred) PHO84 allele

In our work we observed amplification of the S cerevisiaeGRF167 allele of PHO84 in 44 S cerevisiae clones from 4populations and 36 hybrid clones from 6 populations Thisamplification was always preceded by the loss of the S288Callele in S cerevisiae clones and the LOH break points arenever shared with the amplification break points There is a15 fitness difference between carrying two copies of theS288C allele of PHO84 compared with carrying two copiesof the GRF167 allele of PHO84 and additional copies of theGRF167 allele do not provide substantial further fitness gainsThe two alleles differ by several noncoding changes and threenonsynonymous substitutions a mutation from glutamicacid to aspartic acid (E229D) leucine to proline (L259P)and leucine to glutamine (L556Q) the latter two of whichare considered ldquononconservativerdquo protein mutations due tochanges in hydrophobicity and structure (supplementary figS5 Supplementary Material online) Intriguingly the L259Pmutation has actually been previously identified as being re-sponsible for resistance to tetrachloroisophthalonitrile andpartial resistance to pentachlorophenol in a QTL study ofsmall-molecule drugs (Perlstein et al 2007) IndeedPerlstein et al found proline at residue 259 to be conservedacross fungal species and even in orthologous human xeno-biotic transporters likely because proline-induced kinks intransmembrane spans have been shown to be essential toprotein function (Cordes et al 2002 Perlstein et al 2007) Itthus appears that amplification of PHO84 has been less pre-dictable as the S288C allele does not confer a fitness advan-tage unless mutated per Sunshine et al (2015) Togetherthese results imply that strain background can constrainadaptive pathways

In hybrids the amplification of the S cerevisiae segmentoccurred in conjunction with the loss of the S uvarum alleleHybrid strains with the LOH had a 2557 relative fitness gainwhereas hybrid strains with amplification of the S cerevisiaePHO84 allele without the LOH had a 1253 fitness gain ThusLOH confers an additional 1304 fitness gain showing thatselection for LOH has a larger impact on fitness than ampli-fication alone Note that S uvarum does have proline at res-idue 259 like the preferred GRF167 allele and differs fromboth S cerevisiae alleles at the other two coding substitutions(229N and 556K) but the amino acid and noncoding diver-gence is too high to speculate what substitutions are respon-sible for the selection of the GRF167 allele (supplementary figS6 Supplementary Material online) Why the loss of one alleleis more beneficial remains unclear as PHO84 is thought tofunction as a monomer (Bun-Ya et al 1991) but it may bedue to competition for cell wall space or negative interactionswith other genes in the PHO pathway (Mouillon and Persson2006)

The infusion of variation created by hybridization providesnew templates for selection to act upon which can be moreimportant than either point mutations or copy number var-iants alone Our work shows that outcrossing need not becommon to have long-lasting effects on adaptation This im-plication is particularly relevant in yeast where outcrossingmay occur quite rarely followed by thousands of asexual gen-erations (Ruderfer et al 2006 Greig and Leu 2009 Liti 2015)

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Applications to Other Hybrids and CancerThe observation that LOH occurs in hybrid genomes is in-creasingly documented (Louis et al 2012 Borneman et al2014 Soltis et al 2014 Marcet-Houben and Gabaldon 2015Pryszcz et al 2014 Schroder et al 2016) although the rea-son(s) for this type of mutation has been unresolved As mostexamples stem from allopolyploid events that occurred mil-lions of years ago understanding why LOH is important inhybrid genome evolution is difficult Cancer cells are alsoknown to experience LOH sometimes involved in the inac-tivation of tumor suppressor genes leaving only one copy ofthe gene that may be mutated or silenced (Thiagalingam et al2001 Tuna et al 2009 Lapunzina and Monk 2011) Datasupport the conclusion that LOH events are selected for dur-ing tumor development as many LOH events involve specificchromosomal segments (Thiagalingam et al 2001) althoughthe underlying molecular and genetic reasons for selection isan open debate (Ryland et al 2015)

Here we experimentally demonstrate that LOH can occurin homoploid interspecific hybrids as well as in intraspecifichybrids These events occur within a few hundred generationsand are common mutations more common on average inthe intraspecific hybrid (13 eventsclone) than the interspe-cific hybrid (056 eventsclone) Interestingly the LOH eventsdo not share break points with the CNV events in S cerevisiaeinstead they appear to occur independently and to precedeany subsequent amplification (amplification occurs following913 LOH events) Competition assays with the PHO84 locusprovide support that LOH itself may be more beneficial thanamplification or at least increase the selective benefit of am-plification events The observation that LOH in intraspecifichybrids occurs independently from copy number change pro-vides different opportunities for adaptation to novelconditions

Other cases of LOH like the copy number neutral eventsobserved in Ph4 Ph5 and Sh4 all of which favor the retentionof the S uvarum allele (supplementary fig S3 SupplementaryMaterial online) may be due to hybrid incompatibility withina particular protein complex other epistatic interactions(Piatkowska et al 2013) or neutral processes We furthermorediscover examples where one species allele appears to bepreferred over the other without LOH such as the repeatedamplification of the S uvarum high-affinity glucose transpor-ters HXT67 When one species allele is amplified and theother is not amplified one explanation is that the local se-quence context can permit or deny amplification In the caseof HXT67 previous experiments in S cerevisiae have shownthat amplification of HXT67 is quite common (Brown et al1998 Dunham et al 2002 Gresham et al 2008 Kao andSherlock 2008 Kvitek and Sherlock 2011) thus suggestingthat when given a choice between this locus in S cerevisiaeor S uvarum in the hybrid the preferred allele is indeed Suvarum though more subtle differences in rate cannot yet beruled out A similar scenario is observed with S cerevisiaeSUL1 (Sanchez et al 2017) Together our results show thatthe heterozygosity supplied by hybridization is an importantcontributor to adaptive routes explored by populations asthey adapt to novel conditions

Although we cannot generalize our results from thePHO84 locus across the many other LOH events discoveredin our hybrids and S cerevisiae in the future we can usesimilar methodology to explore whether positive selectionalways drives LOH or whether other explanations such asincompatibility resolution contribute as well Future experi-ments might also utilize a high throughput method to ex-plore segmental LOH in hybrids at a genome-wide scalesimilar to ongoing experiments at the gene level (LancasterS Dunham MJ unpublished data) Although our sample sizeis moderate this is a novel and necessary step in understand-ing forces underlying hybrid genome stabilization and high-lighting an underappreciated mechanism of hybridadaptation

ConclusionsThe mutation events we observe in our experimentallyevolved hybrids are in many ways quite representative ofmutations observed in ancient hybrid genomes suggestingthat hybrid genome stabilization and adaptation can occurquite rapidly (within several hundred generations)Furthermore our results illustrate that the infusion of varia-tion introduced by hybridization at both the intra- and inter-species level can increase fitness by providing choices of allelesfor selection to act upon even when sexual reproduction israre This may be particularly important for leveraging existingvariation for agricultural and industrial processes and as cli-mate change potentially increases natural hybridization (Kellyet al 2010 Hoffmann and Sgro 2011 Muhlfeld et al 2014)

Materials and Methods

StrainsA list of strains used in this study is included in supplementarytable S1 Supplementary Material online All interspecific hy-brids were created by crossing a ura3 LYS2 haploid parent to aURA3 lys2 haploid parent of the other mating type plating onmedia lacking both uracil and lysine and selecting for proto-trophs The S cerevisiae strain background known asldquoGRF167rdquo is itself a cross between FL100 and the genomictype strain S288C (data not shown) GRF167 was chosen as astrain background for simultaneous work investigating trans-posable elements during experimental evolution which willbe addressed in a future study

Evolution ExperimentsContinuous cultures were established using media and con-ditions previously described (Gresham et al 2008 Sanchezet al 2017) Detailed protocols and media recipes are availableat httpdunhamgswashingtoneduprotocolsshtml (lastaccessed March 6 2017) Samples were taken daily and mea-sured for optical density at 600 nm and cell count micros-copy was performed to check for contamination andarchival glycerol stocks were made daily An experimentwas terminated when contamination growth in tubing orclumping appeared (number of generations at the endpoint for each population are presented in tables 1 and

Adaptation in Hybrids doi101093molbevmsx098 MBE

13

2) Samples from each end point population were colonypurified to yield two clones for further study

Array Comparative Genomic HybridizationPopulations from the end point of each evolution were ana-lyzed for copy number changes using array comparative ge-nomic hybridization following the protocol used in Sanchezet al (2017)

SequencingDNA was extracted from overnight cultures using theHoffmanndashWinston protocol (Hoffman and Winston 1987)and cleaned using the Clean amp Concentrator kit (ZymoResearch) Nextera libraries were prepared following theNextera library kit protocol and sequenced using paired end150 bp reads on the Illumina NextSeq 500 machine (sequenc-ing coverage in supplementary table S2 SupplementaryMaterial online) The reference genomes used were S cerevi-siae v3 (Engel et al 2014) S uvarum (Scannell et al 2011) anda hybrid reference genome created by concatenating the twogenomes Sequence was aligned to the appropriate referencegenome using bwa v062 (Li and Durbin 2009) and mutationswere called using GATK (McKenna et al 2010) and samtools0119 (Li et al 2009) Mutations in evolved clones were filteredin comparison with the ancestor to obtain de novo mutationsAll mutations were first visually inspected using IntegrativeGenomics Viewer (Robinson et al 2011) Subsequently pointmutations in the hybrids were confirmed with Sanger se-quencing (supplementary table S3 Supplementary Materialonline) Copy number variants were visualized usingDNAcopy for S cerevisiae and S uvarum (Seshan andOlshen 2016) LOH events were called based on sequencingcoverage in the hybrids and by identifying homozygous variantcalls in S cerevisiae All break points were called by visual in-spection of sequencing reads and are thus approximate

Fitness AssaysThe pairwise competition experiments were performed in 20ml chemostats (Miller and Dunham 2013) Each competitorstrain was cultured individually until steady state was reachedand then was mixed 5050 with a GFP-tagged ancestor Eachcompetition was conducted in at least two biological repli-cates for approximately 15 generations after mixing Sampleswere collected and analyzed twice daily The proportion ofGFPthorn cells in the population was detected using a BD AccuriC6 flow cytometer (BD Biosciences) The data were plottedwith ln [dark cellsGFPthorn cells] versus generations The rela-tive fitness coefficient was determined from the slope of thelinear region

Strain ConstructionAllele replacements for the PHO84 locus were done followingthe protocol of the Caudy lab with further modifications de-scribed here The native locus was replaced withKluyveromyces lactis URA3 The pho84DURA3 strain wasgrown overnight in 5 ml of C-URA media then inoculatedin a flask of 100 ml yeast extract peptone dextrose (YPD) andgrown to an optical density of 06ndash08 Cells were washed then

aliquoted 275 ml of transformation mix (35 ml 1 M lithiumacetate 240 ml of 50 3500 polyethylene glycol) 10 ml ofsalmon sperm and approximately 3 mg of polymerase chainreaction product were added to the cell pellet It was incu-bated at 37 C (S uvarum) or 42 C (S cerevisiae) for 45 minthen plated to YPD It was replica plated to 5-fluorooroticacid the following day and colonies were tested for the gain ofthe appropriate species allele The GRF167 allele was clonedinto the pIL37 plasmid using Gibson assembly (Gibson et al2009) Correct assembly was verified by Sanger sequencing Allprimers used can be found in supplementary table S3Supplementary Material online

Supplementary MaterialSupplementary data are available at Molecular Biology andEvolution online

AcknowledgmentsWe thank Noah Hanson and Erica Alcantara for technicalassistance We thank Monica Sanchez and three anonymousreviewers for helpful comments on this manuscript JosephSchacherer kindly confirmed the contribution of FL100 to theGRF167 strain background Thanks to Yixian Zheng and DougKoshland for contributing to the initial experimental designcreating yeast strains and purchasing the oligonucleotidesused for the microarrays This work was supported by theNational Science Foundation (grant number 1516330) andthe National Institutes of Health (grant number R01GM094306) MD is a Senior Fellow in the GeneticNetworks program at the Canadian Institute for AdvancedResearch and a Rita Allen Foundation Scholar CSH wassupported in part by National Institutes of Health (grantnumber T32 HG00035) This work was also supported bythe National Institutes of Health (grant number P50GM071508) to the Lewis-Sigler Institute and from theHoward Hughes Medical Institute to Doug Koshland andYixian Zheng Sequencing read data have been deposited atthe NCBI under BioProject accession PRJNA374049Microarray data are deposited in the Gene ExpressionOmnibus (GEO) repository under accession numbersGSE95086 and GSE87401 and in the Princeton MicroarrayDatabase

ReferencesAbbott R Albach D Ansell S Arntzen JW Baird SJ Bierne N Boughman J

Brelsford A Buerkle CA Buggs R et al 2013 Hybridization andspeciation J Evol Biol 26229ndash246

Ainouche ML Jenczewski E 2010 Focus on polyploidy New Phytol1861ndash4

Albertin W Marullo P 2012 Polyploidy in fungi evolution after whole-genome duplication Proc Biol Sci 2792497ndash2509

Almeida P Goncalves C Teixeira S Libkind D Bontrager M Masneuf-Pomarede I Albertin W Durrens P Sherman DJ Marullo P et al2014 A Gondwanan imprint on global diversity and domesticationof wine and cider yeast Saccharomyces uvarum Nat Commun54044

Antunovics Z Nguyen HV Gaillardin C Sipiczki M 2005 Gradual ge-nome stabilisation by progressive reduction of the Saccharomyces

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uvarum genome in an interspecific hybrid with Saccharomyces cer-evisiae FEMS Yeast Res 51141ndash1150

Baker E Wang B Bellora N Peris D Hulfachor AB Koshalek JA AdamsM Libkind D Hittinger CT 2015 The Genome sequence ofSaccharomyces eubayanus and the domestication of lager-brewingyeasts Mol Biol Evol 322818ndash2831

Barbosa R Almeida P Safar SVB Santos RO Morais PB Nielly-Thibault LLeducq JB Landry CR Goncalves P Rosa CA Sampaio JP 2016Evidence of natural hybridization in Brazilian wild lineages ofSaccharomyces cerevisiae Genome Biol Evol 8317ndash329

Barton NH 2001 The role of hybridization in evolution Mol Ecol10551ndash568

Belloch C Orlic S Barrio E Querol A 2008 Fermentative stress adapta-tion of hybrids within the Saccharomyces sensu stricto complex Int JFood Microbiol 122188ndash195

Bellon JR Yang F Day MP Inglis DL Chambers PJ 2015 Designingand creating Saccharomyces interspecific hybrids for improvedindustry relevant phenotypes Appl Microbiol Biotechnol998597ndash8609

Borneman AR Zeppel R Chambers PJ Curtin CD 2014 Insights into theDekkera bruxellensis genomic landscape comparative genomics re-veals variations in ploidy and nutrient utilisation potential amongstwine isolates PLoS Genet 10e1004161

Brewer BJ Payen C Di Rienzi SC Higgins MM Ong G Dunham MJRaghuraman MK 2015 Origin-dependent inverted-repeat amplifi-cation tests of a model for inverted DNA amplification PLoS Genet11e1005699

Brown CJ Todd KM Rosenzweig RF 1998 Multiple duplications of yeasthexose transport genes in response to selection in a glucose-limitedenvironment Mol Biol Evol 15931ndash942

Bullini L 1994 Origin and evolution of animal hybrid species Trends EcolEvol 9422ndash426

Bun-Ya M Nishimura M Harashima S Oshima Y 1991 The PHO84 geneof Saccharomyces cerevisiae encodes an inorganic phosphate trans-porter Mol Cell Biol 113229ndash3238

Burke MK Dunham JP Shahrestani P Thornton KR Rose MR Long AD2010 Genome-wide analysis of a long-term evolution experimentwith Drosophila Nature 467587ndash590

Burke MK Liti G Long AD 2014 Standing genetic variation drives re-peatable experimental evolution in outcrossing populations ofSaccharomyces cerevisiae Mol Biol Evol 313228ndash3239

Cherry JM Hong EL Amundsen C Balakrishnan R Binkley G Chan ETChristie KR Costanzo MC Dwight SS Engel SR et al 2012Saccharomyces genome database the genomics resource of buddingyeast Nucleic Acids Res 40D700ndashD705

Chester M Riley RK Soltis PS Soltis DE 2015 Patterns of chromosomalvariation in natural populations of the neoallotetraploid Tragopogonmirus (Asteraceae) Heredity (Edinb) 114309ndash317

Chou JY Leu JY 2010 Speciation through cytonuclear incompatibilityinsights from yeast and implications for higher eukaryotes Bioessays32401ndash411

Cliften PF Fulton RS Wilson RK Johnston M 2006 After the duplicationgene loss and adaptation in Saccharomyces genomes Genetics172863ndash872

Conte GL Arnegard ME Peichel CL Schluter D 2012 The probability ofgenetic parallelism and convergence in natural populations Proc BiolSci 2795039ndash5047

Cordes FS Bright JN Sansom MS 2002 Proline-induced distortions oftransmembrane helices J Mol Biol 323951ndash960

Crow JF 1998 90 years ago the beginning of hybrid maize Genetics148923ndash928

Dasmahapatra KK Walters JR Briscoe AD Davey JW Whibley A NadeauNJ Zimin AV Hughes DST Ferguson LC Martin SH et al 2012Butterfly genome reveals promiscuous exchange of mimicry adap-tations among species Nature 48794ndash98

Dowling TE Smith GR Brown WM 1989 Reproductive isolation andintrogression between Notropis-Cornutus and Notropis-Chrysocephalus (Family Cyprinidae)mdashcomparison of morphologyallozymes and mitochondrial-DNA Evolution 43620ndash634

Doyle JJ Flagel LE Paterson AH Rapp RA Soltis DE Soltis PS Wendel JF2008 Evolutionary genetics of genome merger and doubling inplants Annu Rev Genet 42443ndash461

Dunham MJ Badrane H Ferea T Adams J Brown PO Rosenzweig FBotstein D 2002 Characteristic genome rearrangements in experi-mental evolution of Saccharomyces cerevisiae Proc Natl Acad Sci U SA 9916144ndash16149

Dunn B Paulish T Stanbery A Piotrowski J Koniges G Kroll E LouisEJ Liti G Sherlock G Rosenzweig F 2013 Recurrent rearrange-ment during adaptive evolution in an interspecific yeast hybridsuggests a model for rapid introgression PLoS Genet9e1003366

Dunn B Richter C Kvitek DJ Pugh T Sherlock G 2012 Analysis of theSaccharomyces cerevisiae pan-genome reveals a pool of copy num-ber variants distributed in diverse yeast strains from differing indus-trial environments Genome Res 22908ndash924

Dunn B Sherlock G 2008 Reconstruction of the genome origins andevolution of the hybrid lager yeast Saccharomyces pastorianusGenome Res 181610ndash1623

Elmer KR Meyer A 2011 Adaptation in the age of ecological genomicsinsights from parallelism and convergence Trends Ecol Evol26298ndash306

Engel SR Dietrich FS Fisk DG Binkley G Balakrishnan R Costanzo MCDwight SS Hitz BC Karra K Nash RS et al 2014 The referencegenome sequence of Saccharomyces cerevisiae then and now G3(Bethesda) 4389ndash398

Fedoroff NV 2012 Presidential address Transposable elements epige-netics and genome evolution Science 338758ndash767

Ferea TL Botstein D Brown PO Rosenzweig RF 1999 Systematicchanges in gene expression patterns following adaptive evolutionin yeast Proc Natl Acad Sci U S A 969721ndash9726

Gibson B Liti G 2015 Saccharomyces pastorianus genomic insightsinspiring innovation for industry Yeast 3217ndash27

Gibson DG Young L Chuang RY Venter JC Hutchison CA Smith HO2009 Enzymatic assembly of DNA molecules up to several hundredkilobases Nat Methods 6343ndash5

Gonzalez SS Barrio E Gafner J Querol A 2006 Natural hybrids fromSaccharomyces cerevisiae Saccharomyces bayanus andSaccharomyces kudriavzevii in wine fermentations FEMS Yeast Res61221ndash1234

Gonzalez SS Barrio E Querol A 2008 Molecular characterization of newnatural hybrids of Saccharomyces cerevisiae and S kudriavzevii inbrewing Appl Environ Microbiol 742314ndash2320

Grant PR Grant BR 1994 Phenotypic and genetic consequences ofhybridization in Darwinrsquos finches Evolution 48297ndash316

Grant PR Grant BR 2002 Unpredictable evolution in a 30-year study ofDarwinrsquos finches Science 296707ndash711

Grant PR Grant BR 2010 Natural selection speciation and Darwinrsquosfinches Proc Calif Acad Sci 61245ndash260

Grant PR Grant BR Petren K 2005 Hybridization in the recent past AmNat 16656ndash67

Greig D 2009 Reproductive isolation in Saccharomyces Heredity (Edinb)10239ndash44

Greig D Leu JY 2009 Natural history of budding yeast Current Biol19R886ndashR890

Greig D Louis EJ Borts RH Travisano M 2002 Hybrid speciation inexperimental populations of yeast Science 2981773ndash1775

Gresham D Desai MM Tucker CM Jenq HT Pai DA Ward A DeSevoCG Botstein D Dunham MJ 2008 The repertoire and dynamics ofevolutionary adaptations to controlled nutrient-limited environ-ments in yeast PLoS Genet 4e1000303

Hajjar R Hodgkin T 2007 The use of wild relatives in crop improvementa survey of developments over the last 20 years Euphytica 1561ndash13

Heiser CB 1954 Variation and subspeciation in the common sunflowerHelianthus annuus Am Midl Nat 51287ndash305

Hittinger CT 2013 Saccharomyces diversity and evolution a buddingmodel genus Trends Genet 29309ndash317

Hoang ML Tan FJ Lai DC Celniker SE Hoskins RA Dunham MJ ZhengY Koshland D 2010 Competitive repair by naturally dispersed

Adaptation in Hybrids doi101093molbevmsx098 MBE

15

repetitive DNA during non-allelic homologous recombination PLoSGenet 6e1001228

Hoffmann AA Sgro CM 2011 Climate change and evolutionary adap-tation Nature 470479ndash485

Hoffman CS Winston F 1987 A 10-minute DNA preparation from yeastefficiently releases autonomous plasmids for transformation ofEscherichia coli Gene 57267ndash272

Hou J Friedrich A de Montigny J Schacherer J 2014 Chromosomalrearrangements as a major mechanism in the onset of reproductiveisolation in Saccharomyces cerevisiae Curr Biol 241153ndash1159

Hou J Friedrich A Gounot JS Schacherer J 2015 Comprehensive surveyof condition-specific reproductive isolation reveals genetic incom-patibility in yeast Nat Commun 67214

Huerta-Sanchez E Casey FP 2015 Archaic inheritance supporting high-altitude life in Tibet J Appl Physiol (1985) 1191129ndash1134

Jones FC Grabherr MG Chan YF Russell P Mauceli E Johnson JSwofford R Pirun M Zody MC White S et al 2012 The genomicbasis of adaptive evolution in threespine sticklebacks Nature48455ndash61

Kao KC Sherlock G 2008 Molecular characterization of clonal interfer-ence during adaptive evolution in asexual populations ofSaccharomyces cerevisiae Nat Genet 401499ndash1504

Kawecki TJ Lenski RE Ebert D Hollis B Olivieri I Whitlock MC 2012Experimental evolution Trends Ecol Evol 27547ndash560

Kellis M Patterson N Endrizzi M Birren B Lander ES 2003 Sequencingand comparison of yeast species to identify genes and regulatoryelements Nature 423241ndash254

Kelly B Whiteley A Tallmon D 2010 The Arctic melting pot Nature468891ndash891

Kvitek DJ Sherlock G 2011 Reciprocal sign epistasis between frequentlyexperimentally evolved adaptive mutations causes a rugged fitnesslandscape PLoS Genet 7e1002056

Kvitek DJ Sherlock G 2013 Whole genome whole population sequenc-ing reveals that loss of signaling networks is the major adaptivestrategy in a constant environment PLoS Genet 9e1003972

Landry CR Hartl DL Ranz JM 2007 Genome clashes in hybrids insightsfrom gene expression Heredity (Edinb) 99483ndash493

Lang GI Desai MM 2014 The spectrum of adaptive mutations in ex-perimental evolution Genomics 104412ndash416

Lapunzina P Monk D 2011 The consequences of uniparental disomyand copy number neutral loss-of-heterozygosity during human de-velopment and cancer Biol Cell 103303ndash317

Leducq JB Nielly-Thibault L Charron G Eberlein C Verta JP Samani PSylvester K Hittinger CT Bell G Landry CR 2016 Speciation drivenby hybridization and chromosomal plasticity in a wild yeast NatMicrobiol 115003

Lee HY Chou JY Cheong L Chang NH Yang SY Leu JY 2008Incompatibility of nuclear and mitochondrial genomes causes hy-brid sterility between two yeast species Cell 1351065ndash1073

Li H Durbin R 2009 Fast and accurate short read alignment withBurrows-Wheeler transform Bioinformatics 251754ndash1760

Li H Handsaker B Wysoker A Fennell T Ruan J Homer N Marth GAbecasis G Durbin R 2009 The Sequence AlignmentMap formatand SAMtools Bioinformatics 252078ndash2079

Liti G 2015 The fascinating and secret wild life of the budding yeast Scerevisiae Elife 4e05835

Liti G Barton DB Louis EJ 2006 Sequence diversity reproductiveisolation and species concepts in Saccharomyces Genetics174839ndash850

Lloyd AH Ranoux M Vautrin S Glover N Fourment J Charif D ChouletF Lassalle G Marande W Tran J et al 2014 Meiotic gene evolutioncan you teach a new dog new tricks Mol Biol Evol 311724ndash1727

Losos JB Jackman TR Larson A de Queiroz K Rodriguez-Schettino L1998 Contingency and determinism in replicated adaptive radia-tions of island lizards Science 2792115ndash2118

Louis VL Despons L Friedrich A Martin T Durrens P Casaregola SNeuveglise C Fairhead C Marck C Cruz JA et al 2012 Pichia sorbi-tophila an interspecies yeast hybrid reveals early steps of genomeresolution after polyploidization G3 (Bethesda) 2299ndash311

Mallet J 2007 Hybrid speciation Nature 446279ndash283Marcet-Houben M Gabaldon T 2015 Beyond the whole-genome du-

plication phylogenetic evidence for an ancient interspecies hybrid-ization in the bakerrsquos yeast lineage PLoS Biol 13e1002220

Marinoni G Manuel M Petersen RF Hvidtfeldt J Sulo P Piskur J 1999Horizontal transfer of genetic material among Saccharomyces yeastsJ Bacteriol 1816488ndash6496

Martin A Orgogozo V 2013 The loci of repeated evolution a catalog ofgenetic hotspots of phenotypic variation Evolution 671235ndash1250

Masly JP Jones CD Noor MAF Locke J Orr HA 2006 Gene transpositionas a cause of hybrid sterility in Drosophila Science 3131448ndash1450

Mason AS Batley J 2015 Creating new interspecific hybrid and polyploidcrops Trends Biotechnol 33436ndash441

Mavarez J Salazar CA Bermingham E Salcedo C Jiggins CD Linares M2006 Speciation by hybridization in Heliconius butterflies Nature441868ndash871

McKenna A Hanna M Banks E Sivachenko A Cibulskis K Kernytsky AGarimella K Altshuler D Gabriel S Daly M et al 2010 The genomeanalysis toolkit a MapReduce framework for analyzing next-generation DNA sequencing data Genome Res 201297ndash1303

Meiklejohn CD Holmbeck MA Siddiq MA Abt DN Rand DMMontooth KL 2013 An Incompatibility between a mitochondrialtRNA and its nuclear-encoded tRNA synthetase compromises de-velopment and fitness in Drosophila PLoS Genet 9e1003238

Meyer A Salzburger W Schartl M 2006 Hybrid origin of a swordtailspecies (Teleostei Xiphophorus clemenciae) driven by sexual selec-tion Mol Ecol 15721ndash730

Michalak P 2009 Epigenetic transposon and small RNA determinants ofhybrid dysfunctions Heredity (Edinb) 10245ndash50

Miller AW Dunham MJ 2013 Design and use of multiplexed chemostatarrays J Vis Exp 72e50262

Monod J 1949 The growth of bacterial cultures Annu Rev Microbiol3371ndash394

Mouillon JM Persson BL 2006 New aspects on phosphate sensing andsignalling in Saccharomyces cerevisiae FEMS Yeast Res 6171ndash176

Muhlfeld CC Kovach RP Jones LA Al-Chokhachy R Boyer MC Leary RFLowe WH Luikart G Allendorf FW 2014 Invasive hybridization in athreatened species is accelerated by climate change Nat ClimChange 4620ndash624

Muller LAH McCusker JH 2009 A multispecies-based taxonomic mi-croarray reveals interspecies hybridization and introgression inSaccharomyces cerevisiae FEMS Yeast Res 9143ndash152

Nagalakshmi U Wang Z Waern K Shou C Raha D Gerstein M SnyderM 2008 The transcriptional landscape of the yeast genome definedby RNA sequencing Science 3201344ndash1349

Nakao Y Kanamori T Itoh T Kodama Y Rainieri S Nakamura NShimonaga T Hattori M Ashikari T 2009 Genome sequence ofthe lager brewing yeast an interspecies hybrid DNA Res 16115ndash129

Nolte AW Freyhof J Stemshorn KC Tautz D 2005 An invasive lineage ofsculpins Cottus sp (Pisces Teleostei) in the Rhine with new habitatadaptations has originated from hybridization between old phylo-geographic groups Proc Biol Sci 2722379ndash2387

Novick A Szilard L 1950a Description of the chemostat Science112715ndash716

Novick A Szilard L 1950b Experiments with the Chemostat on spon-taneous mutations of bacteria Proc Natl Acad Sci U S A 36708ndash719

Ouyang YD Liu YG Zhang QF 2010 Hybrid sterility in plant storiesfrom rice Curr Opin Plant Biol 13186ndash192

Payen C Di Rienzi SC Ong GT Pogachar JL Sanchez JC SunshineAB Raghuraman MK Brewer BJ Dunham MJ 2014 The dynam-ics of diverse segmental amplifications in populations ofSaccharomyces cerevisiae adapting to strong selection G3(Bethesda) 4399ndash409

Payen C Sunshine AB Ong GT Pogachar JL Zhao W Dunham MJ 2016High-throughput identification of adaptive mutations in experimen-tally evolved yeast populations PLoS Genet 12e1006339

Perez-Prat E van Lookeren Campagne MM 2002 Hybrid seed produc-tion and the challenge of propagating male-sterile plants TrendsPlant Sci 7199ndash203

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Peris D Langdon QK Moriarty RV Sylvester K Bontrager M Charron GLeducq JB Landry CR Libkind D Hittinger CT 2016 Complex ances-tries of lager-brewing hybrids were shaped by standing variation inthe wild yeast Saccharomyces eubayanus PLoS Genet 12e1006155

Perlstein EO Ruderfer DM Roberts DC Schreiber SL Kruglyak L 2007Genetic basis of individual differences in the response to small-molecule drugs in yeast Nat Genet 39496ndash502

Piatkowska EM Naseeb S Knight D Delneri D 2013 Chimeric proteincomplexes in hybrid species generate novel phenotypes PLoS Genet9e1003836

Piotrowski JS Nagarajan S Kroll E Stanbery A Chiotti KE Kruckeberg ALDunn B Sherlock G Rosenzweig F 2012 Different selective pressureslead to different genomic outcomes as newly-formed hybrid yeastsevolve BMC Evol Biol 1246

Pryszcz LP Nemeth T Gacser A Gabaldon T 2014 Genome comparisonof Candida orthopsilosis clinical strains reveals the existence of hy-brids between two distinct subspecies Genome Biol Evol61069ndash1078

Racimo F Sankararaman S Nielsen R Huerta-Sanchez E 2015 Evidencefor archaic adaptive introgression in humans Nat Rev Genet16359ndash371

Rieseberg LH 1991 Homoploid reticulate evolution in Helianthus(Asteraceae)mdashevidence from ribosomal genes Am J Bot781218ndash1237

Rieseberg LH 1997 Hybrid origins of plant species Annu Rev EcolSystemat 28359ndash389

Robinson JT Thorvaldsdottir H Winckler W Guttman M Lander ESGetz G Mesirov JP 2011 Integrative genomics viewer NatBiotechnol 2924ndash26

Rosenthal DM Schwarzbach AE Donovan LA Raymond O RiesebergLH 2002 Phenotypic differentiation between three ancient hybridtaxa and their parental species Int J Plant Sci 163387ndash398

Ruderfer DM Pratt SC Seidel HS Kruglyak L 2006 Population genomicanalysis of outcrossing and recombination in yeast Nat Genet381077ndash1081

Rundle HD Nagel L Boughman JW Schluter D 2000 Natural selectionand parallel speciation in sympatric sticklebacks Science287306ndash308

Ryland GL Doyle MA Goode D Boyle SE Choong DY Rowley SM Li JBowtell DD Tothill RW Campbell IG et al 2015 Australian OvarianCancer Study Group Loss of heterozygosity what is it good for BMCMed Genomics 845

Salverda MLM Dellus E Gorter FA Debets AJM van der Oost J HoekstraRF Tawfik DS de Visser JAGM 2011 Initial mutations direct alter-native pathways of protein evolution PLoS Genet 7e1001321

Sanchez MR Miller AW Liachko I Sunshine AB Lynch B Huang MAlcantara E DeSevo CG Pai DA Tucker CM et al 2017 Differentialparalog divergence modulates genome evolution across yeast spe-cies PLoS Genet 13(2)e1006585

Scannell DR Zill OA Rokas A Payen C Dunham MJ Eisen MB Rine JJohnston M Hittinger CT 2011 The awesome power of yeast evo-lutionary genetics new genome sequences and strain resources forthe Saccharomyces sensu stricto genus G3 (Bethesda) 111ndash25

Schroder MS Martinez de San Vicente K Prandini TH Hammel SHiggins DG Bagagli E Wolfe KH Butler G 2016 Multiple originsof the pathogenic yeast Candida orthopsilosis by separate hybridiza-tions between two parental species PLoS Genet 12e1006404

Schumer M Rosenthal GG Andolfatto P 2014 How common is homo-ploid hybrid speciation Evolution 681553ndash1560

Schwarzbach AE Donovan LA Rieseberg LH 2001 Transgressivecharacter expression in a hybrid sunflower species Am J Bot88270ndash277

Selmecki AM Maruvka YE Richmond PA Guillet M Shoresh NSorenson AL De S Kishony R Michor F Dowell R et al 2015Polyploidy can drive rapid adaptation in yeast Nature 519349ndash52

Seshan VE Olshen A 2016 DNAcopy DNA copy number data analysisVersion R package version 1460

Soltis DE Visger CJ Soltis PS 2014 The polyploidy revolution then andnow Stebbins revisited Am J Bot 1011057ndash1078

Soltis PS 2013 Hybridization speciation and novelty J Evol Biol26291ndash293

Soltis PS Soltis DE 2009 The role of hybridization in plant speciationAnnu Rev Plant Biol 60561ndash588

Sunshine AB Payen C Ong GT Liachko I Tan KM Dunham MJ 2015The fitness consequences of aneuploidy are driven by condition-dependent gene effects PLoS Biol 13e1002155

Tamai Y Momma T Yoshimoto H Kaneko Y 1998 Co-existence of twotypes of chromosome in the bottom fermenting yeastSaccharomyces pastorianus Yeast 14923ndash933

Taylor DJ Hebert PDN 1993 Habitat-dependent hybrid parentage anddifferential introgression between neighboringly sympatric Daphniaspecies Proc Natl Acad Sci U S A 907079ndash7083

Thiagalingam S Laken S Willson JK Markowitz SD Kinzler KWVogelstein B Lengauer C 2001 Mechanisms underlying losses ofheterozygosity in human colorectal cancers Proc Natl Acad Sci US A 982698ndash2702

Tuna M Knuutila S Mills GB 2009 Uniparental disomy in cancer TrendsMol Med 15120ndash128

Walther A Hesselbart A Wendland J 2014 Genome sequence ofSaccharomyces carlsbergensis the worldrsquos first pure culture lageryeast G3 (Bethesda) 4783ndash793

Wayne RK 1993 Molecular evolution of the dog family Trends Genet9218ndash224

Wenger JW Schwartz K Sherlock G 2010 Bulk segregant analysis byhigh-throughput sequencing reveals a novel xylose utilization genefrom Saccharomyces cerevisiae PLoS Genet 6e1000942

Wessinger CA Rausher MD 2014 Predictability and irreversibility ofgenetic changes associated with flower color evolution inPenstemon barbatus Evolution 681058ndash1070

Wolfe KH 2015 Origin of the yeast whole-genome duplication PLoSBiol 13e1002221

Woods R Schneider D Winkworth CL Riley MA Lenski RE 2006 Testsof parallel molecular evolution in a long-term experiment withEscherichia coli Proc Natl Acad Sci U S A 1039107ndash9112

Wykoff DD OrsquoShea EK 2001 Phosphate transport and sensing inSaccharomyces cerevisiae Genetics 1591491ndash1499

Xiong Z Gaeta RT Pires JC 2011 Homoeologous shuffling andchromosome compensation maintain genome balance inresynthesized allopolyploid Brassica napus Proc Natl Acad SciU S A 1087908ndash7913

Yassour M Kapian T Fraser HB Levin JZ Pfiffner J Adiconis X Schroth GLuo SJ Khrebtukova I Gnirke A et al 2009 Ab initio construction ofa eukaryotic transcriptome by massively parallel mRNA sequencingProc Natl Acad Sci U S A 1063264ndash3269

Zhu YO Sherlock G Petrov DA 2016 Whole genome analysis of 132clinical Saccharomyces cerevisiae strains reveals extensive ploidy var-iation G3 (Bethesda) doi101534g3116029397

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contrast copy number variants in the hybrid were rarely facil-itated by repetitive elements perhaps in part because S uva-rum has no full length Ty elements however why S cerevisiaeTy elements and long terminal repeat (LTR) sequences fromeither genome were not utilized remains unknown

Frequently in nutrient-limited evolution experimentscopy number variants involve amplification of the nutrient-specific transporter and indeed we also observed amplifica-tion of these transporters in many of the clones In sulfatelimitation the S cerevisiae allele of the high-affinity sulfatetransporter gene SUL1 is amplified in 77 hybrid clones and 44 S cerevisiae clones (fig 1 tables 1 and 2 supplementary figsS1 and S3 Supplementary Material online) InterestinglySUL2 is the preferred sulfate transporter in S uvarum(Sanchez et al 2017) and was not observed to be amplifiedin the evolved hybrids (supplementary fig S2 and table S2Supplementary Material online) In glucose limitation previ-ous S cerevisiae evolution experiments found frequent am-plification of the high-affinity glucose transporter genesHXT67 (Brown et al 1998 Dunham et al 2002 Greshamet al 2008 Kao and Sherlock 2008 Kvitek and Sherlock2011) In our experiments the S uvarum alleles of theHXT67 transporters are amplified in 33 hybrid clones andboth S uvarum clones but are not amplified in evolved Scerevisiae clones This is suggestive that the S uvarum HXT67alleles confer a greater fitness advantage compared with Scerevisiae alternatively the genomic context could be morepermissive to amplification in S uvarum (fig 1 tables 1 and 2supplementary figs S1ndashS3 Supplementary Material online)Finally in phosphate limitation the S cerevisiae copy of thehigh-affinity phosphate transporter gene PHO84 is amplifiedand the S uvarum allele is lost in 36 hybrid clones in an eventknown as LOH (figs 1 and 2 table 1 supplementary fig 3Supplementary Material online) Intriguingly the evolved Scerevisiae clones also display LOH and accompanied amplifi-cation favoring the allele derived from strain GRF167 over theS288C allele in 44 clones (fig 2 table 2) All hybrid clonescarry the ldquopreferredrdquo GRF167 S cerevisiae allele as this was theallele used to create the de novo hybrid

Loss of Heterozygosity Is a Common Event inHeterozygous Evolving PopulationsSelection on heterozygosity as a LOH event could representis an underappreciated source of adaptation in microbial ex-perimental evolution as typical experiments evolve a haploidor homozygous diploid strain asexually and as a result havelittle variation to select upon LOH is observed in natural andindustrial hybrids (Albertin and Marullo 2012 Louis et al2012 Pryszcz et al 2014 Wolfe 2015 Schroder et al 2016)but here we document its occurrence in both intra- andinterspecific hybrids in the laboratory as a result of short-termevolution (also see (Dunn et al 2013 Burke et al 2014)) LOHis observed across all nutrient conditions with 13 independentLOH events detected in S cerevisiae and 9 independent eventsdocumented in the hybrids (figs 1 and 2 tables 1 and 2 sup-plementary figs S1 and S3 Supplementary Material online) Itthus appears that this type of mutational event is both com-mon and can occur over short evolutionary timescales

The LOH event can result in copy-neutral (where one alleleis lost and the other allele is amplified) or non-neutral chro-mosomal segments (where one allele is lost rendering thestrain hemizygous at that locus) and can favor the retentionof either allele In S cerevisiae there is a bias in resolutionwhere LOH events favor retaining the GRF167 allele over theS288C allele (1013 events Pfrac14 00169 table 2 supplementaryfig S4 Supplementary Material online) One unique case inclone Sc4 has a small approximately 5 kb LOH event onchrXV favoring GRF167 which switches to favoring S288Cfor the rest of the chromosome The retention of S cerevisiaeis slightly more common in the hybrids (59 events Pfrac14 10table 1 supplementary fig S3 Supplementary Material on-line) though not as drastic as the observed genome resolu-tion in the hybrid S pastorianus where LOH favorsS cerevisiae over S eubayanus (Nakao et al 2009) The sizeof the event ranges from approximately 25 kb to the wholechromosome level in the hybrids and from 5 kb to 540 kbin S cerevisiae Where LOH is accompanied by an amplifi-cation event the LOH event always occurs first and doesnot share break points with the amplification event Unlikemany CNV events most LOH events do not appear to bemediated by existing repetitive sequence such as a trans-posable element in the hybrid or S cerevisiae and are mostlikely a product of break-induced replication or mitoticgene conversion with break-induced replication as the fa-vored method as all events extend to the telomere (Hoanget al 2010) The exceptions are in hybrid clones Ph4 Ph5 andSh1 where there is a non-copy neutral loss of S cerevisiaemediated by a Ty element or LTR and S cerevisiae clonesSc1 and Sc4 where there is a 65-kb deletion of the S288Callele flanked by two Ty elements

LOH events in hybrids could signify several ongoing pro-cesses in hybrid genome evolution LOH regions may repre-sent 1) loci with incompatibilities 2) selection on existingvariation or 3) genetic drift eroding genomic segments It isimpossible to definitively rule out any of these hypotheseswithout further experimentation however there are severalarguments disfavoring the incompatibility hypothesis Firstalthough our sample size is modest failing to see repeatedLOH events across nutrient conditions may indicate a lack ofgeneral incompatibility between species (although we cannotpreclude condition-specific incompatibility Hou et al 2015Piatkowska et al 2013) This is consistent with previous stud-ies in yeast which suggest that classic DobzhanksyndashMullerincompatibilities are rare (Liti et al 2006 Greig 2009 Houet al 2014) Furthermore LOH events observed inevolved S cerevisiae suggest that this mutation type isnot unique to interspecific hybrids Instead repeatedevents within a particular condition such as the repeatedLOH at PHO84 in phosphate limitation or the 65 kbsegment on chrXIII in sulfate limitation suggest thatthese events are beneficial and are indeed selection onone allele over the other

LOH Is Driven by Selection on One AlleleTo test the hypothesis that LOH events provide a selectiveadvantage we used allele replacement in which the allele of

Smukowski Heil et al doi101093molbevmsx098 MBE

10

one speciesstrain is replaced with the allele of the otherspeciesstrain in an otherwise isogenic background We testedthis hypothesis using the most commonly seen LOH eventLOH at PHO84 Although the region extends from 25 kb to234 kb in length in the hybrids and from 40 kb to 85 kb in Scerevisiae and thus includes many genes PHO84 was a primecandidate driving this event PHO84 is one of only ten genesencompassed in the region extending from the telomere tothe break point of the shortest LOH event and is included inevery other LOH event on chromosome XIII (fig 2) It is ahigh-affinity phosphate transporter responsible for inorganicphosphate uptake in high and low phosphate conditions(Wykoff and OrsquoShea 2001) and previous work identified apoint mutation in PHO84 (an alanine to valine substitution atthe 50 end of the gene) which increased fitness by 23 inphosphate-limited conditions (Sunshine et al 2015) Finallyprior work with other nutrient transporters has shown am-plification of nutrient transporters to be a key event in adapt-ing to nutrient-limited conditions

We thus selected a region of approximately 25 kb encom-passing the PHO84 ORF its promoter and 30-UTR(Nagalakshmi et al 2008 Yassour et al 2009 Cherry et al2012) We created allele replacement strains using the twoalleles of S cerevisiae in a S cerevisiae diploid background thetwo alleles are 991 identical in this region (supplementaryfig S5 Supplementary Material online) and each strain isidentical to the ancestral strain used in our evolution exper-iments except at the PHO84 locus The S cerevisiae ancestorcarries one copy of GRF167 (preferred) and one copy ofS288C (ldquoun-preferredrdquo) so named due to which allele wasretained and amplified in the evolved clones To measureany resultant changes in fitness we competed each strainindividually against a fluorescent ancestral strain and mea-sured which strain overtook the culture Relative fitness isthus defined as the growth advantage per generation Twocopies of the un-preferred allele decreased relative fitness by531 (6186) whereas two copies of the preferred alleleincreased relative fitness by 993 (6027) This displays anoverall difference in relative fitness of 1524 between the un-preferred and preferred alleles By comparing the fitness ofthese allele replacement strains with the evolved clones (table 2)the allele replacement does not fully recapitulate the fit-ness gain observed in the evolved clone One explanation isthat the additional mutations present in the evolvedstrains also contribute to their total fitness Another expla-nation could be the increased copy number of the PHO84region that we see in these evolved clones To further ex-plore this fitness difference we cloned the GRF167 alleleonto a low copy number plasmid and transformed theallele replacement strain carrying two preferred S cerevi-siae alleles to simulate increased copy number of PHO84and saw only a minimal fitness increase of 176 (6106note all fitness competitions involving plasmids were donein conjunction with empty plasmids to take into accountany fitness effects from the plasmid itself) This supportsthe conclusion that relative fitness gains in the evolvedclone are largely due to the loss of the S288C allele andselection and amplification of the GRF167 allele with little

additional benefit from further amplification It could also bethe case that co-amplification of other genes in the segmentis required to attain the full benefit as previously observedby the contribution of BSD2 to the SUL1 amplicon in sulfatelimitation (Sunshine et al 2015 Payen et al 2016)

To understand the fitness effects of LOH and amplificationin the hybrids we generated hybrid strains with varying num-bers of S cerevisiae GRF167 PHO84 alleles Unfortunately wewere unable to obtain a successful strain carrying the pre-ferred S cerevisiae allele in a S uvarum background howeverwe were able to generate a S uvarum PHO84 knockout strainthus creating a hybrid with one copy of S cerevisiae PHO84When combined with a low copy plasmid carrying the sameallele this strain effectively has two or more copies ofS cerevisiae PHO84 in a hybrid background This hybrid straincan serve as a proxy for the LOH event observed in the evo-lution experiments and has a relative fitness gain of 2557(6288) The ancestral hybrid with the same plasmid (effec-tively 1 S uvarum allele and 2 or more S cerevisiae alleles) hasa relative fitness gain of 1253 (6131) The difference be-tween these two hybrids suggests that while amplification isbeneficial the highest fitness is achieved with the loss of theS uvarum allele (Pfrac14 00061)

Together these results support the conclusion that theS cerevisiae GRF167 allele is preferred over the S288C alleleand that S cerevisiae alleles are preferred over the S uvarumallele in the hybrid and hence that the LOH events seen inboth intra- and interspecific hybrids are the product ofselection

DiscussionIn summary we sought to understand forces underlying ge-nome stabilization and evolution in interspecific and intra-specific hybrids as they adapt to novel environments Weevolved and sequenced clones from 16 hybrid populationsand 16 parental populations to reveal a variety of mutationalevents conferring adaptation to 3 nutrient-limited conditionsOf particular note we find LOH in both evolved intraspecificand interspecific hybrid clones in all nutrient environmentspotentially signifying areas where selection has acted on pre-existing variation present in the ancestral clone We used anallele replacement strategy to test this hypothesis for a com-monly repeated LOH event and show that selection is indeeddriving the homogenization of the genome at this locusAlthough other studies in natural industrial and lab-evolved isolates have observed LOH we present the first em-pirical test of the causal evolutionary forces influencing theseevents This work can begin to help us understand past hy-bridization events and subsequent genome resolution in hy-brids in natural and artificial systems

Similarities and Differences between Intra- andInterspecific HybridsAlthough our sample size is modest we observe several in-teresting trends when comparing S uvarum clones (homo-zygous) S cerevisiae clones (intraspecific hybrid andpreviously published homozygous) and interspecific hybrid

Adaptation in Hybrids doi101093molbevmsx098 MBE

11

clones First theory and previous research predict the inter-specific hybrid may experience more genome instability in theform of chromosomal rearrangements and CNV events(Xiong et al 2011 Lloyd et al 2014 Chester et al 2015Mason and Batley 2015) Instead in our work the interspecifichybrid and S cerevisiae clones experience the same numberof CNV events (both 15 CNVsclone) However the mech-anism of CNV formation seems to differ between the hybridand S cerevisiae clones Whereas S cerevisiae CNV breakpoints typically occur at transposable elements in our studyand previous studies (Dunham et al 2002 Gresham et al2008 Fedoroff 2012) the interspecific hybrid CNVs do notutilize transposable elements although they obviously sharethe same sequence background as the S cerevisiae clones(albeit in one copy) Whether this difference is due to theabsence of full-length transposable elements in S uvarum isunknown but this could potentially explain the lower num-ber of CNV events in S uvarum clones (1 CNVclone) andpresents an intriguing direction for future study

The Predictability of EvolutionWe now have many examples of predictable evolution innatural systems (Losos et al 1998 Rundle et al 2000 Elmerand Meyer 2011 Conte et al 2012 Jones et al 2012 Martinand Orgogozo 2013 Wessinger and Rausher 2014) and inlaboratory experimental evolution in which there often ap-pears to be a limited number of high fitness pathways thatstrains follow when adapting to a particular condition (Fereaet al 1999 Woods et al 2006 Gresham et al 2008 Burke et al2010 Salverda et al 2011 Kawecki et al 2012 Kvitek andSherlock 2013 Lang and Desai 2014) For example it is wellestablished that amplifications of nutrient transporters aredrivers of adaptation in evolution in nutrient-limited condi-tions Previous work in our group has particularly focused onthe amplification of the high-affinity sulfate transporter geneSUL1 in sulfate-limited conditions which occurs in almostevery sulfate-limited evolution experiment and confers a fit-ness advantage of as much as 40 compared with the un-evolved ancestor strain The amplification of phosphatetransporters has been markedly less common and thus driv-ers of adaptation in this condition have been less clearGresham et al (2008) identified a whole chromosome ampli-fication of chrXIII in one population In a follow-up studySunshine et al (2015) found whole or partial amplification ofchrXIII in 38 populations A genome-wide screen for segmen-tal amplifications found a slight increase in fitness for a smalltelomeric segment of chromosome XIII and a A49V pointmutation in PHO84 was observed to increase fitness by 23However screens by Payen et al (2016) showed that althoughPHO84 is recurrently mutated in various experiments itshowed no benefit when amplified or deleted in phos-phate-limited conditions Finally additional evolution exper-iments recapitulated the point mutation seen in Sunshineet al (2015) in 2432 populations and saw amplification ofPHO84 in 832 populations (Miller A Dunham MJ unpub-lished data) It is important to note that all these experimentsused a strain background derived from S288C or CENPKboth of which carry the same (un-preferred) PHO84 allele

In our work we observed amplification of the S cerevisiaeGRF167 allele of PHO84 in 44 S cerevisiae clones from 4populations and 36 hybrid clones from 6 populations Thisamplification was always preceded by the loss of the S288Callele in S cerevisiae clones and the LOH break points arenever shared with the amplification break points There is a15 fitness difference between carrying two copies of theS288C allele of PHO84 compared with carrying two copiesof the GRF167 allele of PHO84 and additional copies of theGRF167 allele do not provide substantial further fitness gainsThe two alleles differ by several noncoding changes and threenonsynonymous substitutions a mutation from glutamicacid to aspartic acid (E229D) leucine to proline (L259P)and leucine to glutamine (L556Q) the latter two of whichare considered ldquononconservativerdquo protein mutations due tochanges in hydrophobicity and structure (supplementary figS5 Supplementary Material online) Intriguingly the L259Pmutation has actually been previously identified as being re-sponsible for resistance to tetrachloroisophthalonitrile andpartial resistance to pentachlorophenol in a QTL study ofsmall-molecule drugs (Perlstein et al 2007) IndeedPerlstein et al found proline at residue 259 to be conservedacross fungal species and even in orthologous human xeno-biotic transporters likely because proline-induced kinks intransmembrane spans have been shown to be essential toprotein function (Cordes et al 2002 Perlstein et al 2007) Itthus appears that amplification of PHO84 has been less pre-dictable as the S288C allele does not confer a fitness advan-tage unless mutated per Sunshine et al (2015) Togetherthese results imply that strain background can constrainadaptive pathways

In hybrids the amplification of the S cerevisiae segmentoccurred in conjunction with the loss of the S uvarum alleleHybrid strains with the LOH had a 2557 relative fitness gainwhereas hybrid strains with amplification of the S cerevisiaePHO84 allele without the LOH had a 1253 fitness gain ThusLOH confers an additional 1304 fitness gain showing thatselection for LOH has a larger impact on fitness than ampli-fication alone Note that S uvarum does have proline at res-idue 259 like the preferred GRF167 allele and differs fromboth S cerevisiae alleles at the other two coding substitutions(229N and 556K) but the amino acid and noncoding diver-gence is too high to speculate what substitutions are respon-sible for the selection of the GRF167 allele (supplementary figS6 Supplementary Material online) Why the loss of one alleleis more beneficial remains unclear as PHO84 is thought tofunction as a monomer (Bun-Ya et al 1991) but it may bedue to competition for cell wall space or negative interactionswith other genes in the PHO pathway (Mouillon and Persson2006)

The infusion of variation created by hybridization providesnew templates for selection to act upon which can be moreimportant than either point mutations or copy number var-iants alone Our work shows that outcrossing need not becommon to have long-lasting effects on adaptation This im-plication is particularly relevant in yeast where outcrossingmay occur quite rarely followed by thousands of asexual gen-erations (Ruderfer et al 2006 Greig and Leu 2009 Liti 2015)

Smukowski Heil et al doi101093molbevmsx098 MBE

12

Applications to Other Hybrids and CancerThe observation that LOH occurs in hybrid genomes is in-creasingly documented (Louis et al 2012 Borneman et al2014 Soltis et al 2014 Marcet-Houben and Gabaldon 2015Pryszcz et al 2014 Schroder et al 2016) although the rea-son(s) for this type of mutation has been unresolved As mostexamples stem from allopolyploid events that occurred mil-lions of years ago understanding why LOH is important inhybrid genome evolution is difficult Cancer cells are alsoknown to experience LOH sometimes involved in the inac-tivation of tumor suppressor genes leaving only one copy ofthe gene that may be mutated or silenced (Thiagalingam et al2001 Tuna et al 2009 Lapunzina and Monk 2011) Datasupport the conclusion that LOH events are selected for dur-ing tumor development as many LOH events involve specificchromosomal segments (Thiagalingam et al 2001) althoughthe underlying molecular and genetic reasons for selection isan open debate (Ryland et al 2015)

Here we experimentally demonstrate that LOH can occurin homoploid interspecific hybrids as well as in intraspecifichybrids These events occur within a few hundred generationsand are common mutations more common on average inthe intraspecific hybrid (13 eventsclone) than the interspe-cific hybrid (056 eventsclone) Interestingly the LOH eventsdo not share break points with the CNV events in S cerevisiaeinstead they appear to occur independently and to precedeany subsequent amplification (amplification occurs following913 LOH events) Competition assays with the PHO84 locusprovide support that LOH itself may be more beneficial thanamplification or at least increase the selective benefit of am-plification events The observation that LOH in intraspecifichybrids occurs independently from copy number change pro-vides different opportunities for adaptation to novelconditions

Other cases of LOH like the copy number neutral eventsobserved in Ph4 Ph5 and Sh4 all of which favor the retentionof the S uvarum allele (supplementary fig S3 SupplementaryMaterial online) may be due to hybrid incompatibility withina particular protein complex other epistatic interactions(Piatkowska et al 2013) or neutral processes We furthermorediscover examples where one species allele appears to bepreferred over the other without LOH such as the repeatedamplification of the S uvarum high-affinity glucose transpor-ters HXT67 When one species allele is amplified and theother is not amplified one explanation is that the local se-quence context can permit or deny amplification In the caseof HXT67 previous experiments in S cerevisiae have shownthat amplification of HXT67 is quite common (Brown et al1998 Dunham et al 2002 Gresham et al 2008 Kao andSherlock 2008 Kvitek and Sherlock 2011) thus suggestingthat when given a choice between this locus in S cerevisiaeor S uvarum in the hybrid the preferred allele is indeed Suvarum though more subtle differences in rate cannot yet beruled out A similar scenario is observed with S cerevisiaeSUL1 (Sanchez et al 2017) Together our results show thatthe heterozygosity supplied by hybridization is an importantcontributor to adaptive routes explored by populations asthey adapt to novel conditions

Although we cannot generalize our results from thePHO84 locus across the many other LOH events discoveredin our hybrids and S cerevisiae in the future we can usesimilar methodology to explore whether positive selectionalways drives LOH or whether other explanations such asincompatibility resolution contribute as well Future experi-ments might also utilize a high throughput method to ex-plore segmental LOH in hybrids at a genome-wide scalesimilar to ongoing experiments at the gene level (LancasterS Dunham MJ unpublished data) Although our sample sizeis moderate this is a novel and necessary step in understand-ing forces underlying hybrid genome stabilization and high-lighting an underappreciated mechanism of hybridadaptation

ConclusionsThe mutation events we observe in our experimentallyevolved hybrids are in many ways quite representative ofmutations observed in ancient hybrid genomes suggestingthat hybrid genome stabilization and adaptation can occurquite rapidly (within several hundred generations)Furthermore our results illustrate that the infusion of varia-tion introduced by hybridization at both the intra- and inter-species level can increase fitness by providing choices of allelesfor selection to act upon even when sexual reproduction israre This may be particularly important for leveraging existingvariation for agricultural and industrial processes and as cli-mate change potentially increases natural hybridization (Kellyet al 2010 Hoffmann and Sgro 2011 Muhlfeld et al 2014)

Materials and Methods

StrainsA list of strains used in this study is included in supplementarytable S1 Supplementary Material online All interspecific hy-brids were created by crossing a ura3 LYS2 haploid parent to aURA3 lys2 haploid parent of the other mating type plating onmedia lacking both uracil and lysine and selecting for proto-trophs The S cerevisiae strain background known asldquoGRF167rdquo is itself a cross between FL100 and the genomictype strain S288C (data not shown) GRF167 was chosen as astrain background for simultaneous work investigating trans-posable elements during experimental evolution which willbe addressed in a future study

Evolution ExperimentsContinuous cultures were established using media and con-ditions previously described (Gresham et al 2008 Sanchezet al 2017) Detailed protocols and media recipes are availableat httpdunhamgswashingtoneduprotocolsshtml (lastaccessed March 6 2017) Samples were taken daily and mea-sured for optical density at 600 nm and cell count micros-copy was performed to check for contamination andarchival glycerol stocks were made daily An experimentwas terminated when contamination growth in tubing orclumping appeared (number of generations at the endpoint for each population are presented in tables 1 and

Adaptation in Hybrids doi101093molbevmsx098 MBE

13

2) Samples from each end point population were colonypurified to yield two clones for further study

Array Comparative Genomic HybridizationPopulations from the end point of each evolution were ana-lyzed for copy number changes using array comparative ge-nomic hybridization following the protocol used in Sanchezet al (2017)

SequencingDNA was extracted from overnight cultures using theHoffmanndashWinston protocol (Hoffman and Winston 1987)and cleaned using the Clean amp Concentrator kit (ZymoResearch) Nextera libraries were prepared following theNextera library kit protocol and sequenced using paired end150 bp reads on the Illumina NextSeq 500 machine (sequenc-ing coverage in supplementary table S2 SupplementaryMaterial online) The reference genomes used were S cerevi-siae v3 (Engel et al 2014) S uvarum (Scannell et al 2011) anda hybrid reference genome created by concatenating the twogenomes Sequence was aligned to the appropriate referencegenome using bwa v062 (Li and Durbin 2009) and mutationswere called using GATK (McKenna et al 2010) and samtools0119 (Li et al 2009) Mutations in evolved clones were filteredin comparison with the ancestor to obtain de novo mutationsAll mutations were first visually inspected using IntegrativeGenomics Viewer (Robinson et al 2011) Subsequently pointmutations in the hybrids were confirmed with Sanger se-quencing (supplementary table S3 Supplementary Materialonline) Copy number variants were visualized usingDNAcopy for S cerevisiae and S uvarum (Seshan andOlshen 2016) LOH events were called based on sequencingcoverage in the hybrids and by identifying homozygous variantcalls in S cerevisiae All break points were called by visual in-spection of sequencing reads and are thus approximate

Fitness AssaysThe pairwise competition experiments were performed in 20ml chemostats (Miller and Dunham 2013) Each competitorstrain was cultured individually until steady state was reachedand then was mixed 5050 with a GFP-tagged ancestor Eachcompetition was conducted in at least two biological repli-cates for approximately 15 generations after mixing Sampleswere collected and analyzed twice daily The proportion ofGFPthorn cells in the population was detected using a BD AccuriC6 flow cytometer (BD Biosciences) The data were plottedwith ln [dark cellsGFPthorn cells] versus generations The rela-tive fitness coefficient was determined from the slope of thelinear region

Strain ConstructionAllele replacements for the PHO84 locus were done followingthe protocol of the Caudy lab with further modifications de-scribed here The native locus was replaced withKluyveromyces lactis URA3 The pho84DURA3 strain wasgrown overnight in 5 ml of C-URA media then inoculatedin a flask of 100 ml yeast extract peptone dextrose (YPD) andgrown to an optical density of 06ndash08 Cells were washed then

aliquoted 275 ml of transformation mix (35 ml 1 M lithiumacetate 240 ml of 50 3500 polyethylene glycol) 10 ml ofsalmon sperm and approximately 3 mg of polymerase chainreaction product were added to the cell pellet It was incu-bated at 37 C (S uvarum) or 42 C (S cerevisiae) for 45 minthen plated to YPD It was replica plated to 5-fluorooroticacid the following day and colonies were tested for the gain ofthe appropriate species allele The GRF167 allele was clonedinto the pIL37 plasmid using Gibson assembly (Gibson et al2009) Correct assembly was verified by Sanger sequencing Allprimers used can be found in supplementary table S3Supplementary Material online

Supplementary MaterialSupplementary data are available at Molecular Biology andEvolution online

AcknowledgmentsWe thank Noah Hanson and Erica Alcantara for technicalassistance We thank Monica Sanchez and three anonymousreviewers for helpful comments on this manuscript JosephSchacherer kindly confirmed the contribution of FL100 to theGRF167 strain background Thanks to Yixian Zheng and DougKoshland for contributing to the initial experimental designcreating yeast strains and purchasing the oligonucleotidesused for the microarrays This work was supported by theNational Science Foundation (grant number 1516330) andthe National Institutes of Health (grant number R01GM094306) MD is a Senior Fellow in the GeneticNetworks program at the Canadian Institute for AdvancedResearch and a Rita Allen Foundation Scholar CSH wassupported in part by National Institutes of Health (grantnumber T32 HG00035) This work was also supported bythe National Institutes of Health (grant number P50GM071508) to the Lewis-Sigler Institute and from theHoward Hughes Medical Institute to Doug Koshland andYixian Zheng Sequencing read data have been deposited atthe NCBI under BioProject accession PRJNA374049Microarray data are deposited in the Gene ExpressionOmnibus (GEO) repository under accession numbersGSE95086 and GSE87401 and in the Princeton MicroarrayDatabase

ReferencesAbbott R Albach D Ansell S Arntzen JW Baird SJ Bierne N Boughman J

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Albertin W Marullo P 2012 Polyploidy in fungi evolution after whole-genome duplication Proc Biol Sci 2792497ndash2509

Almeida P Goncalves C Teixeira S Libkind D Bontrager M Masneuf-Pomarede I Albertin W Durrens P Sherman DJ Marullo P et al2014 A Gondwanan imprint on global diversity and domesticationof wine and cider yeast Saccharomyces uvarum Nat Commun54044

Antunovics Z Nguyen HV Gaillardin C Sipiczki M 2005 Gradual ge-nome stabilisation by progressive reduction of the Saccharomyces

Smukowski Heil et al doi101093molbevmsx098 MBE

14

uvarum genome in an interspecific hybrid with Saccharomyces cer-evisiae FEMS Yeast Res 51141ndash1150

Baker E Wang B Bellora N Peris D Hulfachor AB Koshalek JA AdamsM Libkind D Hittinger CT 2015 The Genome sequence ofSaccharomyces eubayanus and the domestication of lager-brewingyeasts Mol Biol Evol 322818ndash2831

Barbosa R Almeida P Safar SVB Santos RO Morais PB Nielly-Thibault LLeducq JB Landry CR Goncalves P Rosa CA Sampaio JP 2016Evidence of natural hybridization in Brazilian wild lineages ofSaccharomyces cerevisiae Genome Biol Evol 8317ndash329

Barton NH 2001 The role of hybridization in evolution Mol Ecol10551ndash568

Belloch C Orlic S Barrio E Querol A 2008 Fermentative stress adapta-tion of hybrids within the Saccharomyces sensu stricto complex Int JFood Microbiol 122188ndash195

Bellon JR Yang F Day MP Inglis DL Chambers PJ 2015 Designingand creating Saccharomyces interspecific hybrids for improvedindustry relevant phenotypes Appl Microbiol Biotechnol998597ndash8609

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Brewer BJ Payen C Di Rienzi SC Higgins MM Ong G Dunham MJRaghuraman MK 2015 Origin-dependent inverted-repeat amplifi-cation tests of a model for inverted DNA amplification PLoS Genet11e1005699

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Bullini L 1994 Origin and evolution of animal hybrid species Trends EcolEvol 9422ndash426

Bun-Ya M Nishimura M Harashima S Oshima Y 1991 The PHO84 geneof Saccharomyces cerevisiae encodes an inorganic phosphate trans-porter Mol Cell Biol 113229ndash3238

Burke MK Dunham JP Shahrestani P Thornton KR Rose MR Long AD2010 Genome-wide analysis of a long-term evolution experimentwith Drosophila Nature 467587ndash590

Burke MK Liti G Long AD 2014 Standing genetic variation drives re-peatable experimental evolution in outcrossing populations ofSaccharomyces cerevisiae Mol Biol Evol 313228ndash3239

Cherry JM Hong EL Amundsen C Balakrishnan R Binkley G Chan ETChristie KR Costanzo MC Dwight SS Engel SR et al 2012Saccharomyces genome database the genomics resource of buddingyeast Nucleic Acids Res 40D700ndashD705

Chester M Riley RK Soltis PS Soltis DE 2015 Patterns of chromosomalvariation in natural populations of the neoallotetraploid Tragopogonmirus (Asteraceae) Heredity (Edinb) 114309ndash317

Chou JY Leu JY 2010 Speciation through cytonuclear incompatibilityinsights from yeast and implications for higher eukaryotes Bioessays32401ndash411

Cliften PF Fulton RS Wilson RK Johnston M 2006 After the duplicationgene loss and adaptation in Saccharomyces genomes Genetics172863ndash872

Conte GL Arnegard ME Peichel CL Schluter D 2012 The probability ofgenetic parallelism and convergence in natural populations Proc BiolSci 2795039ndash5047

Cordes FS Bright JN Sansom MS 2002 Proline-induced distortions oftransmembrane helices J Mol Biol 323951ndash960

Crow JF 1998 90 years ago the beginning of hybrid maize Genetics148923ndash928

Dasmahapatra KK Walters JR Briscoe AD Davey JW Whibley A NadeauNJ Zimin AV Hughes DST Ferguson LC Martin SH et al 2012Butterfly genome reveals promiscuous exchange of mimicry adap-tations among species Nature 48794ndash98

Dowling TE Smith GR Brown WM 1989 Reproductive isolation andintrogression between Notropis-Cornutus and Notropis-Chrysocephalus (Family Cyprinidae)mdashcomparison of morphologyallozymes and mitochondrial-DNA Evolution 43620ndash634

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Dunham MJ Badrane H Ferea T Adams J Brown PO Rosenzweig FBotstein D 2002 Characteristic genome rearrangements in experi-mental evolution of Saccharomyces cerevisiae Proc Natl Acad Sci U SA 9916144ndash16149

Dunn B Paulish T Stanbery A Piotrowski J Koniges G Kroll E LouisEJ Liti G Sherlock G Rosenzweig F 2013 Recurrent rearrange-ment during adaptive evolution in an interspecific yeast hybridsuggests a model for rapid introgression PLoS Genet9e1003366

Dunn B Richter C Kvitek DJ Pugh T Sherlock G 2012 Analysis of theSaccharomyces cerevisiae pan-genome reveals a pool of copy num-ber variants distributed in diverse yeast strains from differing indus-trial environments Genome Res 22908ndash924

Dunn B Sherlock G 2008 Reconstruction of the genome origins andevolution of the hybrid lager yeast Saccharomyces pastorianusGenome Res 181610ndash1623

Elmer KR Meyer A 2011 Adaptation in the age of ecological genomicsinsights from parallelism and convergence Trends Ecol Evol26298ndash306

Engel SR Dietrich FS Fisk DG Binkley G Balakrishnan R Costanzo MCDwight SS Hitz BC Karra K Nash RS et al 2014 The referencegenome sequence of Saccharomyces cerevisiae then and now G3(Bethesda) 4389ndash398

Fedoroff NV 2012 Presidential address Transposable elements epige-netics and genome evolution Science 338758ndash767

Ferea TL Botstein D Brown PO Rosenzweig RF 1999 Systematicchanges in gene expression patterns following adaptive evolutionin yeast Proc Natl Acad Sci U S A 969721ndash9726

Gibson B Liti G 2015 Saccharomyces pastorianus genomic insightsinspiring innovation for industry Yeast 3217ndash27

Gibson DG Young L Chuang RY Venter JC Hutchison CA Smith HO2009 Enzymatic assembly of DNA molecules up to several hundredkilobases Nat Methods 6343ndash5

Gonzalez SS Barrio E Gafner J Querol A 2006 Natural hybrids fromSaccharomyces cerevisiae Saccharomyces bayanus andSaccharomyces kudriavzevii in wine fermentations FEMS Yeast Res61221ndash1234

Gonzalez SS Barrio E Querol A 2008 Molecular characterization of newnatural hybrids of Saccharomyces cerevisiae and S kudriavzevii inbrewing Appl Environ Microbiol 742314ndash2320

Grant PR Grant BR 1994 Phenotypic and genetic consequences ofhybridization in Darwinrsquos finches Evolution 48297ndash316

Grant PR Grant BR 2002 Unpredictable evolution in a 30-year study ofDarwinrsquos finches Science 296707ndash711

Grant PR Grant BR 2010 Natural selection speciation and Darwinrsquosfinches Proc Calif Acad Sci 61245ndash260

Grant PR Grant BR Petren K 2005 Hybridization in the recent past AmNat 16656ndash67

Greig D 2009 Reproductive isolation in Saccharomyces Heredity (Edinb)10239ndash44

Greig D Leu JY 2009 Natural history of budding yeast Current Biol19R886ndashR890

Greig D Louis EJ Borts RH Travisano M 2002 Hybrid speciation inexperimental populations of yeast Science 2981773ndash1775

Gresham D Desai MM Tucker CM Jenq HT Pai DA Ward A DeSevoCG Botstein D Dunham MJ 2008 The repertoire and dynamics ofevolutionary adaptations to controlled nutrient-limited environ-ments in yeast PLoS Genet 4e1000303

Hajjar R Hodgkin T 2007 The use of wild relatives in crop improvementa survey of developments over the last 20 years Euphytica 1561ndash13

Heiser CB 1954 Variation and subspeciation in the common sunflowerHelianthus annuus Am Midl Nat 51287ndash305

Hittinger CT 2013 Saccharomyces diversity and evolution a buddingmodel genus Trends Genet 29309ndash317

Hoang ML Tan FJ Lai DC Celniker SE Hoskins RA Dunham MJ ZhengY Koshland D 2010 Competitive repair by naturally dispersed

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repetitive DNA during non-allelic homologous recombination PLoSGenet 6e1001228

Hoffmann AA Sgro CM 2011 Climate change and evolutionary adap-tation Nature 470479ndash485

Hoffman CS Winston F 1987 A 10-minute DNA preparation from yeastefficiently releases autonomous plasmids for transformation ofEscherichia coli Gene 57267ndash272

Hou J Friedrich A de Montigny J Schacherer J 2014 Chromosomalrearrangements as a major mechanism in the onset of reproductiveisolation in Saccharomyces cerevisiae Curr Biol 241153ndash1159

Hou J Friedrich A Gounot JS Schacherer J 2015 Comprehensive surveyof condition-specific reproductive isolation reveals genetic incom-patibility in yeast Nat Commun 67214

Huerta-Sanchez E Casey FP 2015 Archaic inheritance supporting high-altitude life in Tibet J Appl Physiol (1985) 1191129ndash1134

Jones FC Grabherr MG Chan YF Russell P Mauceli E Johnson JSwofford R Pirun M Zody MC White S et al 2012 The genomicbasis of adaptive evolution in threespine sticklebacks Nature48455ndash61

Kao KC Sherlock G 2008 Molecular characterization of clonal interfer-ence during adaptive evolution in asexual populations ofSaccharomyces cerevisiae Nat Genet 401499ndash1504

Kawecki TJ Lenski RE Ebert D Hollis B Olivieri I Whitlock MC 2012Experimental evolution Trends Ecol Evol 27547ndash560

Kellis M Patterson N Endrizzi M Birren B Lander ES 2003 Sequencingand comparison of yeast species to identify genes and regulatoryelements Nature 423241ndash254

Kelly B Whiteley A Tallmon D 2010 The Arctic melting pot Nature468891ndash891

Kvitek DJ Sherlock G 2011 Reciprocal sign epistasis between frequentlyexperimentally evolved adaptive mutations causes a rugged fitnesslandscape PLoS Genet 7e1002056

Kvitek DJ Sherlock G 2013 Whole genome whole population sequenc-ing reveals that loss of signaling networks is the major adaptivestrategy in a constant environment PLoS Genet 9e1003972

Landry CR Hartl DL Ranz JM 2007 Genome clashes in hybrids insightsfrom gene expression Heredity (Edinb) 99483ndash493

Lang GI Desai MM 2014 The spectrum of adaptive mutations in ex-perimental evolution Genomics 104412ndash416

Lapunzina P Monk D 2011 The consequences of uniparental disomyand copy number neutral loss-of-heterozygosity during human de-velopment and cancer Biol Cell 103303ndash317

Leducq JB Nielly-Thibault L Charron G Eberlein C Verta JP Samani PSylvester K Hittinger CT Bell G Landry CR 2016 Speciation drivenby hybridization and chromosomal plasticity in a wild yeast NatMicrobiol 115003

Lee HY Chou JY Cheong L Chang NH Yang SY Leu JY 2008Incompatibility of nuclear and mitochondrial genomes causes hy-brid sterility between two yeast species Cell 1351065ndash1073

Li H Durbin R 2009 Fast and accurate short read alignment withBurrows-Wheeler transform Bioinformatics 251754ndash1760

Li H Handsaker B Wysoker A Fennell T Ruan J Homer N Marth GAbecasis G Durbin R 2009 The Sequence AlignmentMap formatand SAMtools Bioinformatics 252078ndash2079

Liti G 2015 The fascinating and secret wild life of the budding yeast Scerevisiae Elife 4e05835

Liti G Barton DB Louis EJ 2006 Sequence diversity reproductiveisolation and species concepts in Saccharomyces Genetics174839ndash850

Lloyd AH Ranoux M Vautrin S Glover N Fourment J Charif D ChouletF Lassalle G Marande W Tran J et al 2014 Meiotic gene evolutioncan you teach a new dog new tricks Mol Biol Evol 311724ndash1727

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Louis VL Despons L Friedrich A Martin T Durrens P Casaregola SNeuveglise C Fairhead C Marck C Cruz JA et al 2012 Pichia sorbi-tophila an interspecies yeast hybrid reveals early steps of genomeresolution after polyploidization G3 (Bethesda) 2299ndash311

Mallet J 2007 Hybrid speciation Nature 446279ndash283Marcet-Houben M Gabaldon T 2015 Beyond the whole-genome du-

plication phylogenetic evidence for an ancient interspecies hybrid-ization in the bakerrsquos yeast lineage PLoS Biol 13e1002220

Marinoni G Manuel M Petersen RF Hvidtfeldt J Sulo P Piskur J 1999Horizontal transfer of genetic material among Saccharomyces yeastsJ Bacteriol 1816488ndash6496

Martin A Orgogozo V 2013 The loci of repeated evolution a catalog ofgenetic hotspots of phenotypic variation Evolution 671235ndash1250

Masly JP Jones CD Noor MAF Locke J Orr HA 2006 Gene transpositionas a cause of hybrid sterility in Drosophila Science 3131448ndash1450

Mason AS Batley J 2015 Creating new interspecific hybrid and polyploidcrops Trends Biotechnol 33436ndash441

Mavarez J Salazar CA Bermingham E Salcedo C Jiggins CD Linares M2006 Speciation by hybridization in Heliconius butterflies Nature441868ndash871

McKenna A Hanna M Banks E Sivachenko A Cibulskis K Kernytsky AGarimella K Altshuler D Gabriel S Daly M et al 2010 The genomeanalysis toolkit a MapReduce framework for analyzing next-generation DNA sequencing data Genome Res 201297ndash1303

Meiklejohn CD Holmbeck MA Siddiq MA Abt DN Rand DMMontooth KL 2013 An Incompatibility between a mitochondrialtRNA and its nuclear-encoded tRNA synthetase compromises de-velopment and fitness in Drosophila PLoS Genet 9e1003238

Meyer A Salzburger W Schartl M 2006 Hybrid origin of a swordtailspecies (Teleostei Xiphophorus clemenciae) driven by sexual selec-tion Mol Ecol 15721ndash730

Michalak P 2009 Epigenetic transposon and small RNA determinants ofhybrid dysfunctions Heredity (Edinb) 10245ndash50

Miller AW Dunham MJ 2013 Design and use of multiplexed chemostatarrays J Vis Exp 72e50262

Monod J 1949 The growth of bacterial cultures Annu Rev Microbiol3371ndash394

Mouillon JM Persson BL 2006 New aspects on phosphate sensing andsignalling in Saccharomyces cerevisiae FEMS Yeast Res 6171ndash176

Muhlfeld CC Kovach RP Jones LA Al-Chokhachy R Boyer MC Leary RFLowe WH Luikart G Allendorf FW 2014 Invasive hybridization in athreatened species is accelerated by climate change Nat ClimChange 4620ndash624

Muller LAH McCusker JH 2009 A multispecies-based taxonomic mi-croarray reveals interspecies hybridization and introgression inSaccharomyces cerevisiae FEMS Yeast Res 9143ndash152

Nagalakshmi U Wang Z Waern K Shou C Raha D Gerstein M SnyderM 2008 The transcriptional landscape of the yeast genome definedby RNA sequencing Science 3201344ndash1349

Nakao Y Kanamori T Itoh T Kodama Y Rainieri S Nakamura NShimonaga T Hattori M Ashikari T 2009 Genome sequence ofthe lager brewing yeast an interspecies hybrid DNA Res 16115ndash129

Nolte AW Freyhof J Stemshorn KC Tautz D 2005 An invasive lineage ofsculpins Cottus sp (Pisces Teleostei) in the Rhine with new habitatadaptations has originated from hybridization between old phylo-geographic groups Proc Biol Sci 2722379ndash2387

Novick A Szilard L 1950a Description of the chemostat Science112715ndash716

Novick A Szilard L 1950b Experiments with the Chemostat on spon-taneous mutations of bacteria Proc Natl Acad Sci U S A 36708ndash719

Ouyang YD Liu YG Zhang QF 2010 Hybrid sterility in plant storiesfrom rice Curr Opin Plant Biol 13186ndash192

Payen C Di Rienzi SC Ong GT Pogachar JL Sanchez JC SunshineAB Raghuraman MK Brewer BJ Dunham MJ 2014 The dynam-ics of diverse segmental amplifications in populations ofSaccharomyces cerevisiae adapting to strong selection G3(Bethesda) 4399ndash409

Payen C Sunshine AB Ong GT Pogachar JL Zhao W Dunham MJ 2016High-throughput identification of adaptive mutations in experimen-tally evolved yeast populations PLoS Genet 12e1006339

Perez-Prat E van Lookeren Campagne MM 2002 Hybrid seed produc-tion and the challenge of propagating male-sterile plants TrendsPlant Sci 7199ndash203

Smukowski Heil et al doi101093molbevmsx098 MBE

16

Peris D Langdon QK Moriarty RV Sylvester K Bontrager M Charron GLeducq JB Landry CR Libkind D Hittinger CT 2016 Complex ances-tries of lager-brewing hybrids were shaped by standing variation inthe wild yeast Saccharomyces eubayanus PLoS Genet 12e1006155

Perlstein EO Ruderfer DM Roberts DC Schreiber SL Kruglyak L 2007Genetic basis of individual differences in the response to small-molecule drugs in yeast Nat Genet 39496ndash502

Piatkowska EM Naseeb S Knight D Delneri D 2013 Chimeric proteincomplexes in hybrid species generate novel phenotypes PLoS Genet9e1003836

Piotrowski JS Nagarajan S Kroll E Stanbery A Chiotti KE Kruckeberg ALDunn B Sherlock G Rosenzweig F 2012 Different selective pressureslead to different genomic outcomes as newly-formed hybrid yeastsevolve BMC Evol Biol 1246

Pryszcz LP Nemeth T Gacser A Gabaldon T 2014 Genome comparisonof Candida orthopsilosis clinical strains reveals the existence of hy-brids between two distinct subspecies Genome Biol Evol61069ndash1078

Racimo F Sankararaman S Nielsen R Huerta-Sanchez E 2015 Evidencefor archaic adaptive introgression in humans Nat Rev Genet16359ndash371

Rieseberg LH 1991 Homoploid reticulate evolution in Helianthus(Asteraceae)mdashevidence from ribosomal genes Am J Bot781218ndash1237

Rieseberg LH 1997 Hybrid origins of plant species Annu Rev EcolSystemat 28359ndash389

Robinson JT Thorvaldsdottir H Winckler W Guttman M Lander ESGetz G Mesirov JP 2011 Integrative genomics viewer NatBiotechnol 2924ndash26

Rosenthal DM Schwarzbach AE Donovan LA Raymond O RiesebergLH 2002 Phenotypic differentiation between three ancient hybridtaxa and their parental species Int J Plant Sci 163387ndash398

Ruderfer DM Pratt SC Seidel HS Kruglyak L 2006 Population genomicanalysis of outcrossing and recombination in yeast Nat Genet381077ndash1081

Rundle HD Nagel L Boughman JW Schluter D 2000 Natural selectionand parallel speciation in sympatric sticklebacks Science287306ndash308

Ryland GL Doyle MA Goode D Boyle SE Choong DY Rowley SM Li JBowtell DD Tothill RW Campbell IG et al 2015 Australian OvarianCancer Study Group Loss of heterozygosity what is it good for BMCMed Genomics 845

Salverda MLM Dellus E Gorter FA Debets AJM van der Oost J HoekstraRF Tawfik DS de Visser JAGM 2011 Initial mutations direct alter-native pathways of protein evolution PLoS Genet 7e1001321

Sanchez MR Miller AW Liachko I Sunshine AB Lynch B Huang MAlcantara E DeSevo CG Pai DA Tucker CM et al 2017 Differentialparalog divergence modulates genome evolution across yeast spe-cies PLoS Genet 13(2)e1006585

Scannell DR Zill OA Rokas A Payen C Dunham MJ Eisen MB Rine JJohnston M Hittinger CT 2011 The awesome power of yeast evo-lutionary genetics new genome sequences and strain resources forthe Saccharomyces sensu stricto genus G3 (Bethesda) 111ndash25

Schroder MS Martinez de San Vicente K Prandini TH Hammel SHiggins DG Bagagli E Wolfe KH Butler G 2016 Multiple originsof the pathogenic yeast Candida orthopsilosis by separate hybridiza-tions between two parental species PLoS Genet 12e1006404

Schumer M Rosenthal GG Andolfatto P 2014 How common is homo-ploid hybrid speciation Evolution 681553ndash1560

Schwarzbach AE Donovan LA Rieseberg LH 2001 Transgressivecharacter expression in a hybrid sunflower species Am J Bot88270ndash277

Selmecki AM Maruvka YE Richmond PA Guillet M Shoresh NSorenson AL De S Kishony R Michor F Dowell R et al 2015Polyploidy can drive rapid adaptation in yeast Nature 519349ndash52

Seshan VE Olshen A 2016 DNAcopy DNA copy number data analysisVersion R package version 1460

Soltis DE Visger CJ Soltis PS 2014 The polyploidy revolution then andnow Stebbins revisited Am J Bot 1011057ndash1078

Soltis PS 2013 Hybridization speciation and novelty J Evol Biol26291ndash293

Soltis PS Soltis DE 2009 The role of hybridization in plant speciationAnnu Rev Plant Biol 60561ndash588

Sunshine AB Payen C Ong GT Liachko I Tan KM Dunham MJ 2015The fitness consequences of aneuploidy are driven by condition-dependent gene effects PLoS Biol 13e1002155

Tamai Y Momma T Yoshimoto H Kaneko Y 1998 Co-existence of twotypes of chromosome in the bottom fermenting yeastSaccharomyces pastorianus Yeast 14923ndash933

Taylor DJ Hebert PDN 1993 Habitat-dependent hybrid parentage anddifferential introgression between neighboringly sympatric Daphniaspecies Proc Natl Acad Sci U S A 907079ndash7083

Thiagalingam S Laken S Willson JK Markowitz SD Kinzler KWVogelstein B Lengauer C 2001 Mechanisms underlying losses ofheterozygosity in human colorectal cancers Proc Natl Acad Sci US A 982698ndash2702

Tuna M Knuutila S Mills GB 2009 Uniparental disomy in cancer TrendsMol Med 15120ndash128

Walther A Hesselbart A Wendland J 2014 Genome sequence ofSaccharomyces carlsbergensis the worldrsquos first pure culture lageryeast G3 (Bethesda) 4783ndash793

Wayne RK 1993 Molecular evolution of the dog family Trends Genet9218ndash224

Wenger JW Schwartz K Sherlock G 2010 Bulk segregant analysis byhigh-throughput sequencing reveals a novel xylose utilization genefrom Saccharomyces cerevisiae PLoS Genet 6e1000942

Wessinger CA Rausher MD 2014 Predictability and irreversibility ofgenetic changes associated with flower color evolution inPenstemon barbatus Evolution 681058ndash1070

Wolfe KH 2015 Origin of the yeast whole-genome duplication PLoSBiol 13e1002221

Woods R Schneider D Winkworth CL Riley MA Lenski RE 2006 Testsof parallel molecular evolution in a long-term experiment withEscherichia coli Proc Natl Acad Sci U S A 1039107ndash9112

Wykoff DD OrsquoShea EK 2001 Phosphate transport and sensing inSaccharomyces cerevisiae Genetics 1591491ndash1499

Xiong Z Gaeta RT Pires JC 2011 Homoeologous shuffling andchromosome compensation maintain genome balance inresynthesized allopolyploid Brassica napus Proc Natl Acad SciU S A 1087908ndash7913

Yassour M Kapian T Fraser HB Levin JZ Pfiffner J Adiconis X Schroth GLuo SJ Khrebtukova I Gnirke A et al 2009 Ab initio construction ofa eukaryotic transcriptome by massively parallel mRNA sequencingProc Natl Acad Sci U S A 1063264ndash3269

Zhu YO Sherlock G Petrov DA 2016 Whole genome analysis of 132clinical Saccharomyces cerevisiae strains reveals extensive ploidy var-iation G3 (Bethesda) doi101534g3116029397

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one speciesstrain is replaced with the allele of the otherspeciesstrain in an otherwise isogenic background We testedthis hypothesis using the most commonly seen LOH eventLOH at PHO84 Although the region extends from 25 kb to234 kb in length in the hybrids and from 40 kb to 85 kb in Scerevisiae and thus includes many genes PHO84 was a primecandidate driving this event PHO84 is one of only ten genesencompassed in the region extending from the telomere tothe break point of the shortest LOH event and is included inevery other LOH event on chromosome XIII (fig 2) It is ahigh-affinity phosphate transporter responsible for inorganicphosphate uptake in high and low phosphate conditions(Wykoff and OrsquoShea 2001) and previous work identified apoint mutation in PHO84 (an alanine to valine substitution atthe 50 end of the gene) which increased fitness by 23 inphosphate-limited conditions (Sunshine et al 2015) Finallyprior work with other nutrient transporters has shown am-plification of nutrient transporters to be a key event in adapt-ing to nutrient-limited conditions

We thus selected a region of approximately 25 kb encom-passing the PHO84 ORF its promoter and 30-UTR(Nagalakshmi et al 2008 Yassour et al 2009 Cherry et al2012) We created allele replacement strains using the twoalleles of S cerevisiae in a S cerevisiae diploid background thetwo alleles are 991 identical in this region (supplementaryfig S5 Supplementary Material online) and each strain isidentical to the ancestral strain used in our evolution exper-iments except at the PHO84 locus The S cerevisiae ancestorcarries one copy of GRF167 (preferred) and one copy ofS288C (ldquoun-preferredrdquo) so named due to which allele wasretained and amplified in the evolved clones To measureany resultant changes in fitness we competed each strainindividually against a fluorescent ancestral strain and mea-sured which strain overtook the culture Relative fitness isthus defined as the growth advantage per generation Twocopies of the un-preferred allele decreased relative fitness by531 (6186) whereas two copies of the preferred alleleincreased relative fitness by 993 (6027) This displays anoverall difference in relative fitness of 1524 between the un-preferred and preferred alleles By comparing the fitness ofthese allele replacement strains with the evolved clones (table 2)the allele replacement does not fully recapitulate the fit-ness gain observed in the evolved clone One explanation isthat the additional mutations present in the evolvedstrains also contribute to their total fitness Another expla-nation could be the increased copy number of the PHO84region that we see in these evolved clones To further ex-plore this fitness difference we cloned the GRF167 alleleonto a low copy number plasmid and transformed theallele replacement strain carrying two preferred S cerevi-siae alleles to simulate increased copy number of PHO84and saw only a minimal fitness increase of 176 (6106note all fitness competitions involving plasmids were donein conjunction with empty plasmids to take into accountany fitness effects from the plasmid itself) This supportsthe conclusion that relative fitness gains in the evolvedclone are largely due to the loss of the S288C allele andselection and amplification of the GRF167 allele with little

additional benefit from further amplification It could also bethe case that co-amplification of other genes in the segmentis required to attain the full benefit as previously observedby the contribution of BSD2 to the SUL1 amplicon in sulfatelimitation (Sunshine et al 2015 Payen et al 2016)

To understand the fitness effects of LOH and amplificationin the hybrids we generated hybrid strains with varying num-bers of S cerevisiae GRF167 PHO84 alleles Unfortunately wewere unable to obtain a successful strain carrying the pre-ferred S cerevisiae allele in a S uvarum background howeverwe were able to generate a S uvarum PHO84 knockout strainthus creating a hybrid with one copy of S cerevisiae PHO84When combined with a low copy plasmid carrying the sameallele this strain effectively has two or more copies ofS cerevisiae PHO84 in a hybrid background This hybrid straincan serve as a proxy for the LOH event observed in the evo-lution experiments and has a relative fitness gain of 2557(6288) The ancestral hybrid with the same plasmid (effec-tively 1 S uvarum allele and 2 or more S cerevisiae alleles) hasa relative fitness gain of 1253 (6131) The difference be-tween these two hybrids suggests that while amplification isbeneficial the highest fitness is achieved with the loss of theS uvarum allele (Pfrac14 00061)

Together these results support the conclusion that theS cerevisiae GRF167 allele is preferred over the S288C alleleand that S cerevisiae alleles are preferred over the S uvarumallele in the hybrid and hence that the LOH events seen inboth intra- and interspecific hybrids are the product ofselection

DiscussionIn summary we sought to understand forces underlying ge-nome stabilization and evolution in interspecific and intra-specific hybrids as they adapt to novel environments Weevolved and sequenced clones from 16 hybrid populationsand 16 parental populations to reveal a variety of mutationalevents conferring adaptation to 3 nutrient-limited conditionsOf particular note we find LOH in both evolved intraspecificand interspecific hybrid clones in all nutrient environmentspotentially signifying areas where selection has acted on pre-existing variation present in the ancestral clone We used anallele replacement strategy to test this hypothesis for a com-monly repeated LOH event and show that selection is indeeddriving the homogenization of the genome at this locusAlthough other studies in natural industrial and lab-evolved isolates have observed LOH we present the first em-pirical test of the causal evolutionary forces influencing theseevents This work can begin to help us understand past hy-bridization events and subsequent genome resolution in hy-brids in natural and artificial systems

Similarities and Differences between Intra- andInterspecific HybridsAlthough our sample size is modest we observe several in-teresting trends when comparing S uvarum clones (homo-zygous) S cerevisiae clones (intraspecific hybrid andpreviously published homozygous) and interspecific hybrid

Adaptation in Hybrids doi101093molbevmsx098 MBE

11

clones First theory and previous research predict the inter-specific hybrid may experience more genome instability in theform of chromosomal rearrangements and CNV events(Xiong et al 2011 Lloyd et al 2014 Chester et al 2015Mason and Batley 2015) Instead in our work the interspecifichybrid and S cerevisiae clones experience the same numberof CNV events (both 15 CNVsclone) However the mech-anism of CNV formation seems to differ between the hybridand S cerevisiae clones Whereas S cerevisiae CNV breakpoints typically occur at transposable elements in our studyand previous studies (Dunham et al 2002 Gresham et al2008 Fedoroff 2012) the interspecific hybrid CNVs do notutilize transposable elements although they obviously sharethe same sequence background as the S cerevisiae clones(albeit in one copy) Whether this difference is due to theabsence of full-length transposable elements in S uvarum isunknown but this could potentially explain the lower num-ber of CNV events in S uvarum clones (1 CNVclone) andpresents an intriguing direction for future study

The Predictability of EvolutionWe now have many examples of predictable evolution innatural systems (Losos et al 1998 Rundle et al 2000 Elmerand Meyer 2011 Conte et al 2012 Jones et al 2012 Martinand Orgogozo 2013 Wessinger and Rausher 2014) and inlaboratory experimental evolution in which there often ap-pears to be a limited number of high fitness pathways thatstrains follow when adapting to a particular condition (Fereaet al 1999 Woods et al 2006 Gresham et al 2008 Burke et al2010 Salverda et al 2011 Kawecki et al 2012 Kvitek andSherlock 2013 Lang and Desai 2014) For example it is wellestablished that amplifications of nutrient transporters aredrivers of adaptation in evolution in nutrient-limited condi-tions Previous work in our group has particularly focused onthe amplification of the high-affinity sulfate transporter geneSUL1 in sulfate-limited conditions which occurs in almostevery sulfate-limited evolution experiment and confers a fit-ness advantage of as much as 40 compared with the un-evolved ancestor strain The amplification of phosphatetransporters has been markedly less common and thus driv-ers of adaptation in this condition have been less clearGresham et al (2008) identified a whole chromosome ampli-fication of chrXIII in one population In a follow-up studySunshine et al (2015) found whole or partial amplification ofchrXIII in 38 populations A genome-wide screen for segmen-tal amplifications found a slight increase in fitness for a smalltelomeric segment of chromosome XIII and a A49V pointmutation in PHO84 was observed to increase fitness by 23However screens by Payen et al (2016) showed that althoughPHO84 is recurrently mutated in various experiments itshowed no benefit when amplified or deleted in phos-phate-limited conditions Finally additional evolution exper-iments recapitulated the point mutation seen in Sunshineet al (2015) in 2432 populations and saw amplification ofPHO84 in 832 populations (Miller A Dunham MJ unpub-lished data) It is important to note that all these experimentsused a strain background derived from S288C or CENPKboth of which carry the same (un-preferred) PHO84 allele

In our work we observed amplification of the S cerevisiaeGRF167 allele of PHO84 in 44 S cerevisiae clones from 4populations and 36 hybrid clones from 6 populations Thisamplification was always preceded by the loss of the S288Callele in S cerevisiae clones and the LOH break points arenever shared with the amplification break points There is a15 fitness difference between carrying two copies of theS288C allele of PHO84 compared with carrying two copiesof the GRF167 allele of PHO84 and additional copies of theGRF167 allele do not provide substantial further fitness gainsThe two alleles differ by several noncoding changes and threenonsynonymous substitutions a mutation from glutamicacid to aspartic acid (E229D) leucine to proline (L259P)and leucine to glutamine (L556Q) the latter two of whichare considered ldquononconservativerdquo protein mutations due tochanges in hydrophobicity and structure (supplementary figS5 Supplementary Material online) Intriguingly the L259Pmutation has actually been previously identified as being re-sponsible for resistance to tetrachloroisophthalonitrile andpartial resistance to pentachlorophenol in a QTL study ofsmall-molecule drugs (Perlstein et al 2007) IndeedPerlstein et al found proline at residue 259 to be conservedacross fungal species and even in orthologous human xeno-biotic transporters likely because proline-induced kinks intransmembrane spans have been shown to be essential toprotein function (Cordes et al 2002 Perlstein et al 2007) Itthus appears that amplification of PHO84 has been less pre-dictable as the S288C allele does not confer a fitness advan-tage unless mutated per Sunshine et al (2015) Togetherthese results imply that strain background can constrainadaptive pathways

In hybrids the amplification of the S cerevisiae segmentoccurred in conjunction with the loss of the S uvarum alleleHybrid strains with the LOH had a 2557 relative fitness gainwhereas hybrid strains with amplification of the S cerevisiaePHO84 allele without the LOH had a 1253 fitness gain ThusLOH confers an additional 1304 fitness gain showing thatselection for LOH has a larger impact on fitness than ampli-fication alone Note that S uvarum does have proline at res-idue 259 like the preferred GRF167 allele and differs fromboth S cerevisiae alleles at the other two coding substitutions(229N and 556K) but the amino acid and noncoding diver-gence is too high to speculate what substitutions are respon-sible for the selection of the GRF167 allele (supplementary figS6 Supplementary Material online) Why the loss of one alleleis more beneficial remains unclear as PHO84 is thought tofunction as a monomer (Bun-Ya et al 1991) but it may bedue to competition for cell wall space or negative interactionswith other genes in the PHO pathway (Mouillon and Persson2006)

The infusion of variation created by hybridization providesnew templates for selection to act upon which can be moreimportant than either point mutations or copy number var-iants alone Our work shows that outcrossing need not becommon to have long-lasting effects on adaptation This im-plication is particularly relevant in yeast where outcrossingmay occur quite rarely followed by thousands of asexual gen-erations (Ruderfer et al 2006 Greig and Leu 2009 Liti 2015)

Smukowski Heil et al doi101093molbevmsx098 MBE

12

Applications to Other Hybrids and CancerThe observation that LOH occurs in hybrid genomes is in-creasingly documented (Louis et al 2012 Borneman et al2014 Soltis et al 2014 Marcet-Houben and Gabaldon 2015Pryszcz et al 2014 Schroder et al 2016) although the rea-son(s) for this type of mutation has been unresolved As mostexamples stem from allopolyploid events that occurred mil-lions of years ago understanding why LOH is important inhybrid genome evolution is difficult Cancer cells are alsoknown to experience LOH sometimes involved in the inac-tivation of tumor suppressor genes leaving only one copy ofthe gene that may be mutated or silenced (Thiagalingam et al2001 Tuna et al 2009 Lapunzina and Monk 2011) Datasupport the conclusion that LOH events are selected for dur-ing tumor development as many LOH events involve specificchromosomal segments (Thiagalingam et al 2001) althoughthe underlying molecular and genetic reasons for selection isan open debate (Ryland et al 2015)

Here we experimentally demonstrate that LOH can occurin homoploid interspecific hybrids as well as in intraspecifichybrids These events occur within a few hundred generationsand are common mutations more common on average inthe intraspecific hybrid (13 eventsclone) than the interspe-cific hybrid (056 eventsclone) Interestingly the LOH eventsdo not share break points with the CNV events in S cerevisiaeinstead they appear to occur independently and to precedeany subsequent amplification (amplification occurs following913 LOH events) Competition assays with the PHO84 locusprovide support that LOH itself may be more beneficial thanamplification or at least increase the selective benefit of am-plification events The observation that LOH in intraspecifichybrids occurs independently from copy number change pro-vides different opportunities for adaptation to novelconditions

Other cases of LOH like the copy number neutral eventsobserved in Ph4 Ph5 and Sh4 all of which favor the retentionof the S uvarum allele (supplementary fig S3 SupplementaryMaterial online) may be due to hybrid incompatibility withina particular protein complex other epistatic interactions(Piatkowska et al 2013) or neutral processes We furthermorediscover examples where one species allele appears to bepreferred over the other without LOH such as the repeatedamplification of the S uvarum high-affinity glucose transpor-ters HXT67 When one species allele is amplified and theother is not amplified one explanation is that the local se-quence context can permit or deny amplification In the caseof HXT67 previous experiments in S cerevisiae have shownthat amplification of HXT67 is quite common (Brown et al1998 Dunham et al 2002 Gresham et al 2008 Kao andSherlock 2008 Kvitek and Sherlock 2011) thus suggestingthat when given a choice between this locus in S cerevisiaeor S uvarum in the hybrid the preferred allele is indeed Suvarum though more subtle differences in rate cannot yet beruled out A similar scenario is observed with S cerevisiaeSUL1 (Sanchez et al 2017) Together our results show thatthe heterozygosity supplied by hybridization is an importantcontributor to adaptive routes explored by populations asthey adapt to novel conditions

Although we cannot generalize our results from thePHO84 locus across the many other LOH events discoveredin our hybrids and S cerevisiae in the future we can usesimilar methodology to explore whether positive selectionalways drives LOH or whether other explanations such asincompatibility resolution contribute as well Future experi-ments might also utilize a high throughput method to ex-plore segmental LOH in hybrids at a genome-wide scalesimilar to ongoing experiments at the gene level (LancasterS Dunham MJ unpublished data) Although our sample sizeis moderate this is a novel and necessary step in understand-ing forces underlying hybrid genome stabilization and high-lighting an underappreciated mechanism of hybridadaptation

ConclusionsThe mutation events we observe in our experimentallyevolved hybrids are in many ways quite representative ofmutations observed in ancient hybrid genomes suggestingthat hybrid genome stabilization and adaptation can occurquite rapidly (within several hundred generations)Furthermore our results illustrate that the infusion of varia-tion introduced by hybridization at both the intra- and inter-species level can increase fitness by providing choices of allelesfor selection to act upon even when sexual reproduction israre This may be particularly important for leveraging existingvariation for agricultural and industrial processes and as cli-mate change potentially increases natural hybridization (Kellyet al 2010 Hoffmann and Sgro 2011 Muhlfeld et al 2014)

Materials and Methods

StrainsA list of strains used in this study is included in supplementarytable S1 Supplementary Material online All interspecific hy-brids were created by crossing a ura3 LYS2 haploid parent to aURA3 lys2 haploid parent of the other mating type plating onmedia lacking both uracil and lysine and selecting for proto-trophs The S cerevisiae strain background known asldquoGRF167rdquo is itself a cross between FL100 and the genomictype strain S288C (data not shown) GRF167 was chosen as astrain background for simultaneous work investigating trans-posable elements during experimental evolution which willbe addressed in a future study

Evolution ExperimentsContinuous cultures were established using media and con-ditions previously described (Gresham et al 2008 Sanchezet al 2017) Detailed protocols and media recipes are availableat httpdunhamgswashingtoneduprotocolsshtml (lastaccessed March 6 2017) Samples were taken daily and mea-sured for optical density at 600 nm and cell count micros-copy was performed to check for contamination andarchival glycerol stocks were made daily An experimentwas terminated when contamination growth in tubing orclumping appeared (number of generations at the endpoint for each population are presented in tables 1 and

Adaptation in Hybrids doi101093molbevmsx098 MBE

13

2) Samples from each end point population were colonypurified to yield two clones for further study

Array Comparative Genomic HybridizationPopulations from the end point of each evolution were ana-lyzed for copy number changes using array comparative ge-nomic hybridization following the protocol used in Sanchezet al (2017)

SequencingDNA was extracted from overnight cultures using theHoffmanndashWinston protocol (Hoffman and Winston 1987)and cleaned using the Clean amp Concentrator kit (ZymoResearch) Nextera libraries were prepared following theNextera library kit protocol and sequenced using paired end150 bp reads on the Illumina NextSeq 500 machine (sequenc-ing coverage in supplementary table S2 SupplementaryMaterial online) The reference genomes used were S cerevi-siae v3 (Engel et al 2014) S uvarum (Scannell et al 2011) anda hybrid reference genome created by concatenating the twogenomes Sequence was aligned to the appropriate referencegenome using bwa v062 (Li and Durbin 2009) and mutationswere called using GATK (McKenna et al 2010) and samtools0119 (Li et al 2009) Mutations in evolved clones were filteredin comparison with the ancestor to obtain de novo mutationsAll mutations were first visually inspected using IntegrativeGenomics Viewer (Robinson et al 2011) Subsequently pointmutations in the hybrids were confirmed with Sanger se-quencing (supplementary table S3 Supplementary Materialonline) Copy number variants were visualized usingDNAcopy for S cerevisiae and S uvarum (Seshan andOlshen 2016) LOH events were called based on sequencingcoverage in the hybrids and by identifying homozygous variantcalls in S cerevisiae All break points were called by visual in-spection of sequencing reads and are thus approximate

Fitness AssaysThe pairwise competition experiments were performed in 20ml chemostats (Miller and Dunham 2013) Each competitorstrain was cultured individually until steady state was reachedand then was mixed 5050 with a GFP-tagged ancestor Eachcompetition was conducted in at least two biological repli-cates for approximately 15 generations after mixing Sampleswere collected and analyzed twice daily The proportion ofGFPthorn cells in the population was detected using a BD AccuriC6 flow cytometer (BD Biosciences) The data were plottedwith ln [dark cellsGFPthorn cells] versus generations The rela-tive fitness coefficient was determined from the slope of thelinear region

Strain ConstructionAllele replacements for the PHO84 locus were done followingthe protocol of the Caudy lab with further modifications de-scribed here The native locus was replaced withKluyveromyces lactis URA3 The pho84DURA3 strain wasgrown overnight in 5 ml of C-URA media then inoculatedin a flask of 100 ml yeast extract peptone dextrose (YPD) andgrown to an optical density of 06ndash08 Cells were washed then

aliquoted 275 ml of transformation mix (35 ml 1 M lithiumacetate 240 ml of 50 3500 polyethylene glycol) 10 ml ofsalmon sperm and approximately 3 mg of polymerase chainreaction product were added to the cell pellet It was incu-bated at 37 C (S uvarum) or 42 C (S cerevisiae) for 45 minthen plated to YPD It was replica plated to 5-fluorooroticacid the following day and colonies were tested for the gain ofthe appropriate species allele The GRF167 allele was clonedinto the pIL37 plasmid using Gibson assembly (Gibson et al2009) Correct assembly was verified by Sanger sequencing Allprimers used can be found in supplementary table S3Supplementary Material online

Supplementary MaterialSupplementary data are available at Molecular Biology andEvolution online

AcknowledgmentsWe thank Noah Hanson and Erica Alcantara for technicalassistance We thank Monica Sanchez and three anonymousreviewers for helpful comments on this manuscript JosephSchacherer kindly confirmed the contribution of FL100 to theGRF167 strain background Thanks to Yixian Zheng and DougKoshland for contributing to the initial experimental designcreating yeast strains and purchasing the oligonucleotidesused for the microarrays This work was supported by theNational Science Foundation (grant number 1516330) andthe National Institutes of Health (grant number R01GM094306) MD is a Senior Fellow in the GeneticNetworks program at the Canadian Institute for AdvancedResearch and a Rita Allen Foundation Scholar CSH wassupported in part by National Institutes of Health (grantnumber T32 HG00035) This work was also supported bythe National Institutes of Health (grant number P50GM071508) to the Lewis-Sigler Institute and from theHoward Hughes Medical Institute to Doug Koshland andYixian Zheng Sequencing read data have been deposited atthe NCBI under BioProject accession PRJNA374049Microarray data are deposited in the Gene ExpressionOmnibus (GEO) repository under accession numbersGSE95086 and GSE87401 and in the Princeton MicroarrayDatabase

ReferencesAbbott R Albach D Ansell S Arntzen JW Baird SJ Bierne N Boughman J

Brelsford A Buerkle CA Buggs R et al 2013 Hybridization andspeciation J Evol Biol 26229ndash246

Ainouche ML Jenczewski E 2010 Focus on polyploidy New Phytol1861ndash4

Albertin W Marullo P 2012 Polyploidy in fungi evolution after whole-genome duplication Proc Biol Sci 2792497ndash2509

Almeida P Goncalves C Teixeira S Libkind D Bontrager M Masneuf-Pomarede I Albertin W Durrens P Sherman DJ Marullo P et al2014 A Gondwanan imprint on global diversity and domesticationof wine and cider yeast Saccharomyces uvarum Nat Commun54044

Antunovics Z Nguyen HV Gaillardin C Sipiczki M 2005 Gradual ge-nome stabilisation by progressive reduction of the Saccharomyces

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14

uvarum genome in an interspecific hybrid with Saccharomyces cer-evisiae FEMS Yeast Res 51141ndash1150

Baker E Wang B Bellora N Peris D Hulfachor AB Koshalek JA AdamsM Libkind D Hittinger CT 2015 The Genome sequence ofSaccharomyces eubayanus and the domestication of lager-brewingyeasts Mol Biol Evol 322818ndash2831

Barbosa R Almeida P Safar SVB Santos RO Morais PB Nielly-Thibault LLeducq JB Landry CR Goncalves P Rosa CA Sampaio JP 2016Evidence of natural hybridization in Brazilian wild lineages ofSaccharomyces cerevisiae Genome Biol Evol 8317ndash329

Barton NH 2001 The role of hybridization in evolution Mol Ecol10551ndash568

Belloch C Orlic S Barrio E Querol A 2008 Fermentative stress adapta-tion of hybrids within the Saccharomyces sensu stricto complex Int JFood Microbiol 122188ndash195

Bellon JR Yang F Day MP Inglis DL Chambers PJ 2015 Designingand creating Saccharomyces interspecific hybrids for improvedindustry relevant phenotypes Appl Microbiol Biotechnol998597ndash8609

Borneman AR Zeppel R Chambers PJ Curtin CD 2014 Insights into theDekkera bruxellensis genomic landscape comparative genomics re-veals variations in ploidy and nutrient utilisation potential amongstwine isolates PLoS Genet 10e1004161

Brewer BJ Payen C Di Rienzi SC Higgins MM Ong G Dunham MJRaghuraman MK 2015 Origin-dependent inverted-repeat amplifi-cation tests of a model for inverted DNA amplification PLoS Genet11e1005699

Brown CJ Todd KM Rosenzweig RF 1998 Multiple duplications of yeasthexose transport genes in response to selection in a glucose-limitedenvironment Mol Biol Evol 15931ndash942

Bullini L 1994 Origin and evolution of animal hybrid species Trends EcolEvol 9422ndash426

Bun-Ya M Nishimura M Harashima S Oshima Y 1991 The PHO84 geneof Saccharomyces cerevisiae encodes an inorganic phosphate trans-porter Mol Cell Biol 113229ndash3238

Burke MK Dunham JP Shahrestani P Thornton KR Rose MR Long AD2010 Genome-wide analysis of a long-term evolution experimentwith Drosophila Nature 467587ndash590

Burke MK Liti G Long AD 2014 Standing genetic variation drives re-peatable experimental evolution in outcrossing populations ofSaccharomyces cerevisiae Mol Biol Evol 313228ndash3239

Cherry JM Hong EL Amundsen C Balakrishnan R Binkley G Chan ETChristie KR Costanzo MC Dwight SS Engel SR et al 2012Saccharomyces genome database the genomics resource of buddingyeast Nucleic Acids Res 40D700ndashD705

Chester M Riley RK Soltis PS Soltis DE 2015 Patterns of chromosomalvariation in natural populations of the neoallotetraploid Tragopogonmirus (Asteraceae) Heredity (Edinb) 114309ndash317

Chou JY Leu JY 2010 Speciation through cytonuclear incompatibilityinsights from yeast and implications for higher eukaryotes Bioessays32401ndash411

Cliften PF Fulton RS Wilson RK Johnston M 2006 After the duplicationgene loss and adaptation in Saccharomyces genomes Genetics172863ndash872

Conte GL Arnegard ME Peichel CL Schluter D 2012 The probability ofgenetic parallelism and convergence in natural populations Proc BiolSci 2795039ndash5047

Cordes FS Bright JN Sansom MS 2002 Proline-induced distortions oftransmembrane helices J Mol Biol 323951ndash960

Crow JF 1998 90 years ago the beginning of hybrid maize Genetics148923ndash928

Dasmahapatra KK Walters JR Briscoe AD Davey JW Whibley A NadeauNJ Zimin AV Hughes DST Ferguson LC Martin SH et al 2012Butterfly genome reveals promiscuous exchange of mimicry adap-tations among species Nature 48794ndash98

Dowling TE Smith GR Brown WM 1989 Reproductive isolation andintrogression between Notropis-Cornutus and Notropis-Chrysocephalus (Family Cyprinidae)mdashcomparison of morphologyallozymes and mitochondrial-DNA Evolution 43620ndash634

Doyle JJ Flagel LE Paterson AH Rapp RA Soltis DE Soltis PS Wendel JF2008 Evolutionary genetics of genome merger and doubling inplants Annu Rev Genet 42443ndash461

Dunham MJ Badrane H Ferea T Adams J Brown PO Rosenzweig FBotstein D 2002 Characteristic genome rearrangements in experi-mental evolution of Saccharomyces cerevisiae Proc Natl Acad Sci U SA 9916144ndash16149

Dunn B Paulish T Stanbery A Piotrowski J Koniges G Kroll E LouisEJ Liti G Sherlock G Rosenzweig F 2013 Recurrent rearrange-ment during adaptive evolution in an interspecific yeast hybridsuggests a model for rapid introgression PLoS Genet9e1003366

Dunn B Richter C Kvitek DJ Pugh T Sherlock G 2012 Analysis of theSaccharomyces cerevisiae pan-genome reveals a pool of copy num-ber variants distributed in diverse yeast strains from differing indus-trial environments Genome Res 22908ndash924

Dunn B Sherlock G 2008 Reconstruction of the genome origins andevolution of the hybrid lager yeast Saccharomyces pastorianusGenome Res 181610ndash1623

Elmer KR Meyer A 2011 Adaptation in the age of ecological genomicsinsights from parallelism and convergence Trends Ecol Evol26298ndash306

Engel SR Dietrich FS Fisk DG Binkley G Balakrishnan R Costanzo MCDwight SS Hitz BC Karra K Nash RS et al 2014 The referencegenome sequence of Saccharomyces cerevisiae then and now G3(Bethesda) 4389ndash398

Fedoroff NV 2012 Presidential address Transposable elements epige-netics and genome evolution Science 338758ndash767

Ferea TL Botstein D Brown PO Rosenzweig RF 1999 Systematicchanges in gene expression patterns following adaptive evolutionin yeast Proc Natl Acad Sci U S A 969721ndash9726

Gibson B Liti G 2015 Saccharomyces pastorianus genomic insightsinspiring innovation for industry Yeast 3217ndash27

Gibson DG Young L Chuang RY Venter JC Hutchison CA Smith HO2009 Enzymatic assembly of DNA molecules up to several hundredkilobases Nat Methods 6343ndash5

Gonzalez SS Barrio E Gafner J Querol A 2006 Natural hybrids fromSaccharomyces cerevisiae Saccharomyces bayanus andSaccharomyces kudriavzevii in wine fermentations FEMS Yeast Res61221ndash1234

Gonzalez SS Barrio E Querol A 2008 Molecular characterization of newnatural hybrids of Saccharomyces cerevisiae and S kudriavzevii inbrewing Appl Environ Microbiol 742314ndash2320

Grant PR Grant BR 1994 Phenotypic and genetic consequences ofhybridization in Darwinrsquos finches Evolution 48297ndash316

Grant PR Grant BR 2002 Unpredictable evolution in a 30-year study ofDarwinrsquos finches Science 296707ndash711

Grant PR Grant BR 2010 Natural selection speciation and Darwinrsquosfinches Proc Calif Acad Sci 61245ndash260

Grant PR Grant BR Petren K 2005 Hybridization in the recent past AmNat 16656ndash67

Greig D 2009 Reproductive isolation in Saccharomyces Heredity (Edinb)10239ndash44

Greig D Leu JY 2009 Natural history of budding yeast Current Biol19R886ndashR890

Greig D Louis EJ Borts RH Travisano M 2002 Hybrid speciation inexperimental populations of yeast Science 2981773ndash1775

Gresham D Desai MM Tucker CM Jenq HT Pai DA Ward A DeSevoCG Botstein D Dunham MJ 2008 The repertoire and dynamics ofevolutionary adaptations to controlled nutrient-limited environ-ments in yeast PLoS Genet 4e1000303

Hajjar R Hodgkin T 2007 The use of wild relatives in crop improvementa survey of developments over the last 20 years Euphytica 1561ndash13

Heiser CB 1954 Variation and subspeciation in the common sunflowerHelianthus annuus Am Midl Nat 51287ndash305

Hittinger CT 2013 Saccharomyces diversity and evolution a buddingmodel genus Trends Genet 29309ndash317

Hoang ML Tan FJ Lai DC Celniker SE Hoskins RA Dunham MJ ZhengY Koshland D 2010 Competitive repair by naturally dispersed

Adaptation in Hybrids doi101093molbevmsx098 MBE

15

repetitive DNA during non-allelic homologous recombination PLoSGenet 6e1001228

Hoffmann AA Sgro CM 2011 Climate change and evolutionary adap-tation Nature 470479ndash485

Hoffman CS Winston F 1987 A 10-minute DNA preparation from yeastefficiently releases autonomous plasmids for transformation ofEscherichia coli Gene 57267ndash272

Hou J Friedrich A de Montigny J Schacherer J 2014 Chromosomalrearrangements as a major mechanism in the onset of reproductiveisolation in Saccharomyces cerevisiae Curr Biol 241153ndash1159

Hou J Friedrich A Gounot JS Schacherer J 2015 Comprehensive surveyof condition-specific reproductive isolation reveals genetic incom-patibility in yeast Nat Commun 67214

Huerta-Sanchez E Casey FP 2015 Archaic inheritance supporting high-altitude life in Tibet J Appl Physiol (1985) 1191129ndash1134

Jones FC Grabherr MG Chan YF Russell P Mauceli E Johnson JSwofford R Pirun M Zody MC White S et al 2012 The genomicbasis of adaptive evolution in threespine sticklebacks Nature48455ndash61

Kao KC Sherlock G 2008 Molecular characterization of clonal interfer-ence during adaptive evolution in asexual populations ofSaccharomyces cerevisiae Nat Genet 401499ndash1504

Kawecki TJ Lenski RE Ebert D Hollis B Olivieri I Whitlock MC 2012Experimental evolution Trends Ecol Evol 27547ndash560

Kellis M Patterson N Endrizzi M Birren B Lander ES 2003 Sequencingand comparison of yeast species to identify genes and regulatoryelements Nature 423241ndash254

Kelly B Whiteley A Tallmon D 2010 The Arctic melting pot Nature468891ndash891

Kvitek DJ Sherlock G 2011 Reciprocal sign epistasis between frequentlyexperimentally evolved adaptive mutations causes a rugged fitnesslandscape PLoS Genet 7e1002056

Kvitek DJ Sherlock G 2013 Whole genome whole population sequenc-ing reveals that loss of signaling networks is the major adaptivestrategy in a constant environment PLoS Genet 9e1003972

Landry CR Hartl DL Ranz JM 2007 Genome clashes in hybrids insightsfrom gene expression Heredity (Edinb) 99483ndash493

Lang GI Desai MM 2014 The spectrum of adaptive mutations in ex-perimental evolution Genomics 104412ndash416

Lapunzina P Monk D 2011 The consequences of uniparental disomyand copy number neutral loss-of-heterozygosity during human de-velopment and cancer Biol Cell 103303ndash317

Leducq JB Nielly-Thibault L Charron G Eberlein C Verta JP Samani PSylvester K Hittinger CT Bell G Landry CR 2016 Speciation drivenby hybridization and chromosomal plasticity in a wild yeast NatMicrobiol 115003

Lee HY Chou JY Cheong L Chang NH Yang SY Leu JY 2008Incompatibility of nuclear and mitochondrial genomes causes hy-brid sterility between two yeast species Cell 1351065ndash1073

Li H Durbin R 2009 Fast and accurate short read alignment withBurrows-Wheeler transform Bioinformatics 251754ndash1760

Li H Handsaker B Wysoker A Fennell T Ruan J Homer N Marth GAbecasis G Durbin R 2009 The Sequence AlignmentMap formatand SAMtools Bioinformatics 252078ndash2079

Liti G 2015 The fascinating and secret wild life of the budding yeast Scerevisiae Elife 4e05835

Liti G Barton DB Louis EJ 2006 Sequence diversity reproductiveisolation and species concepts in Saccharomyces Genetics174839ndash850

Lloyd AH Ranoux M Vautrin S Glover N Fourment J Charif D ChouletF Lassalle G Marande W Tran J et al 2014 Meiotic gene evolutioncan you teach a new dog new tricks Mol Biol Evol 311724ndash1727

Losos JB Jackman TR Larson A de Queiroz K Rodriguez-Schettino L1998 Contingency and determinism in replicated adaptive radia-tions of island lizards Science 2792115ndash2118

Louis VL Despons L Friedrich A Martin T Durrens P Casaregola SNeuveglise C Fairhead C Marck C Cruz JA et al 2012 Pichia sorbi-tophila an interspecies yeast hybrid reveals early steps of genomeresolution after polyploidization G3 (Bethesda) 2299ndash311

Mallet J 2007 Hybrid speciation Nature 446279ndash283Marcet-Houben M Gabaldon T 2015 Beyond the whole-genome du-

plication phylogenetic evidence for an ancient interspecies hybrid-ization in the bakerrsquos yeast lineage PLoS Biol 13e1002220

Marinoni G Manuel M Petersen RF Hvidtfeldt J Sulo P Piskur J 1999Horizontal transfer of genetic material among Saccharomyces yeastsJ Bacteriol 1816488ndash6496

Martin A Orgogozo V 2013 The loci of repeated evolution a catalog ofgenetic hotspots of phenotypic variation Evolution 671235ndash1250

Masly JP Jones CD Noor MAF Locke J Orr HA 2006 Gene transpositionas a cause of hybrid sterility in Drosophila Science 3131448ndash1450

Mason AS Batley J 2015 Creating new interspecific hybrid and polyploidcrops Trends Biotechnol 33436ndash441

Mavarez J Salazar CA Bermingham E Salcedo C Jiggins CD Linares M2006 Speciation by hybridization in Heliconius butterflies Nature441868ndash871

McKenna A Hanna M Banks E Sivachenko A Cibulskis K Kernytsky AGarimella K Altshuler D Gabriel S Daly M et al 2010 The genomeanalysis toolkit a MapReduce framework for analyzing next-generation DNA sequencing data Genome Res 201297ndash1303

Meiklejohn CD Holmbeck MA Siddiq MA Abt DN Rand DMMontooth KL 2013 An Incompatibility between a mitochondrialtRNA and its nuclear-encoded tRNA synthetase compromises de-velopment and fitness in Drosophila PLoS Genet 9e1003238

Meyer A Salzburger W Schartl M 2006 Hybrid origin of a swordtailspecies (Teleostei Xiphophorus clemenciae) driven by sexual selec-tion Mol Ecol 15721ndash730

Michalak P 2009 Epigenetic transposon and small RNA determinants ofhybrid dysfunctions Heredity (Edinb) 10245ndash50

Miller AW Dunham MJ 2013 Design and use of multiplexed chemostatarrays J Vis Exp 72e50262

Monod J 1949 The growth of bacterial cultures Annu Rev Microbiol3371ndash394

Mouillon JM Persson BL 2006 New aspects on phosphate sensing andsignalling in Saccharomyces cerevisiae FEMS Yeast Res 6171ndash176

Muhlfeld CC Kovach RP Jones LA Al-Chokhachy R Boyer MC Leary RFLowe WH Luikart G Allendorf FW 2014 Invasive hybridization in athreatened species is accelerated by climate change Nat ClimChange 4620ndash624

Muller LAH McCusker JH 2009 A multispecies-based taxonomic mi-croarray reveals interspecies hybridization and introgression inSaccharomyces cerevisiae FEMS Yeast Res 9143ndash152

Nagalakshmi U Wang Z Waern K Shou C Raha D Gerstein M SnyderM 2008 The transcriptional landscape of the yeast genome definedby RNA sequencing Science 3201344ndash1349

Nakao Y Kanamori T Itoh T Kodama Y Rainieri S Nakamura NShimonaga T Hattori M Ashikari T 2009 Genome sequence ofthe lager brewing yeast an interspecies hybrid DNA Res 16115ndash129

Nolte AW Freyhof J Stemshorn KC Tautz D 2005 An invasive lineage ofsculpins Cottus sp (Pisces Teleostei) in the Rhine with new habitatadaptations has originated from hybridization between old phylo-geographic groups Proc Biol Sci 2722379ndash2387

Novick A Szilard L 1950a Description of the chemostat Science112715ndash716

Novick A Szilard L 1950b Experiments with the Chemostat on spon-taneous mutations of bacteria Proc Natl Acad Sci U S A 36708ndash719

Ouyang YD Liu YG Zhang QF 2010 Hybrid sterility in plant storiesfrom rice Curr Opin Plant Biol 13186ndash192

Payen C Di Rienzi SC Ong GT Pogachar JL Sanchez JC SunshineAB Raghuraman MK Brewer BJ Dunham MJ 2014 The dynam-ics of diverse segmental amplifications in populations ofSaccharomyces cerevisiae adapting to strong selection G3(Bethesda) 4399ndash409

Payen C Sunshine AB Ong GT Pogachar JL Zhao W Dunham MJ 2016High-throughput identification of adaptive mutations in experimen-tally evolved yeast populations PLoS Genet 12e1006339

Perez-Prat E van Lookeren Campagne MM 2002 Hybrid seed produc-tion and the challenge of propagating male-sterile plants TrendsPlant Sci 7199ndash203

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16

Peris D Langdon QK Moriarty RV Sylvester K Bontrager M Charron GLeducq JB Landry CR Libkind D Hittinger CT 2016 Complex ances-tries of lager-brewing hybrids were shaped by standing variation inthe wild yeast Saccharomyces eubayanus PLoS Genet 12e1006155

Perlstein EO Ruderfer DM Roberts DC Schreiber SL Kruglyak L 2007Genetic basis of individual differences in the response to small-molecule drugs in yeast Nat Genet 39496ndash502

Piatkowska EM Naseeb S Knight D Delneri D 2013 Chimeric proteincomplexes in hybrid species generate novel phenotypes PLoS Genet9e1003836

Piotrowski JS Nagarajan S Kroll E Stanbery A Chiotti KE Kruckeberg ALDunn B Sherlock G Rosenzweig F 2012 Different selective pressureslead to different genomic outcomes as newly-formed hybrid yeastsevolve BMC Evol Biol 1246

Pryszcz LP Nemeth T Gacser A Gabaldon T 2014 Genome comparisonof Candida orthopsilosis clinical strains reveals the existence of hy-brids between two distinct subspecies Genome Biol Evol61069ndash1078

Racimo F Sankararaman S Nielsen R Huerta-Sanchez E 2015 Evidencefor archaic adaptive introgression in humans Nat Rev Genet16359ndash371

Rieseberg LH 1991 Homoploid reticulate evolution in Helianthus(Asteraceae)mdashevidence from ribosomal genes Am J Bot781218ndash1237

Rieseberg LH 1997 Hybrid origins of plant species Annu Rev EcolSystemat 28359ndash389

Robinson JT Thorvaldsdottir H Winckler W Guttman M Lander ESGetz G Mesirov JP 2011 Integrative genomics viewer NatBiotechnol 2924ndash26

Rosenthal DM Schwarzbach AE Donovan LA Raymond O RiesebergLH 2002 Phenotypic differentiation between three ancient hybridtaxa and their parental species Int J Plant Sci 163387ndash398

Ruderfer DM Pratt SC Seidel HS Kruglyak L 2006 Population genomicanalysis of outcrossing and recombination in yeast Nat Genet381077ndash1081

Rundle HD Nagel L Boughman JW Schluter D 2000 Natural selectionand parallel speciation in sympatric sticklebacks Science287306ndash308

Ryland GL Doyle MA Goode D Boyle SE Choong DY Rowley SM Li JBowtell DD Tothill RW Campbell IG et al 2015 Australian OvarianCancer Study Group Loss of heterozygosity what is it good for BMCMed Genomics 845

Salverda MLM Dellus E Gorter FA Debets AJM van der Oost J HoekstraRF Tawfik DS de Visser JAGM 2011 Initial mutations direct alter-native pathways of protein evolution PLoS Genet 7e1001321

Sanchez MR Miller AW Liachko I Sunshine AB Lynch B Huang MAlcantara E DeSevo CG Pai DA Tucker CM et al 2017 Differentialparalog divergence modulates genome evolution across yeast spe-cies PLoS Genet 13(2)e1006585

Scannell DR Zill OA Rokas A Payen C Dunham MJ Eisen MB Rine JJohnston M Hittinger CT 2011 The awesome power of yeast evo-lutionary genetics new genome sequences and strain resources forthe Saccharomyces sensu stricto genus G3 (Bethesda) 111ndash25

Schroder MS Martinez de San Vicente K Prandini TH Hammel SHiggins DG Bagagli E Wolfe KH Butler G 2016 Multiple originsof the pathogenic yeast Candida orthopsilosis by separate hybridiza-tions between two parental species PLoS Genet 12e1006404

Schumer M Rosenthal GG Andolfatto P 2014 How common is homo-ploid hybrid speciation Evolution 681553ndash1560

Schwarzbach AE Donovan LA Rieseberg LH 2001 Transgressivecharacter expression in a hybrid sunflower species Am J Bot88270ndash277

Selmecki AM Maruvka YE Richmond PA Guillet M Shoresh NSorenson AL De S Kishony R Michor F Dowell R et al 2015Polyploidy can drive rapid adaptation in yeast Nature 519349ndash52

Seshan VE Olshen A 2016 DNAcopy DNA copy number data analysisVersion R package version 1460

Soltis DE Visger CJ Soltis PS 2014 The polyploidy revolution then andnow Stebbins revisited Am J Bot 1011057ndash1078

Soltis PS 2013 Hybridization speciation and novelty J Evol Biol26291ndash293

Soltis PS Soltis DE 2009 The role of hybridization in plant speciationAnnu Rev Plant Biol 60561ndash588

Sunshine AB Payen C Ong GT Liachko I Tan KM Dunham MJ 2015The fitness consequences of aneuploidy are driven by condition-dependent gene effects PLoS Biol 13e1002155

Tamai Y Momma T Yoshimoto H Kaneko Y 1998 Co-existence of twotypes of chromosome in the bottom fermenting yeastSaccharomyces pastorianus Yeast 14923ndash933

Taylor DJ Hebert PDN 1993 Habitat-dependent hybrid parentage anddifferential introgression between neighboringly sympatric Daphniaspecies Proc Natl Acad Sci U S A 907079ndash7083

Thiagalingam S Laken S Willson JK Markowitz SD Kinzler KWVogelstein B Lengauer C 2001 Mechanisms underlying losses ofheterozygosity in human colorectal cancers Proc Natl Acad Sci US A 982698ndash2702

Tuna M Knuutila S Mills GB 2009 Uniparental disomy in cancer TrendsMol Med 15120ndash128

Walther A Hesselbart A Wendland J 2014 Genome sequence ofSaccharomyces carlsbergensis the worldrsquos first pure culture lageryeast G3 (Bethesda) 4783ndash793

Wayne RK 1993 Molecular evolution of the dog family Trends Genet9218ndash224

Wenger JW Schwartz K Sherlock G 2010 Bulk segregant analysis byhigh-throughput sequencing reveals a novel xylose utilization genefrom Saccharomyces cerevisiae PLoS Genet 6e1000942

Wessinger CA Rausher MD 2014 Predictability and irreversibility ofgenetic changes associated with flower color evolution inPenstemon barbatus Evolution 681058ndash1070

Wolfe KH 2015 Origin of the yeast whole-genome duplication PLoSBiol 13e1002221

Woods R Schneider D Winkworth CL Riley MA Lenski RE 2006 Testsof parallel molecular evolution in a long-term experiment withEscherichia coli Proc Natl Acad Sci U S A 1039107ndash9112

Wykoff DD OrsquoShea EK 2001 Phosphate transport and sensing inSaccharomyces cerevisiae Genetics 1591491ndash1499

Xiong Z Gaeta RT Pires JC 2011 Homoeologous shuffling andchromosome compensation maintain genome balance inresynthesized allopolyploid Brassica napus Proc Natl Acad SciU S A 1087908ndash7913

Yassour M Kapian T Fraser HB Levin JZ Pfiffner J Adiconis X Schroth GLuo SJ Khrebtukova I Gnirke A et al 2009 Ab initio construction ofa eukaryotic transcriptome by massively parallel mRNA sequencingProc Natl Acad Sci U S A 1063264ndash3269

Zhu YO Sherlock G Petrov DA 2016 Whole genome analysis of 132clinical Saccharomyces cerevisiae strains reveals extensive ploidy var-iation G3 (Bethesda) doi101534g3116029397

Adaptation in Hybrids doi101093molbevmsx098 MBE

17

  • msx098-TF1
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Page 12: Loss of Heterozygosity Drives Adaptation in Hybrid Yeastdunham.gs.washington.edu/SmukowskiHeil.pdf · 2017-05-20 · Loss of Heterozygosity Drives Adaptation in Hybrid Yeast Caiti

clones First theory and previous research predict the inter-specific hybrid may experience more genome instability in theform of chromosomal rearrangements and CNV events(Xiong et al 2011 Lloyd et al 2014 Chester et al 2015Mason and Batley 2015) Instead in our work the interspecifichybrid and S cerevisiae clones experience the same numberof CNV events (both 15 CNVsclone) However the mech-anism of CNV formation seems to differ between the hybridand S cerevisiae clones Whereas S cerevisiae CNV breakpoints typically occur at transposable elements in our studyand previous studies (Dunham et al 2002 Gresham et al2008 Fedoroff 2012) the interspecific hybrid CNVs do notutilize transposable elements although they obviously sharethe same sequence background as the S cerevisiae clones(albeit in one copy) Whether this difference is due to theabsence of full-length transposable elements in S uvarum isunknown but this could potentially explain the lower num-ber of CNV events in S uvarum clones (1 CNVclone) andpresents an intriguing direction for future study

The Predictability of EvolutionWe now have many examples of predictable evolution innatural systems (Losos et al 1998 Rundle et al 2000 Elmerand Meyer 2011 Conte et al 2012 Jones et al 2012 Martinand Orgogozo 2013 Wessinger and Rausher 2014) and inlaboratory experimental evolution in which there often ap-pears to be a limited number of high fitness pathways thatstrains follow when adapting to a particular condition (Fereaet al 1999 Woods et al 2006 Gresham et al 2008 Burke et al2010 Salverda et al 2011 Kawecki et al 2012 Kvitek andSherlock 2013 Lang and Desai 2014) For example it is wellestablished that amplifications of nutrient transporters aredrivers of adaptation in evolution in nutrient-limited condi-tions Previous work in our group has particularly focused onthe amplification of the high-affinity sulfate transporter geneSUL1 in sulfate-limited conditions which occurs in almostevery sulfate-limited evolution experiment and confers a fit-ness advantage of as much as 40 compared with the un-evolved ancestor strain The amplification of phosphatetransporters has been markedly less common and thus driv-ers of adaptation in this condition have been less clearGresham et al (2008) identified a whole chromosome ampli-fication of chrXIII in one population In a follow-up studySunshine et al (2015) found whole or partial amplification ofchrXIII in 38 populations A genome-wide screen for segmen-tal amplifications found a slight increase in fitness for a smalltelomeric segment of chromosome XIII and a A49V pointmutation in PHO84 was observed to increase fitness by 23However screens by Payen et al (2016) showed that althoughPHO84 is recurrently mutated in various experiments itshowed no benefit when amplified or deleted in phos-phate-limited conditions Finally additional evolution exper-iments recapitulated the point mutation seen in Sunshineet al (2015) in 2432 populations and saw amplification ofPHO84 in 832 populations (Miller A Dunham MJ unpub-lished data) It is important to note that all these experimentsused a strain background derived from S288C or CENPKboth of which carry the same (un-preferred) PHO84 allele

In our work we observed amplification of the S cerevisiaeGRF167 allele of PHO84 in 44 S cerevisiae clones from 4populations and 36 hybrid clones from 6 populations Thisamplification was always preceded by the loss of the S288Callele in S cerevisiae clones and the LOH break points arenever shared with the amplification break points There is a15 fitness difference between carrying two copies of theS288C allele of PHO84 compared with carrying two copiesof the GRF167 allele of PHO84 and additional copies of theGRF167 allele do not provide substantial further fitness gainsThe two alleles differ by several noncoding changes and threenonsynonymous substitutions a mutation from glutamicacid to aspartic acid (E229D) leucine to proline (L259P)and leucine to glutamine (L556Q) the latter two of whichare considered ldquononconservativerdquo protein mutations due tochanges in hydrophobicity and structure (supplementary figS5 Supplementary Material online) Intriguingly the L259Pmutation has actually been previously identified as being re-sponsible for resistance to tetrachloroisophthalonitrile andpartial resistance to pentachlorophenol in a QTL study ofsmall-molecule drugs (Perlstein et al 2007) IndeedPerlstein et al found proline at residue 259 to be conservedacross fungal species and even in orthologous human xeno-biotic transporters likely because proline-induced kinks intransmembrane spans have been shown to be essential toprotein function (Cordes et al 2002 Perlstein et al 2007) Itthus appears that amplification of PHO84 has been less pre-dictable as the S288C allele does not confer a fitness advan-tage unless mutated per Sunshine et al (2015) Togetherthese results imply that strain background can constrainadaptive pathways

In hybrids the amplification of the S cerevisiae segmentoccurred in conjunction with the loss of the S uvarum alleleHybrid strains with the LOH had a 2557 relative fitness gainwhereas hybrid strains with amplification of the S cerevisiaePHO84 allele without the LOH had a 1253 fitness gain ThusLOH confers an additional 1304 fitness gain showing thatselection for LOH has a larger impact on fitness than ampli-fication alone Note that S uvarum does have proline at res-idue 259 like the preferred GRF167 allele and differs fromboth S cerevisiae alleles at the other two coding substitutions(229N and 556K) but the amino acid and noncoding diver-gence is too high to speculate what substitutions are respon-sible for the selection of the GRF167 allele (supplementary figS6 Supplementary Material online) Why the loss of one alleleis more beneficial remains unclear as PHO84 is thought tofunction as a monomer (Bun-Ya et al 1991) but it may bedue to competition for cell wall space or negative interactionswith other genes in the PHO pathway (Mouillon and Persson2006)

The infusion of variation created by hybridization providesnew templates for selection to act upon which can be moreimportant than either point mutations or copy number var-iants alone Our work shows that outcrossing need not becommon to have long-lasting effects on adaptation This im-plication is particularly relevant in yeast where outcrossingmay occur quite rarely followed by thousands of asexual gen-erations (Ruderfer et al 2006 Greig and Leu 2009 Liti 2015)

Smukowski Heil et al doi101093molbevmsx098 MBE

12

Applications to Other Hybrids and CancerThe observation that LOH occurs in hybrid genomes is in-creasingly documented (Louis et al 2012 Borneman et al2014 Soltis et al 2014 Marcet-Houben and Gabaldon 2015Pryszcz et al 2014 Schroder et al 2016) although the rea-son(s) for this type of mutation has been unresolved As mostexamples stem from allopolyploid events that occurred mil-lions of years ago understanding why LOH is important inhybrid genome evolution is difficult Cancer cells are alsoknown to experience LOH sometimes involved in the inac-tivation of tumor suppressor genes leaving only one copy ofthe gene that may be mutated or silenced (Thiagalingam et al2001 Tuna et al 2009 Lapunzina and Monk 2011) Datasupport the conclusion that LOH events are selected for dur-ing tumor development as many LOH events involve specificchromosomal segments (Thiagalingam et al 2001) althoughthe underlying molecular and genetic reasons for selection isan open debate (Ryland et al 2015)

Here we experimentally demonstrate that LOH can occurin homoploid interspecific hybrids as well as in intraspecifichybrids These events occur within a few hundred generationsand are common mutations more common on average inthe intraspecific hybrid (13 eventsclone) than the interspe-cific hybrid (056 eventsclone) Interestingly the LOH eventsdo not share break points with the CNV events in S cerevisiaeinstead they appear to occur independently and to precedeany subsequent amplification (amplification occurs following913 LOH events) Competition assays with the PHO84 locusprovide support that LOH itself may be more beneficial thanamplification or at least increase the selective benefit of am-plification events The observation that LOH in intraspecifichybrids occurs independently from copy number change pro-vides different opportunities for adaptation to novelconditions

Other cases of LOH like the copy number neutral eventsobserved in Ph4 Ph5 and Sh4 all of which favor the retentionof the S uvarum allele (supplementary fig S3 SupplementaryMaterial online) may be due to hybrid incompatibility withina particular protein complex other epistatic interactions(Piatkowska et al 2013) or neutral processes We furthermorediscover examples where one species allele appears to bepreferred over the other without LOH such as the repeatedamplification of the S uvarum high-affinity glucose transpor-ters HXT67 When one species allele is amplified and theother is not amplified one explanation is that the local se-quence context can permit or deny amplification In the caseof HXT67 previous experiments in S cerevisiae have shownthat amplification of HXT67 is quite common (Brown et al1998 Dunham et al 2002 Gresham et al 2008 Kao andSherlock 2008 Kvitek and Sherlock 2011) thus suggestingthat when given a choice between this locus in S cerevisiaeor S uvarum in the hybrid the preferred allele is indeed Suvarum though more subtle differences in rate cannot yet beruled out A similar scenario is observed with S cerevisiaeSUL1 (Sanchez et al 2017) Together our results show thatthe heterozygosity supplied by hybridization is an importantcontributor to adaptive routes explored by populations asthey adapt to novel conditions

Although we cannot generalize our results from thePHO84 locus across the many other LOH events discoveredin our hybrids and S cerevisiae in the future we can usesimilar methodology to explore whether positive selectionalways drives LOH or whether other explanations such asincompatibility resolution contribute as well Future experi-ments might also utilize a high throughput method to ex-plore segmental LOH in hybrids at a genome-wide scalesimilar to ongoing experiments at the gene level (LancasterS Dunham MJ unpublished data) Although our sample sizeis moderate this is a novel and necessary step in understand-ing forces underlying hybrid genome stabilization and high-lighting an underappreciated mechanism of hybridadaptation

ConclusionsThe mutation events we observe in our experimentallyevolved hybrids are in many ways quite representative ofmutations observed in ancient hybrid genomes suggestingthat hybrid genome stabilization and adaptation can occurquite rapidly (within several hundred generations)Furthermore our results illustrate that the infusion of varia-tion introduced by hybridization at both the intra- and inter-species level can increase fitness by providing choices of allelesfor selection to act upon even when sexual reproduction israre This may be particularly important for leveraging existingvariation for agricultural and industrial processes and as cli-mate change potentially increases natural hybridization (Kellyet al 2010 Hoffmann and Sgro 2011 Muhlfeld et al 2014)

Materials and Methods

StrainsA list of strains used in this study is included in supplementarytable S1 Supplementary Material online All interspecific hy-brids were created by crossing a ura3 LYS2 haploid parent to aURA3 lys2 haploid parent of the other mating type plating onmedia lacking both uracil and lysine and selecting for proto-trophs The S cerevisiae strain background known asldquoGRF167rdquo is itself a cross between FL100 and the genomictype strain S288C (data not shown) GRF167 was chosen as astrain background for simultaneous work investigating trans-posable elements during experimental evolution which willbe addressed in a future study

Evolution ExperimentsContinuous cultures were established using media and con-ditions previously described (Gresham et al 2008 Sanchezet al 2017) Detailed protocols and media recipes are availableat httpdunhamgswashingtoneduprotocolsshtml (lastaccessed March 6 2017) Samples were taken daily and mea-sured for optical density at 600 nm and cell count micros-copy was performed to check for contamination andarchival glycerol stocks were made daily An experimentwas terminated when contamination growth in tubing orclumping appeared (number of generations at the endpoint for each population are presented in tables 1 and

Adaptation in Hybrids doi101093molbevmsx098 MBE

13

2) Samples from each end point population were colonypurified to yield two clones for further study

Array Comparative Genomic HybridizationPopulations from the end point of each evolution were ana-lyzed for copy number changes using array comparative ge-nomic hybridization following the protocol used in Sanchezet al (2017)

SequencingDNA was extracted from overnight cultures using theHoffmanndashWinston protocol (Hoffman and Winston 1987)and cleaned using the Clean amp Concentrator kit (ZymoResearch) Nextera libraries were prepared following theNextera library kit protocol and sequenced using paired end150 bp reads on the Illumina NextSeq 500 machine (sequenc-ing coverage in supplementary table S2 SupplementaryMaterial online) The reference genomes used were S cerevi-siae v3 (Engel et al 2014) S uvarum (Scannell et al 2011) anda hybrid reference genome created by concatenating the twogenomes Sequence was aligned to the appropriate referencegenome using bwa v062 (Li and Durbin 2009) and mutationswere called using GATK (McKenna et al 2010) and samtools0119 (Li et al 2009) Mutations in evolved clones were filteredin comparison with the ancestor to obtain de novo mutationsAll mutations were first visually inspected using IntegrativeGenomics Viewer (Robinson et al 2011) Subsequently pointmutations in the hybrids were confirmed with Sanger se-quencing (supplementary table S3 Supplementary Materialonline) Copy number variants were visualized usingDNAcopy for S cerevisiae and S uvarum (Seshan andOlshen 2016) LOH events were called based on sequencingcoverage in the hybrids and by identifying homozygous variantcalls in S cerevisiae All break points were called by visual in-spection of sequencing reads and are thus approximate

Fitness AssaysThe pairwise competition experiments were performed in 20ml chemostats (Miller and Dunham 2013) Each competitorstrain was cultured individually until steady state was reachedand then was mixed 5050 with a GFP-tagged ancestor Eachcompetition was conducted in at least two biological repli-cates for approximately 15 generations after mixing Sampleswere collected and analyzed twice daily The proportion ofGFPthorn cells in the population was detected using a BD AccuriC6 flow cytometer (BD Biosciences) The data were plottedwith ln [dark cellsGFPthorn cells] versus generations The rela-tive fitness coefficient was determined from the slope of thelinear region

Strain ConstructionAllele replacements for the PHO84 locus were done followingthe protocol of the Caudy lab with further modifications de-scribed here The native locus was replaced withKluyveromyces lactis URA3 The pho84DURA3 strain wasgrown overnight in 5 ml of C-URA media then inoculatedin a flask of 100 ml yeast extract peptone dextrose (YPD) andgrown to an optical density of 06ndash08 Cells were washed then

aliquoted 275 ml of transformation mix (35 ml 1 M lithiumacetate 240 ml of 50 3500 polyethylene glycol) 10 ml ofsalmon sperm and approximately 3 mg of polymerase chainreaction product were added to the cell pellet It was incu-bated at 37 C (S uvarum) or 42 C (S cerevisiae) for 45 minthen plated to YPD It was replica plated to 5-fluorooroticacid the following day and colonies were tested for the gain ofthe appropriate species allele The GRF167 allele was clonedinto the pIL37 plasmid using Gibson assembly (Gibson et al2009) Correct assembly was verified by Sanger sequencing Allprimers used can be found in supplementary table S3Supplementary Material online

Supplementary MaterialSupplementary data are available at Molecular Biology andEvolution online

AcknowledgmentsWe thank Noah Hanson and Erica Alcantara for technicalassistance We thank Monica Sanchez and three anonymousreviewers for helpful comments on this manuscript JosephSchacherer kindly confirmed the contribution of FL100 to theGRF167 strain background Thanks to Yixian Zheng and DougKoshland for contributing to the initial experimental designcreating yeast strains and purchasing the oligonucleotidesused for the microarrays This work was supported by theNational Science Foundation (grant number 1516330) andthe National Institutes of Health (grant number R01GM094306) MD is a Senior Fellow in the GeneticNetworks program at the Canadian Institute for AdvancedResearch and a Rita Allen Foundation Scholar CSH wassupported in part by National Institutes of Health (grantnumber T32 HG00035) This work was also supported bythe National Institutes of Health (grant number P50GM071508) to the Lewis-Sigler Institute and from theHoward Hughes Medical Institute to Doug Koshland andYixian Zheng Sequencing read data have been deposited atthe NCBI under BioProject accession PRJNA374049Microarray data are deposited in the Gene ExpressionOmnibus (GEO) repository under accession numbersGSE95086 and GSE87401 and in the Princeton MicroarrayDatabase

ReferencesAbbott R Albach D Ansell S Arntzen JW Baird SJ Bierne N Boughman J

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Albertin W Marullo P 2012 Polyploidy in fungi evolution after whole-genome duplication Proc Biol Sci 2792497ndash2509

Almeida P Goncalves C Teixeira S Libkind D Bontrager M Masneuf-Pomarede I Albertin W Durrens P Sherman DJ Marullo P et al2014 A Gondwanan imprint on global diversity and domesticationof wine and cider yeast Saccharomyces uvarum Nat Commun54044

Antunovics Z Nguyen HV Gaillardin C Sipiczki M 2005 Gradual ge-nome stabilisation by progressive reduction of the Saccharomyces

Smukowski Heil et al doi101093molbevmsx098 MBE

14

uvarum genome in an interspecific hybrid with Saccharomyces cer-evisiae FEMS Yeast Res 51141ndash1150

Baker E Wang B Bellora N Peris D Hulfachor AB Koshalek JA AdamsM Libkind D Hittinger CT 2015 The Genome sequence ofSaccharomyces eubayanus and the domestication of lager-brewingyeasts Mol Biol Evol 322818ndash2831

Barbosa R Almeida P Safar SVB Santos RO Morais PB Nielly-Thibault LLeducq JB Landry CR Goncalves P Rosa CA Sampaio JP 2016Evidence of natural hybridization in Brazilian wild lineages ofSaccharomyces cerevisiae Genome Biol Evol 8317ndash329

Barton NH 2001 The role of hybridization in evolution Mol Ecol10551ndash568

Belloch C Orlic S Barrio E Querol A 2008 Fermentative stress adapta-tion of hybrids within the Saccharomyces sensu stricto complex Int JFood Microbiol 122188ndash195

Bellon JR Yang F Day MP Inglis DL Chambers PJ 2015 Designingand creating Saccharomyces interspecific hybrids for improvedindustry relevant phenotypes Appl Microbiol Biotechnol998597ndash8609

Borneman AR Zeppel R Chambers PJ Curtin CD 2014 Insights into theDekkera bruxellensis genomic landscape comparative genomics re-veals variations in ploidy and nutrient utilisation potential amongstwine isolates PLoS Genet 10e1004161

Brewer BJ Payen C Di Rienzi SC Higgins MM Ong G Dunham MJRaghuraman MK 2015 Origin-dependent inverted-repeat amplifi-cation tests of a model for inverted DNA amplification PLoS Genet11e1005699

Brown CJ Todd KM Rosenzweig RF 1998 Multiple duplications of yeasthexose transport genes in response to selection in a glucose-limitedenvironment Mol Biol Evol 15931ndash942

Bullini L 1994 Origin and evolution of animal hybrid species Trends EcolEvol 9422ndash426

Bun-Ya M Nishimura M Harashima S Oshima Y 1991 The PHO84 geneof Saccharomyces cerevisiae encodes an inorganic phosphate trans-porter Mol Cell Biol 113229ndash3238

Burke MK Dunham JP Shahrestani P Thornton KR Rose MR Long AD2010 Genome-wide analysis of a long-term evolution experimentwith Drosophila Nature 467587ndash590

Burke MK Liti G Long AD 2014 Standing genetic variation drives re-peatable experimental evolution in outcrossing populations ofSaccharomyces cerevisiae Mol Biol Evol 313228ndash3239

Cherry JM Hong EL Amundsen C Balakrishnan R Binkley G Chan ETChristie KR Costanzo MC Dwight SS Engel SR et al 2012Saccharomyces genome database the genomics resource of buddingyeast Nucleic Acids Res 40D700ndashD705

Chester M Riley RK Soltis PS Soltis DE 2015 Patterns of chromosomalvariation in natural populations of the neoallotetraploid Tragopogonmirus (Asteraceae) Heredity (Edinb) 114309ndash317

Chou JY Leu JY 2010 Speciation through cytonuclear incompatibilityinsights from yeast and implications for higher eukaryotes Bioessays32401ndash411

Cliften PF Fulton RS Wilson RK Johnston M 2006 After the duplicationgene loss and adaptation in Saccharomyces genomes Genetics172863ndash872

Conte GL Arnegard ME Peichel CL Schluter D 2012 The probability ofgenetic parallelism and convergence in natural populations Proc BiolSci 2795039ndash5047

Cordes FS Bright JN Sansom MS 2002 Proline-induced distortions oftransmembrane helices J Mol Biol 323951ndash960

Crow JF 1998 90 years ago the beginning of hybrid maize Genetics148923ndash928

Dasmahapatra KK Walters JR Briscoe AD Davey JW Whibley A NadeauNJ Zimin AV Hughes DST Ferguson LC Martin SH et al 2012Butterfly genome reveals promiscuous exchange of mimicry adap-tations among species Nature 48794ndash98

Dowling TE Smith GR Brown WM 1989 Reproductive isolation andintrogression between Notropis-Cornutus and Notropis-Chrysocephalus (Family Cyprinidae)mdashcomparison of morphologyallozymes and mitochondrial-DNA Evolution 43620ndash634

Doyle JJ Flagel LE Paterson AH Rapp RA Soltis DE Soltis PS Wendel JF2008 Evolutionary genetics of genome merger and doubling inplants Annu Rev Genet 42443ndash461

Dunham MJ Badrane H Ferea T Adams J Brown PO Rosenzweig FBotstein D 2002 Characteristic genome rearrangements in experi-mental evolution of Saccharomyces cerevisiae Proc Natl Acad Sci U SA 9916144ndash16149

Dunn B Paulish T Stanbery A Piotrowski J Koniges G Kroll E LouisEJ Liti G Sherlock G Rosenzweig F 2013 Recurrent rearrange-ment during adaptive evolution in an interspecific yeast hybridsuggests a model for rapid introgression PLoS Genet9e1003366

Dunn B Richter C Kvitek DJ Pugh T Sherlock G 2012 Analysis of theSaccharomyces cerevisiae pan-genome reveals a pool of copy num-ber variants distributed in diverse yeast strains from differing indus-trial environments Genome Res 22908ndash924

Dunn B Sherlock G 2008 Reconstruction of the genome origins andevolution of the hybrid lager yeast Saccharomyces pastorianusGenome Res 181610ndash1623

Elmer KR Meyer A 2011 Adaptation in the age of ecological genomicsinsights from parallelism and convergence Trends Ecol Evol26298ndash306

Engel SR Dietrich FS Fisk DG Binkley G Balakrishnan R Costanzo MCDwight SS Hitz BC Karra K Nash RS et al 2014 The referencegenome sequence of Saccharomyces cerevisiae then and now G3(Bethesda) 4389ndash398

Fedoroff NV 2012 Presidential address Transposable elements epige-netics and genome evolution Science 338758ndash767

Ferea TL Botstein D Brown PO Rosenzweig RF 1999 Systematicchanges in gene expression patterns following adaptive evolutionin yeast Proc Natl Acad Sci U S A 969721ndash9726

Gibson B Liti G 2015 Saccharomyces pastorianus genomic insightsinspiring innovation for industry Yeast 3217ndash27

Gibson DG Young L Chuang RY Venter JC Hutchison CA Smith HO2009 Enzymatic assembly of DNA molecules up to several hundredkilobases Nat Methods 6343ndash5

Gonzalez SS Barrio E Gafner J Querol A 2006 Natural hybrids fromSaccharomyces cerevisiae Saccharomyces bayanus andSaccharomyces kudriavzevii in wine fermentations FEMS Yeast Res61221ndash1234

Gonzalez SS Barrio E Querol A 2008 Molecular characterization of newnatural hybrids of Saccharomyces cerevisiae and S kudriavzevii inbrewing Appl Environ Microbiol 742314ndash2320

Grant PR Grant BR 1994 Phenotypic and genetic consequences ofhybridization in Darwinrsquos finches Evolution 48297ndash316

Grant PR Grant BR 2002 Unpredictable evolution in a 30-year study ofDarwinrsquos finches Science 296707ndash711

Grant PR Grant BR 2010 Natural selection speciation and Darwinrsquosfinches Proc Calif Acad Sci 61245ndash260

Grant PR Grant BR Petren K 2005 Hybridization in the recent past AmNat 16656ndash67

Greig D 2009 Reproductive isolation in Saccharomyces Heredity (Edinb)10239ndash44

Greig D Leu JY 2009 Natural history of budding yeast Current Biol19R886ndashR890

Greig D Louis EJ Borts RH Travisano M 2002 Hybrid speciation inexperimental populations of yeast Science 2981773ndash1775

Gresham D Desai MM Tucker CM Jenq HT Pai DA Ward A DeSevoCG Botstein D Dunham MJ 2008 The repertoire and dynamics ofevolutionary adaptations to controlled nutrient-limited environ-ments in yeast PLoS Genet 4e1000303

Hajjar R Hodgkin T 2007 The use of wild relatives in crop improvementa survey of developments over the last 20 years Euphytica 1561ndash13

Heiser CB 1954 Variation and subspeciation in the common sunflowerHelianthus annuus Am Midl Nat 51287ndash305

Hittinger CT 2013 Saccharomyces diversity and evolution a buddingmodel genus Trends Genet 29309ndash317

Hoang ML Tan FJ Lai DC Celniker SE Hoskins RA Dunham MJ ZhengY Koshland D 2010 Competitive repair by naturally dispersed

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repetitive DNA during non-allelic homologous recombination PLoSGenet 6e1001228

Hoffmann AA Sgro CM 2011 Climate change and evolutionary adap-tation Nature 470479ndash485

Hoffman CS Winston F 1987 A 10-minute DNA preparation from yeastefficiently releases autonomous plasmids for transformation ofEscherichia coli Gene 57267ndash272

Hou J Friedrich A de Montigny J Schacherer J 2014 Chromosomalrearrangements as a major mechanism in the onset of reproductiveisolation in Saccharomyces cerevisiae Curr Biol 241153ndash1159

Hou J Friedrich A Gounot JS Schacherer J 2015 Comprehensive surveyof condition-specific reproductive isolation reveals genetic incom-patibility in yeast Nat Commun 67214

Huerta-Sanchez E Casey FP 2015 Archaic inheritance supporting high-altitude life in Tibet J Appl Physiol (1985) 1191129ndash1134

Jones FC Grabherr MG Chan YF Russell P Mauceli E Johnson JSwofford R Pirun M Zody MC White S et al 2012 The genomicbasis of adaptive evolution in threespine sticklebacks Nature48455ndash61

Kao KC Sherlock G 2008 Molecular characterization of clonal interfer-ence during adaptive evolution in asexual populations ofSaccharomyces cerevisiae Nat Genet 401499ndash1504

Kawecki TJ Lenski RE Ebert D Hollis B Olivieri I Whitlock MC 2012Experimental evolution Trends Ecol Evol 27547ndash560

Kellis M Patterson N Endrizzi M Birren B Lander ES 2003 Sequencingand comparison of yeast species to identify genes and regulatoryelements Nature 423241ndash254

Kelly B Whiteley A Tallmon D 2010 The Arctic melting pot Nature468891ndash891

Kvitek DJ Sherlock G 2011 Reciprocal sign epistasis between frequentlyexperimentally evolved adaptive mutations causes a rugged fitnesslandscape PLoS Genet 7e1002056

Kvitek DJ Sherlock G 2013 Whole genome whole population sequenc-ing reveals that loss of signaling networks is the major adaptivestrategy in a constant environment PLoS Genet 9e1003972

Landry CR Hartl DL Ranz JM 2007 Genome clashes in hybrids insightsfrom gene expression Heredity (Edinb) 99483ndash493

Lang GI Desai MM 2014 The spectrum of adaptive mutations in ex-perimental evolution Genomics 104412ndash416

Lapunzina P Monk D 2011 The consequences of uniparental disomyand copy number neutral loss-of-heterozygosity during human de-velopment and cancer Biol Cell 103303ndash317

Leducq JB Nielly-Thibault L Charron G Eberlein C Verta JP Samani PSylvester K Hittinger CT Bell G Landry CR 2016 Speciation drivenby hybridization and chromosomal plasticity in a wild yeast NatMicrobiol 115003

Lee HY Chou JY Cheong L Chang NH Yang SY Leu JY 2008Incompatibility of nuclear and mitochondrial genomes causes hy-brid sterility between two yeast species Cell 1351065ndash1073

Li H Durbin R 2009 Fast and accurate short read alignment withBurrows-Wheeler transform Bioinformatics 251754ndash1760

Li H Handsaker B Wysoker A Fennell T Ruan J Homer N Marth GAbecasis G Durbin R 2009 The Sequence AlignmentMap formatand SAMtools Bioinformatics 252078ndash2079

Liti G 2015 The fascinating and secret wild life of the budding yeast Scerevisiae Elife 4e05835

Liti G Barton DB Louis EJ 2006 Sequence diversity reproductiveisolation and species concepts in Saccharomyces Genetics174839ndash850

Lloyd AH Ranoux M Vautrin S Glover N Fourment J Charif D ChouletF Lassalle G Marande W Tran J et al 2014 Meiotic gene evolutioncan you teach a new dog new tricks Mol Biol Evol 311724ndash1727

Losos JB Jackman TR Larson A de Queiroz K Rodriguez-Schettino L1998 Contingency and determinism in replicated adaptive radia-tions of island lizards Science 2792115ndash2118

Louis VL Despons L Friedrich A Martin T Durrens P Casaregola SNeuveglise C Fairhead C Marck C Cruz JA et al 2012 Pichia sorbi-tophila an interspecies yeast hybrid reveals early steps of genomeresolution after polyploidization G3 (Bethesda) 2299ndash311

Mallet J 2007 Hybrid speciation Nature 446279ndash283Marcet-Houben M Gabaldon T 2015 Beyond the whole-genome du-

plication phylogenetic evidence for an ancient interspecies hybrid-ization in the bakerrsquos yeast lineage PLoS Biol 13e1002220

Marinoni G Manuel M Petersen RF Hvidtfeldt J Sulo P Piskur J 1999Horizontal transfer of genetic material among Saccharomyces yeastsJ Bacteriol 1816488ndash6496

Martin A Orgogozo V 2013 The loci of repeated evolution a catalog ofgenetic hotspots of phenotypic variation Evolution 671235ndash1250

Masly JP Jones CD Noor MAF Locke J Orr HA 2006 Gene transpositionas a cause of hybrid sterility in Drosophila Science 3131448ndash1450

Mason AS Batley J 2015 Creating new interspecific hybrid and polyploidcrops Trends Biotechnol 33436ndash441

Mavarez J Salazar CA Bermingham E Salcedo C Jiggins CD Linares M2006 Speciation by hybridization in Heliconius butterflies Nature441868ndash871

McKenna A Hanna M Banks E Sivachenko A Cibulskis K Kernytsky AGarimella K Altshuler D Gabriel S Daly M et al 2010 The genomeanalysis toolkit a MapReduce framework for analyzing next-generation DNA sequencing data Genome Res 201297ndash1303

Meiklejohn CD Holmbeck MA Siddiq MA Abt DN Rand DMMontooth KL 2013 An Incompatibility between a mitochondrialtRNA and its nuclear-encoded tRNA synthetase compromises de-velopment and fitness in Drosophila PLoS Genet 9e1003238

Meyer A Salzburger W Schartl M 2006 Hybrid origin of a swordtailspecies (Teleostei Xiphophorus clemenciae) driven by sexual selec-tion Mol Ecol 15721ndash730

Michalak P 2009 Epigenetic transposon and small RNA determinants ofhybrid dysfunctions Heredity (Edinb) 10245ndash50

Miller AW Dunham MJ 2013 Design and use of multiplexed chemostatarrays J Vis Exp 72e50262

Monod J 1949 The growth of bacterial cultures Annu Rev Microbiol3371ndash394

Mouillon JM Persson BL 2006 New aspects on phosphate sensing andsignalling in Saccharomyces cerevisiae FEMS Yeast Res 6171ndash176

Muhlfeld CC Kovach RP Jones LA Al-Chokhachy R Boyer MC Leary RFLowe WH Luikart G Allendorf FW 2014 Invasive hybridization in athreatened species is accelerated by climate change Nat ClimChange 4620ndash624

Muller LAH McCusker JH 2009 A multispecies-based taxonomic mi-croarray reveals interspecies hybridization and introgression inSaccharomyces cerevisiae FEMS Yeast Res 9143ndash152

Nagalakshmi U Wang Z Waern K Shou C Raha D Gerstein M SnyderM 2008 The transcriptional landscape of the yeast genome definedby RNA sequencing Science 3201344ndash1349

Nakao Y Kanamori T Itoh T Kodama Y Rainieri S Nakamura NShimonaga T Hattori M Ashikari T 2009 Genome sequence ofthe lager brewing yeast an interspecies hybrid DNA Res 16115ndash129

Nolte AW Freyhof J Stemshorn KC Tautz D 2005 An invasive lineage ofsculpins Cottus sp (Pisces Teleostei) in the Rhine with new habitatadaptations has originated from hybridization between old phylo-geographic groups Proc Biol Sci 2722379ndash2387

Novick A Szilard L 1950a Description of the chemostat Science112715ndash716

Novick A Szilard L 1950b Experiments with the Chemostat on spon-taneous mutations of bacteria Proc Natl Acad Sci U S A 36708ndash719

Ouyang YD Liu YG Zhang QF 2010 Hybrid sterility in plant storiesfrom rice Curr Opin Plant Biol 13186ndash192

Payen C Di Rienzi SC Ong GT Pogachar JL Sanchez JC SunshineAB Raghuraman MK Brewer BJ Dunham MJ 2014 The dynam-ics of diverse segmental amplifications in populations ofSaccharomyces cerevisiae adapting to strong selection G3(Bethesda) 4399ndash409

Payen C Sunshine AB Ong GT Pogachar JL Zhao W Dunham MJ 2016High-throughput identification of adaptive mutations in experimen-tally evolved yeast populations PLoS Genet 12e1006339

Perez-Prat E van Lookeren Campagne MM 2002 Hybrid seed produc-tion and the challenge of propagating male-sterile plants TrendsPlant Sci 7199ndash203

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Peris D Langdon QK Moriarty RV Sylvester K Bontrager M Charron GLeducq JB Landry CR Libkind D Hittinger CT 2016 Complex ances-tries of lager-brewing hybrids were shaped by standing variation inthe wild yeast Saccharomyces eubayanus PLoS Genet 12e1006155

Perlstein EO Ruderfer DM Roberts DC Schreiber SL Kruglyak L 2007Genetic basis of individual differences in the response to small-molecule drugs in yeast Nat Genet 39496ndash502

Piatkowska EM Naseeb S Knight D Delneri D 2013 Chimeric proteincomplexes in hybrid species generate novel phenotypes PLoS Genet9e1003836

Piotrowski JS Nagarajan S Kroll E Stanbery A Chiotti KE Kruckeberg ALDunn B Sherlock G Rosenzweig F 2012 Different selective pressureslead to different genomic outcomes as newly-formed hybrid yeastsevolve BMC Evol Biol 1246

Pryszcz LP Nemeth T Gacser A Gabaldon T 2014 Genome comparisonof Candida orthopsilosis clinical strains reveals the existence of hy-brids between two distinct subspecies Genome Biol Evol61069ndash1078

Racimo F Sankararaman S Nielsen R Huerta-Sanchez E 2015 Evidencefor archaic adaptive introgression in humans Nat Rev Genet16359ndash371

Rieseberg LH 1991 Homoploid reticulate evolution in Helianthus(Asteraceae)mdashevidence from ribosomal genes Am J Bot781218ndash1237

Rieseberg LH 1997 Hybrid origins of plant species Annu Rev EcolSystemat 28359ndash389

Robinson JT Thorvaldsdottir H Winckler W Guttman M Lander ESGetz G Mesirov JP 2011 Integrative genomics viewer NatBiotechnol 2924ndash26

Rosenthal DM Schwarzbach AE Donovan LA Raymond O RiesebergLH 2002 Phenotypic differentiation between three ancient hybridtaxa and their parental species Int J Plant Sci 163387ndash398

Ruderfer DM Pratt SC Seidel HS Kruglyak L 2006 Population genomicanalysis of outcrossing and recombination in yeast Nat Genet381077ndash1081

Rundle HD Nagel L Boughman JW Schluter D 2000 Natural selectionand parallel speciation in sympatric sticklebacks Science287306ndash308

Ryland GL Doyle MA Goode D Boyle SE Choong DY Rowley SM Li JBowtell DD Tothill RW Campbell IG et al 2015 Australian OvarianCancer Study Group Loss of heterozygosity what is it good for BMCMed Genomics 845

Salverda MLM Dellus E Gorter FA Debets AJM van der Oost J HoekstraRF Tawfik DS de Visser JAGM 2011 Initial mutations direct alter-native pathways of protein evolution PLoS Genet 7e1001321

Sanchez MR Miller AW Liachko I Sunshine AB Lynch B Huang MAlcantara E DeSevo CG Pai DA Tucker CM et al 2017 Differentialparalog divergence modulates genome evolution across yeast spe-cies PLoS Genet 13(2)e1006585

Scannell DR Zill OA Rokas A Payen C Dunham MJ Eisen MB Rine JJohnston M Hittinger CT 2011 The awesome power of yeast evo-lutionary genetics new genome sequences and strain resources forthe Saccharomyces sensu stricto genus G3 (Bethesda) 111ndash25

Schroder MS Martinez de San Vicente K Prandini TH Hammel SHiggins DG Bagagli E Wolfe KH Butler G 2016 Multiple originsof the pathogenic yeast Candida orthopsilosis by separate hybridiza-tions between two parental species PLoS Genet 12e1006404

Schumer M Rosenthal GG Andolfatto P 2014 How common is homo-ploid hybrid speciation Evolution 681553ndash1560

Schwarzbach AE Donovan LA Rieseberg LH 2001 Transgressivecharacter expression in a hybrid sunflower species Am J Bot88270ndash277

Selmecki AM Maruvka YE Richmond PA Guillet M Shoresh NSorenson AL De S Kishony R Michor F Dowell R et al 2015Polyploidy can drive rapid adaptation in yeast Nature 519349ndash52

Seshan VE Olshen A 2016 DNAcopy DNA copy number data analysisVersion R package version 1460

Soltis DE Visger CJ Soltis PS 2014 The polyploidy revolution then andnow Stebbins revisited Am J Bot 1011057ndash1078

Soltis PS 2013 Hybridization speciation and novelty J Evol Biol26291ndash293

Soltis PS Soltis DE 2009 The role of hybridization in plant speciationAnnu Rev Plant Biol 60561ndash588

Sunshine AB Payen C Ong GT Liachko I Tan KM Dunham MJ 2015The fitness consequences of aneuploidy are driven by condition-dependent gene effects PLoS Biol 13e1002155

Tamai Y Momma T Yoshimoto H Kaneko Y 1998 Co-existence of twotypes of chromosome in the bottom fermenting yeastSaccharomyces pastorianus Yeast 14923ndash933

Taylor DJ Hebert PDN 1993 Habitat-dependent hybrid parentage anddifferential introgression between neighboringly sympatric Daphniaspecies Proc Natl Acad Sci U S A 907079ndash7083

Thiagalingam S Laken S Willson JK Markowitz SD Kinzler KWVogelstein B Lengauer C 2001 Mechanisms underlying losses ofheterozygosity in human colorectal cancers Proc Natl Acad Sci US A 982698ndash2702

Tuna M Knuutila S Mills GB 2009 Uniparental disomy in cancer TrendsMol Med 15120ndash128

Walther A Hesselbart A Wendland J 2014 Genome sequence ofSaccharomyces carlsbergensis the worldrsquos first pure culture lageryeast G3 (Bethesda) 4783ndash793

Wayne RK 1993 Molecular evolution of the dog family Trends Genet9218ndash224

Wenger JW Schwartz K Sherlock G 2010 Bulk segregant analysis byhigh-throughput sequencing reveals a novel xylose utilization genefrom Saccharomyces cerevisiae PLoS Genet 6e1000942

Wessinger CA Rausher MD 2014 Predictability and irreversibility ofgenetic changes associated with flower color evolution inPenstemon barbatus Evolution 681058ndash1070

Wolfe KH 2015 Origin of the yeast whole-genome duplication PLoSBiol 13e1002221

Woods R Schneider D Winkworth CL Riley MA Lenski RE 2006 Testsof parallel molecular evolution in a long-term experiment withEscherichia coli Proc Natl Acad Sci U S A 1039107ndash9112

Wykoff DD OrsquoShea EK 2001 Phosphate transport and sensing inSaccharomyces cerevisiae Genetics 1591491ndash1499

Xiong Z Gaeta RT Pires JC 2011 Homoeologous shuffling andchromosome compensation maintain genome balance inresynthesized allopolyploid Brassica napus Proc Natl Acad SciU S A 1087908ndash7913

Yassour M Kapian T Fraser HB Levin JZ Pfiffner J Adiconis X Schroth GLuo SJ Khrebtukova I Gnirke A et al 2009 Ab initio construction ofa eukaryotic transcriptome by massively parallel mRNA sequencingProc Natl Acad Sci U S A 1063264ndash3269

Zhu YO Sherlock G Petrov DA 2016 Whole genome analysis of 132clinical Saccharomyces cerevisiae strains reveals extensive ploidy var-iation G3 (Bethesda) doi101534g3116029397

Adaptation in Hybrids doi101093molbevmsx098 MBE

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Page 13: Loss of Heterozygosity Drives Adaptation in Hybrid Yeastdunham.gs.washington.edu/SmukowskiHeil.pdf · 2017-05-20 · Loss of Heterozygosity Drives Adaptation in Hybrid Yeast Caiti

Applications to Other Hybrids and CancerThe observation that LOH occurs in hybrid genomes is in-creasingly documented (Louis et al 2012 Borneman et al2014 Soltis et al 2014 Marcet-Houben and Gabaldon 2015Pryszcz et al 2014 Schroder et al 2016) although the rea-son(s) for this type of mutation has been unresolved As mostexamples stem from allopolyploid events that occurred mil-lions of years ago understanding why LOH is important inhybrid genome evolution is difficult Cancer cells are alsoknown to experience LOH sometimes involved in the inac-tivation of tumor suppressor genes leaving only one copy ofthe gene that may be mutated or silenced (Thiagalingam et al2001 Tuna et al 2009 Lapunzina and Monk 2011) Datasupport the conclusion that LOH events are selected for dur-ing tumor development as many LOH events involve specificchromosomal segments (Thiagalingam et al 2001) althoughthe underlying molecular and genetic reasons for selection isan open debate (Ryland et al 2015)

Here we experimentally demonstrate that LOH can occurin homoploid interspecific hybrids as well as in intraspecifichybrids These events occur within a few hundred generationsand are common mutations more common on average inthe intraspecific hybrid (13 eventsclone) than the interspe-cific hybrid (056 eventsclone) Interestingly the LOH eventsdo not share break points with the CNV events in S cerevisiaeinstead they appear to occur independently and to precedeany subsequent amplification (amplification occurs following913 LOH events) Competition assays with the PHO84 locusprovide support that LOH itself may be more beneficial thanamplification or at least increase the selective benefit of am-plification events The observation that LOH in intraspecifichybrids occurs independently from copy number change pro-vides different opportunities for adaptation to novelconditions

Other cases of LOH like the copy number neutral eventsobserved in Ph4 Ph5 and Sh4 all of which favor the retentionof the S uvarum allele (supplementary fig S3 SupplementaryMaterial online) may be due to hybrid incompatibility withina particular protein complex other epistatic interactions(Piatkowska et al 2013) or neutral processes We furthermorediscover examples where one species allele appears to bepreferred over the other without LOH such as the repeatedamplification of the S uvarum high-affinity glucose transpor-ters HXT67 When one species allele is amplified and theother is not amplified one explanation is that the local se-quence context can permit or deny amplification In the caseof HXT67 previous experiments in S cerevisiae have shownthat amplification of HXT67 is quite common (Brown et al1998 Dunham et al 2002 Gresham et al 2008 Kao andSherlock 2008 Kvitek and Sherlock 2011) thus suggestingthat when given a choice between this locus in S cerevisiaeor S uvarum in the hybrid the preferred allele is indeed Suvarum though more subtle differences in rate cannot yet beruled out A similar scenario is observed with S cerevisiaeSUL1 (Sanchez et al 2017) Together our results show thatthe heterozygosity supplied by hybridization is an importantcontributor to adaptive routes explored by populations asthey adapt to novel conditions

Although we cannot generalize our results from thePHO84 locus across the many other LOH events discoveredin our hybrids and S cerevisiae in the future we can usesimilar methodology to explore whether positive selectionalways drives LOH or whether other explanations such asincompatibility resolution contribute as well Future experi-ments might also utilize a high throughput method to ex-plore segmental LOH in hybrids at a genome-wide scalesimilar to ongoing experiments at the gene level (LancasterS Dunham MJ unpublished data) Although our sample sizeis moderate this is a novel and necessary step in understand-ing forces underlying hybrid genome stabilization and high-lighting an underappreciated mechanism of hybridadaptation

ConclusionsThe mutation events we observe in our experimentallyevolved hybrids are in many ways quite representative ofmutations observed in ancient hybrid genomes suggestingthat hybrid genome stabilization and adaptation can occurquite rapidly (within several hundred generations)Furthermore our results illustrate that the infusion of varia-tion introduced by hybridization at both the intra- and inter-species level can increase fitness by providing choices of allelesfor selection to act upon even when sexual reproduction israre This may be particularly important for leveraging existingvariation for agricultural and industrial processes and as cli-mate change potentially increases natural hybridization (Kellyet al 2010 Hoffmann and Sgro 2011 Muhlfeld et al 2014)

Materials and Methods

StrainsA list of strains used in this study is included in supplementarytable S1 Supplementary Material online All interspecific hy-brids were created by crossing a ura3 LYS2 haploid parent to aURA3 lys2 haploid parent of the other mating type plating onmedia lacking both uracil and lysine and selecting for proto-trophs The S cerevisiae strain background known asldquoGRF167rdquo is itself a cross between FL100 and the genomictype strain S288C (data not shown) GRF167 was chosen as astrain background for simultaneous work investigating trans-posable elements during experimental evolution which willbe addressed in a future study

Evolution ExperimentsContinuous cultures were established using media and con-ditions previously described (Gresham et al 2008 Sanchezet al 2017) Detailed protocols and media recipes are availableat httpdunhamgswashingtoneduprotocolsshtml (lastaccessed March 6 2017) Samples were taken daily and mea-sured for optical density at 600 nm and cell count micros-copy was performed to check for contamination andarchival glycerol stocks were made daily An experimentwas terminated when contamination growth in tubing orclumping appeared (number of generations at the endpoint for each population are presented in tables 1 and

Adaptation in Hybrids doi101093molbevmsx098 MBE

13

2) Samples from each end point population were colonypurified to yield two clones for further study

Array Comparative Genomic HybridizationPopulations from the end point of each evolution were ana-lyzed for copy number changes using array comparative ge-nomic hybridization following the protocol used in Sanchezet al (2017)

SequencingDNA was extracted from overnight cultures using theHoffmanndashWinston protocol (Hoffman and Winston 1987)and cleaned using the Clean amp Concentrator kit (ZymoResearch) Nextera libraries were prepared following theNextera library kit protocol and sequenced using paired end150 bp reads on the Illumina NextSeq 500 machine (sequenc-ing coverage in supplementary table S2 SupplementaryMaterial online) The reference genomes used were S cerevi-siae v3 (Engel et al 2014) S uvarum (Scannell et al 2011) anda hybrid reference genome created by concatenating the twogenomes Sequence was aligned to the appropriate referencegenome using bwa v062 (Li and Durbin 2009) and mutationswere called using GATK (McKenna et al 2010) and samtools0119 (Li et al 2009) Mutations in evolved clones were filteredin comparison with the ancestor to obtain de novo mutationsAll mutations were first visually inspected using IntegrativeGenomics Viewer (Robinson et al 2011) Subsequently pointmutations in the hybrids were confirmed with Sanger se-quencing (supplementary table S3 Supplementary Materialonline) Copy number variants were visualized usingDNAcopy for S cerevisiae and S uvarum (Seshan andOlshen 2016) LOH events were called based on sequencingcoverage in the hybrids and by identifying homozygous variantcalls in S cerevisiae All break points were called by visual in-spection of sequencing reads and are thus approximate

Fitness AssaysThe pairwise competition experiments were performed in 20ml chemostats (Miller and Dunham 2013) Each competitorstrain was cultured individually until steady state was reachedand then was mixed 5050 with a GFP-tagged ancestor Eachcompetition was conducted in at least two biological repli-cates for approximately 15 generations after mixing Sampleswere collected and analyzed twice daily The proportion ofGFPthorn cells in the population was detected using a BD AccuriC6 flow cytometer (BD Biosciences) The data were plottedwith ln [dark cellsGFPthorn cells] versus generations The rela-tive fitness coefficient was determined from the slope of thelinear region

Strain ConstructionAllele replacements for the PHO84 locus were done followingthe protocol of the Caudy lab with further modifications de-scribed here The native locus was replaced withKluyveromyces lactis URA3 The pho84DURA3 strain wasgrown overnight in 5 ml of C-URA media then inoculatedin a flask of 100 ml yeast extract peptone dextrose (YPD) andgrown to an optical density of 06ndash08 Cells were washed then

aliquoted 275 ml of transformation mix (35 ml 1 M lithiumacetate 240 ml of 50 3500 polyethylene glycol) 10 ml ofsalmon sperm and approximately 3 mg of polymerase chainreaction product were added to the cell pellet It was incu-bated at 37 C (S uvarum) or 42 C (S cerevisiae) for 45 minthen plated to YPD It was replica plated to 5-fluorooroticacid the following day and colonies were tested for the gain ofthe appropriate species allele The GRF167 allele was clonedinto the pIL37 plasmid using Gibson assembly (Gibson et al2009) Correct assembly was verified by Sanger sequencing Allprimers used can be found in supplementary table S3Supplementary Material online

Supplementary MaterialSupplementary data are available at Molecular Biology andEvolution online

AcknowledgmentsWe thank Noah Hanson and Erica Alcantara for technicalassistance We thank Monica Sanchez and three anonymousreviewers for helpful comments on this manuscript JosephSchacherer kindly confirmed the contribution of FL100 to theGRF167 strain background Thanks to Yixian Zheng and DougKoshland for contributing to the initial experimental designcreating yeast strains and purchasing the oligonucleotidesused for the microarrays This work was supported by theNational Science Foundation (grant number 1516330) andthe National Institutes of Health (grant number R01GM094306) MD is a Senior Fellow in the GeneticNetworks program at the Canadian Institute for AdvancedResearch and a Rita Allen Foundation Scholar CSH wassupported in part by National Institutes of Health (grantnumber T32 HG00035) This work was also supported bythe National Institutes of Health (grant number P50GM071508) to the Lewis-Sigler Institute and from theHoward Hughes Medical Institute to Doug Koshland andYixian Zheng Sequencing read data have been deposited atthe NCBI under BioProject accession PRJNA374049Microarray data are deposited in the Gene ExpressionOmnibus (GEO) repository under accession numbersGSE95086 and GSE87401 and in the Princeton MicroarrayDatabase

ReferencesAbbott R Albach D Ansell S Arntzen JW Baird SJ Bierne N Boughman J

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Albertin W Marullo P 2012 Polyploidy in fungi evolution after whole-genome duplication Proc Biol Sci 2792497ndash2509

Almeida P Goncalves C Teixeira S Libkind D Bontrager M Masneuf-Pomarede I Albertin W Durrens P Sherman DJ Marullo P et al2014 A Gondwanan imprint on global diversity and domesticationof wine and cider yeast Saccharomyces uvarum Nat Commun54044

Antunovics Z Nguyen HV Gaillardin C Sipiczki M 2005 Gradual ge-nome stabilisation by progressive reduction of the Saccharomyces

Smukowski Heil et al doi101093molbevmsx098 MBE

14

uvarum genome in an interspecific hybrid with Saccharomyces cer-evisiae FEMS Yeast Res 51141ndash1150

Baker E Wang B Bellora N Peris D Hulfachor AB Koshalek JA AdamsM Libkind D Hittinger CT 2015 The Genome sequence ofSaccharomyces eubayanus and the domestication of lager-brewingyeasts Mol Biol Evol 322818ndash2831

Barbosa R Almeida P Safar SVB Santos RO Morais PB Nielly-Thibault LLeducq JB Landry CR Goncalves P Rosa CA Sampaio JP 2016Evidence of natural hybridization in Brazilian wild lineages ofSaccharomyces cerevisiae Genome Biol Evol 8317ndash329

Barton NH 2001 The role of hybridization in evolution Mol Ecol10551ndash568

Belloch C Orlic S Barrio E Querol A 2008 Fermentative stress adapta-tion of hybrids within the Saccharomyces sensu stricto complex Int JFood Microbiol 122188ndash195

Bellon JR Yang F Day MP Inglis DL Chambers PJ 2015 Designingand creating Saccharomyces interspecific hybrids for improvedindustry relevant phenotypes Appl Microbiol Biotechnol998597ndash8609

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Brewer BJ Payen C Di Rienzi SC Higgins MM Ong G Dunham MJRaghuraman MK 2015 Origin-dependent inverted-repeat amplifi-cation tests of a model for inverted DNA amplification PLoS Genet11e1005699

Brown CJ Todd KM Rosenzweig RF 1998 Multiple duplications of yeasthexose transport genes in response to selection in a glucose-limitedenvironment Mol Biol Evol 15931ndash942

Bullini L 1994 Origin and evolution of animal hybrid species Trends EcolEvol 9422ndash426

Bun-Ya M Nishimura M Harashima S Oshima Y 1991 The PHO84 geneof Saccharomyces cerevisiae encodes an inorganic phosphate trans-porter Mol Cell Biol 113229ndash3238

Burke MK Dunham JP Shahrestani P Thornton KR Rose MR Long AD2010 Genome-wide analysis of a long-term evolution experimentwith Drosophila Nature 467587ndash590

Burke MK Liti G Long AD 2014 Standing genetic variation drives re-peatable experimental evolution in outcrossing populations ofSaccharomyces cerevisiae Mol Biol Evol 313228ndash3239

Cherry JM Hong EL Amundsen C Balakrishnan R Binkley G Chan ETChristie KR Costanzo MC Dwight SS Engel SR et al 2012Saccharomyces genome database the genomics resource of buddingyeast Nucleic Acids Res 40D700ndashD705

Chester M Riley RK Soltis PS Soltis DE 2015 Patterns of chromosomalvariation in natural populations of the neoallotetraploid Tragopogonmirus (Asteraceae) Heredity (Edinb) 114309ndash317

Chou JY Leu JY 2010 Speciation through cytonuclear incompatibilityinsights from yeast and implications for higher eukaryotes Bioessays32401ndash411

Cliften PF Fulton RS Wilson RK Johnston M 2006 After the duplicationgene loss and adaptation in Saccharomyces genomes Genetics172863ndash872

Conte GL Arnegard ME Peichel CL Schluter D 2012 The probability ofgenetic parallelism and convergence in natural populations Proc BiolSci 2795039ndash5047

Cordes FS Bright JN Sansom MS 2002 Proline-induced distortions oftransmembrane helices J Mol Biol 323951ndash960

Crow JF 1998 90 years ago the beginning of hybrid maize Genetics148923ndash928

Dasmahapatra KK Walters JR Briscoe AD Davey JW Whibley A NadeauNJ Zimin AV Hughes DST Ferguson LC Martin SH et al 2012Butterfly genome reveals promiscuous exchange of mimicry adap-tations among species Nature 48794ndash98

Dowling TE Smith GR Brown WM 1989 Reproductive isolation andintrogression between Notropis-Cornutus and Notropis-Chrysocephalus (Family Cyprinidae)mdashcomparison of morphologyallozymes and mitochondrial-DNA Evolution 43620ndash634

Doyle JJ Flagel LE Paterson AH Rapp RA Soltis DE Soltis PS Wendel JF2008 Evolutionary genetics of genome merger and doubling inplants Annu Rev Genet 42443ndash461

Dunham MJ Badrane H Ferea T Adams J Brown PO Rosenzweig FBotstein D 2002 Characteristic genome rearrangements in experi-mental evolution of Saccharomyces cerevisiae Proc Natl Acad Sci U SA 9916144ndash16149

Dunn B Paulish T Stanbery A Piotrowski J Koniges G Kroll E LouisEJ Liti G Sherlock G Rosenzweig F 2013 Recurrent rearrange-ment during adaptive evolution in an interspecific yeast hybridsuggests a model for rapid introgression PLoS Genet9e1003366

Dunn B Richter C Kvitek DJ Pugh T Sherlock G 2012 Analysis of theSaccharomyces cerevisiae pan-genome reveals a pool of copy num-ber variants distributed in diverse yeast strains from differing indus-trial environments Genome Res 22908ndash924

Dunn B Sherlock G 2008 Reconstruction of the genome origins andevolution of the hybrid lager yeast Saccharomyces pastorianusGenome Res 181610ndash1623

Elmer KR Meyer A 2011 Adaptation in the age of ecological genomicsinsights from parallelism and convergence Trends Ecol Evol26298ndash306

Engel SR Dietrich FS Fisk DG Binkley G Balakrishnan R Costanzo MCDwight SS Hitz BC Karra K Nash RS et al 2014 The referencegenome sequence of Saccharomyces cerevisiae then and now G3(Bethesda) 4389ndash398

Fedoroff NV 2012 Presidential address Transposable elements epige-netics and genome evolution Science 338758ndash767

Ferea TL Botstein D Brown PO Rosenzweig RF 1999 Systematicchanges in gene expression patterns following adaptive evolutionin yeast Proc Natl Acad Sci U S A 969721ndash9726

Gibson B Liti G 2015 Saccharomyces pastorianus genomic insightsinspiring innovation for industry Yeast 3217ndash27

Gibson DG Young L Chuang RY Venter JC Hutchison CA Smith HO2009 Enzymatic assembly of DNA molecules up to several hundredkilobases Nat Methods 6343ndash5

Gonzalez SS Barrio E Gafner J Querol A 2006 Natural hybrids fromSaccharomyces cerevisiae Saccharomyces bayanus andSaccharomyces kudriavzevii in wine fermentations FEMS Yeast Res61221ndash1234

Gonzalez SS Barrio E Querol A 2008 Molecular characterization of newnatural hybrids of Saccharomyces cerevisiae and S kudriavzevii inbrewing Appl Environ Microbiol 742314ndash2320

Grant PR Grant BR 1994 Phenotypic and genetic consequences ofhybridization in Darwinrsquos finches Evolution 48297ndash316

Grant PR Grant BR 2002 Unpredictable evolution in a 30-year study ofDarwinrsquos finches Science 296707ndash711

Grant PR Grant BR 2010 Natural selection speciation and Darwinrsquosfinches Proc Calif Acad Sci 61245ndash260

Grant PR Grant BR Petren K 2005 Hybridization in the recent past AmNat 16656ndash67

Greig D 2009 Reproductive isolation in Saccharomyces Heredity (Edinb)10239ndash44

Greig D Leu JY 2009 Natural history of budding yeast Current Biol19R886ndashR890

Greig D Louis EJ Borts RH Travisano M 2002 Hybrid speciation inexperimental populations of yeast Science 2981773ndash1775

Gresham D Desai MM Tucker CM Jenq HT Pai DA Ward A DeSevoCG Botstein D Dunham MJ 2008 The repertoire and dynamics ofevolutionary adaptations to controlled nutrient-limited environ-ments in yeast PLoS Genet 4e1000303

Hajjar R Hodgkin T 2007 The use of wild relatives in crop improvementa survey of developments over the last 20 years Euphytica 1561ndash13

Heiser CB 1954 Variation and subspeciation in the common sunflowerHelianthus annuus Am Midl Nat 51287ndash305

Hittinger CT 2013 Saccharomyces diversity and evolution a buddingmodel genus Trends Genet 29309ndash317

Hoang ML Tan FJ Lai DC Celniker SE Hoskins RA Dunham MJ ZhengY Koshland D 2010 Competitive repair by naturally dispersed

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15

repetitive DNA during non-allelic homologous recombination PLoSGenet 6e1001228

Hoffmann AA Sgro CM 2011 Climate change and evolutionary adap-tation Nature 470479ndash485

Hoffman CS Winston F 1987 A 10-minute DNA preparation from yeastefficiently releases autonomous plasmids for transformation ofEscherichia coli Gene 57267ndash272

Hou J Friedrich A de Montigny J Schacherer J 2014 Chromosomalrearrangements as a major mechanism in the onset of reproductiveisolation in Saccharomyces cerevisiae Curr Biol 241153ndash1159

Hou J Friedrich A Gounot JS Schacherer J 2015 Comprehensive surveyof condition-specific reproductive isolation reveals genetic incom-patibility in yeast Nat Commun 67214

Huerta-Sanchez E Casey FP 2015 Archaic inheritance supporting high-altitude life in Tibet J Appl Physiol (1985) 1191129ndash1134

Jones FC Grabherr MG Chan YF Russell P Mauceli E Johnson JSwofford R Pirun M Zody MC White S et al 2012 The genomicbasis of adaptive evolution in threespine sticklebacks Nature48455ndash61

Kao KC Sherlock G 2008 Molecular characterization of clonal interfer-ence during adaptive evolution in asexual populations ofSaccharomyces cerevisiae Nat Genet 401499ndash1504

Kawecki TJ Lenski RE Ebert D Hollis B Olivieri I Whitlock MC 2012Experimental evolution Trends Ecol Evol 27547ndash560

Kellis M Patterson N Endrizzi M Birren B Lander ES 2003 Sequencingand comparison of yeast species to identify genes and regulatoryelements Nature 423241ndash254

Kelly B Whiteley A Tallmon D 2010 The Arctic melting pot Nature468891ndash891

Kvitek DJ Sherlock G 2011 Reciprocal sign epistasis between frequentlyexperimentally evolved adaptive mutations causes a rugged fitnesslandscape PLoS Genet 7e1002056

Kvitek DJ Sherlock G 2013 Whole genome whole population sequenc-ing reveals that loss of signaling networks is the major adaptivestrategy in a constant environment PLoS Genet 9e1003972

Landry CR Hartl DL Ranz JM 2007 Genome clashes in hybrids insightsfrom gene expression Heredity (Edinb) 99483ndash493

Lang GI Desai MM 2014 The spectrum of adaptive mutations in ex-perimental evolution Genomics 104412ndash416

Lapunzina P Monk D 2011 The consequences of uniparental disomyand copy number neutral loss-of-heterozygosity during human de-velopment and cancer Biol Cell 103303ndash317

Leducq JB Nielly-Thibault L Charron G Eberlein C Verta JP Samani PSylvester K Hittinger CT Bell G Landry CR 2016 Speciation drivenby hybridization and chromosomal plasticity in a wild yeast NatMicrobiol 115003

Lee HY Chou JY Cheong L Chang NH Yang SY Leu JY 2008Incompatibility of nuclear and mitochondrial genomes causes hy-brid sterility between two yeast species Cell 1351065ndash1073

Li H Durbin R 2009 Fast and accurate short read alignment withBurrows-Wheeler transform Bioinformatics 251754ndash1760

Li H Handsaker B Wysoker A Fennell T Ruan J Homer N Marth GAbecasis G Durbin R 2009 The Sequence AlignmentMap formatand SAMtools Bioinformatics 252078ndash2079

Liti G 2015 The fascinating and secret wild life of the budding yeast Scerevisiae Elife 4e05835

Liti G Barton DB Louis EJ 2006 Sequence diversity reproductiveisolation and species concepts in Saccharomyces Genetics174839ndash850

Lloyd AH Ranoux M Vautrin S Glover N Fourment J Charif D ChouletF Lassalle G Marande W Tran J et al 2014 Meiotic gene evolutioncan you teach a new dog new tricks Mol Biol Evol 311724ndash1727

Losos JB Jackman TR Larson A de Queiroz K Rodriguez-Schettino L1998 Contingency and determinism in replicated adaptive radia-tions of island lizards Science 2792115ndash2118

Louis VL Despons L Friedrich A Martin T Durrens P Casaregola SNeuveglise C Fairhead C Marck C Cruz JA et al 2012 Pichia sorbi-tophila an interspecies yeast hybrid reveals early steps of genomeresolution after polyploidization G3 (Bethesda) 2299ndash311

Mallet J 2007 Hybrid speciation Nature 446279ndash283Marcet-Houben M Gabaldon T 2015 Beyond the whole-genome du-

plication phylogenetic evidence for an ancient interspecies hybrid-ization in the bakerrsquos yeast lineage PLoS Biol 13e1002220

Marinoni G Manuel M Petersen RF Hvidtfeldt J Sulo P Piskur J 1999Horizontal transfer of genetic material among Saccharomyces yeastsJ Bacteriol 1816488ndash6496

Martin A Orgogozo V 2013 The loci of repeated evolution a catalog ofgenetic hotspots of phenotypic variation Evolution 671235ndash1250

Masly JP Jones CD Noor MAF Locke J Orr HA 2006 Gene transpositionas a cause of hybrid sterility in Drosophila Science 3131448ndash1450

Mason AS Batley J 2015 Creating new interspecific hybrid and polyploidcrops Trends Biotechnol 33436ndash441

Mavarez J Salazar CA Bermingham E Salcedo C Jiggins CD Linares M2006 Speciation by hybridization in Heliconius butterflies Nature441868ndash871

McKenna A Hanna M Banks E Sivachenko A Cibulskis K Kernytsky AGarimella K Altshuler D Gabriel S Daly M et al 2010 The genomeanalysis toolkit a MapReduce framework for analyzing next-generation DNA sequencing data Genome Res 201297ndash1303

Meiklejohn CD Holmbeck MA Siddiq MA Abt DN Rand DMMontooth KL 2013 An Incompatibility between a mitochondrialtRNA and its nuclear-encoded tRNA synthetase compromises de-velopment and fitness in Drosophila PLoS Genet 9e1003238

Meyer A Salzburger W Schartl M 2006 Hybrid origin of a swordtailspecies (Teleostei Xiphophorus clemenciae) driven by sexual selec-tion Mol Ecol 15721ndash730

Michalak P 2009 Epigenetic transposon and small RNA determinants ofhybrid dysfunctions Heredity (Edinb) 10245ndash50

Miller AW Dunham MJ 2013 Design and use of multiplexed chemostatarrays J Vis Exp 72e50262

Monod J 1949 The growth of bacterial cultures Annu Rev Microbiol3371ndash394

Mouillon JM Persson BL 2006 New aspects on phosphate sensing andsignalling in Saccharomyces cerevisiae FEMS Yeast Res 6171ndash176

Muhlfeld CC Kovach RP Jones LA Al-Chokhachy R Boyer MC Leary RFLowe WH Luikart G Allendorf FW 2014 Invasive hybridization in athreatened species is accelerated by climate change Nat ClimChange 4620ndash624

Muller LAH McCusker JH 2009 A multispecies-based taxonomic mi-croarray reveals interspecies hybridization and introgression inSaccharomyces cerevisiae FEMS Yeast Res 9143ndash152

Nagalakshmi U Wang Z Waern K Shou C Raha D Gerstein M SnyderM 2008 The transcriptional landscape of the yeast genome definedby RNA sequencing Science 3201344ndash1349

Nakao Y Kanamori T Itoh T Kodama Y Rainieri S Nakamura NShimonaga T Hattori M Ashikari T 2009 Genome sequence ofthe lager brewing yeast an interspecies hybrid DNA Res 16115ndash129

Nolte AW Freyhof J Stemshorn KC Tautz D 2005 An invasive lineage ofsculpins Cottus sp (Pisces Teleostei) in the Rhine with new habitatadaptations has originated from hybridization between old phylo-geographic groups Proc Biol Sci 2722379ndash2387

Novick A Szilard L 1950a Description of the chemostat Science112715ndash716

Novick A Szilard L 1950b Experiments with the Chemostat on spon-taneous mutations of bacteria Proc Natl Acad Sci U S A 36708ndash719

Ouyang YD Liu YG Zhang QF 2010 Hybrid sterility in plant storiesfrom rice Curr Opin Plant Biol 13186ndash192

Payen C Di Rienzi SC Ong GT Pogachar JL Sanchez JC SunshineAB Raghuraman MK Brewer BJ Dunham MJ 2014 The dynam-ics of diverse segmental amplifications in populations ofSaccharomyces cerevisiae adapting to strong selection G3(Bethesda) 4399ndash409

Payen C Sunshine AB Ong GT Pogachar JL Zhao W Dunham MJ 2016High-throughput identification of adaptive mutations in experimen-tally evolved yeast populations PLoS Genet 12e1006339

Perez-Prat E van Lookeren Campagne MM 2002 Hybrid seed produc-tion and the challenge of propagating male-sterile plants TrendsPlant Sci 7199ndash203

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16

Peris D Langdon QK Moriarty RV Sylvester K Bontrager M Charron GLeducq JB Landry CR Libkind D Hittinger CT 2016 Complex ances-tries of lager-brewing hybrids were shaped by standing variation inthe wild yeast Saccharomyces eubayanus PLoS Genet 12e1006155

Perlstein EO Ruderfer DM Roberts DC Schreiber SL Kruglyak L 2007Genetic basis of individual differences in the response to small-molecule drugs in yeast Nat Genet 39496ndash502

Piatkowska EM Naseeb S Knight D Delneri D 2013 Chimeric proteincomplexes in hybrid species generate novel phenotypes PLoS Genet9e1003836

Piotrowski JS Nagarajan S Kroll E Stanbery A Chiotti KE Kruckeberg ALDunn B Sherlock G Rosenzweig F 2012 Different selective pressureslead to different genomic outcomes as newly-formed hybrid yeastsevolve BMC Evol Biol 1246

Pryszcz LP Nemeth T Gacser A Gabaldon T 2014 Genome comparisonof Candida orthopsilosis clinical strains reveals the existence of hy-brids between two distinct subspecies Genome Biol Evol61069ndash1078

Racimo F Sankararaman S Nielsen R Huerta-Sanchez E 2015 Evidencefor archaic adaptive introgression in humans Nat Rev Genet16359ndash371

Rieseberg LH 1991 Homoploid reticulate evolution in Helianthus(Asteraceae)mdashevidence from ribosomal genes Am J Bot781218ndash1237

Rieseberg LH 1997 Hybrid origins of plant species Annu Rev EcolSystemat 28359ndash389

Robinson JT Thorvaldsdottir H Winckler W Guttman M Lander ESGetz G Mesirov JP 2011 Integrative genomics viewer NatBiotechnol 2924ndash26

Rosenthal DM Schwarzbach AE Donovan LA Raymond O RiesebergLH 2002 Phenotypic differentiation between three ancient hybridtaxa and their parental species Int J Plant Sci 163387ndash398

Ruderfer DM Pratt SC Seidel HS Kruglyak L 2006 Population genomicanalysis of outcrossing and recombination in yeast Nat Genet381077ndash1081

Rundle HD Nagel L Boughman JW Schluter D 2000 Natural selectionand parallel speciation in sympatric sticklebacks Science287306ndash308

Ryland GL Doyle MA Goode D Boyle SE Choong DY Rowley SM Li JBowtell DD Tothill RW Campbell IG et al 2015 Australian OvarianCancer Study Group Loss of heterozygosity what is it good for BMCMed Genomics 845

Salverda MLM Dellus E Gorter FA Debets AJM van der Oost J HoekstraRF Tawfik DS de Visser JAGM 2011 Initial mutations direct alter-native pathways of protein evolution PLoS Genet 7e1001321

Sanchez MR Miller AW Liachko I Sunshine AB Lynch B Huang MAlcantara E DeSevo CG Pai DA Tucker CM et al 2017 Differentialparalog divergence modulates genome evolution across yeast spe-cies PLoS Genet 13(2)e1006585

Scannell DR Zill OA Rokas A Payen C Dunham MJ Eisen MB Rine JJohnston M Hittinger CT 2011 The awesome power of yeast evo-lutionary genetics new genome sequences and strain resources forthe Saccharomyces sensu stricto genus G3 (Bethesda) 111ndash25

Schroder MS Martinez de San Vicente K Prandini TH Hammel SHiggins DG Bagagli E Wolfe KH Butler G 2016 Multiple originsof the pathogenic yeast Candida orthopsilosis by separate hybridiza-tions between two parental species PLoS Genet 12e1006404

Schumer M Rosenthal GG Andolfatto P 2014 How common is homo-ploid hybrid speciation Evolution 681553ndash1560

Schwarzbach AE Donovan LA Rieseberg LH 2001 Transgressivecharacter expression in a hybrid sunflower species Am J Bot88270ndash277

Selmecki AM Maruvka YE Richmond PA Guillet M Shoresh NSorenson AL De S Kishony R Michor F Dowell R et al 2015Polyploidy can drive rapid adaptation in yeast Nature 519349ndash52

Seshan VE Olshen A 2016 DNAcopy DNA copy number data analysisVersion R package version 1460

Soltis DE Visger CJ Soltis PS 2014 The polyploidy revolution then andnow Stebbins revisited Am J Bot 1011057ndash1078

Soltis PS 2013 Hybridization speciation and novelty J Evol Biol26291ndash293

Soltis PS Soltis DE 2009 The role of hybridization in plant speciationAnnu Rev Plant Biol 60561ndash588

Sunshine AB Payen C Ong GT Liachko I Tan KM Dunham MJ 2015The fitness consequences of aneuploidy are driven by condition-dependent gene effects PLoS Biol 13e1002155

Tamai Y Momma T Yoshimoto H Kaneko Y 1998 Co-existence of twotypes of chromosome in the bottom fermenting yeastSaccharomyces pastorianus Yeast 14923ndash933

Taylor DJ Hebert PDN 1993 Habitat-dependent hybrid parentage anddifferential introgression between neighboringly sympatric Daphniaspecies Proc Natl Acad Sci U S A 907079ndash7083

Thiagalingam S Laken S Willson JK Markowitz SD Kinzler KWVogelstein B Lengauer C 2001 Mechanisms underlying losses ofheterozygosity in human colorectal cancers Proc Natl Acad Sci US A 982698ndash2702

Tuna M Knuutila S Mills GB 2009 Uniparental disomy in cancer TrendsMol Med 15120ndash128

Walther A Hesselbart A Wendland J 2014 Genome sequence ofSaccharomyces carlsbergensis the worldrsquos first pure culture lageryeast G3 (Bethesda) 4783ndash793

Wayne RK 1993 Molecular evolution of the dog family Trends Genet9218ndash224

Wenger JW Schwartz K Sherlock G 2010 Bulk segregant analysis byhigh-throughput sequencing reveals a novel xylose utilization genefrom Saccharomyces cerevisiae PLoS Genet 6e1000942

Wessinger CA Rausher MD 2014 Predictability and irreversibility ofgenetic changes associated with flower color evolution inPenstemon barbatus Evolution 681058ndash1070

Wolfe KH 2015 Origin of the yeast whole-genome duplication PLoSBiol 13e1002221

Woods R Schneider D Winkworth CL Riley MA Lenski RE 2006 Testsof parallel molecular evolution in a long-term experiment withEscherichia coli Proc Natl Acad Sci U S A 1039107ndash9112

Wykoff DD OrsquoShea EK 2001 Phosphate transport and sensing inSaccharomyces cerevisiae Genetics 1591491ndash1499

Xiong Z Gaeta RT Pires JC 2011 Homoeologous shuffling andchromosome compensation maintain genome balance inresynthesized allopolyploid Brassica napus Proc Natl Acad SciU S A 1087908ndash7913

Yassour M Kapian T Fraser HB Levin JZ Pfiffner J Adiconis X Schroth GLuo SJ Khrebtukova I Gnirke A et al 2009 Ab initio construction ofa eukaryotic transcriptome by massively parallel mRNA sequencingProc Natl Acad Sci U S A 1063264ndash3269

Zhu YO Sherlock G Petrov DA 2016 Whole genome analysis of 132clinical Saccharomyces cerevisiae strains reveals extensive ploidy var-iation G3 (Bethesda) doi101534g3116029397

Adaptation in Hybrids doi101093molbevmsx098 MBE

17

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Page 14: Loss of Heterozygosity Drives Adaptation in Hybrid Yeastdunham.gs.washington.edu/SmukowskiHeil.pdf · 2017-05-20 · Loss of Heterozygosity Drives Adaptation in Hybrid Yeast Caiti

2) Samples from each end point population were colonypurified to yield two clones for further study

Array Comparative Genomic HybridizationPopulations from the end point of each evolution were ana-lyzed for copy number changes using array comparative ge-nomic hybridization following the protocol used in Sanchezet al (2017)

SequencingDNA was extracted from overnight cultures using theHoffmanndashWinston protocol (Hoffman and Winston 1987)and cleaned using the Clean amp Concentrator kit (ZymoResearch) Nextera libraries were prepared following theNextera library kit protocol and sequenced using paired end150 bp reads on the Illumina NextSeq 500 machine (sequenc-ing coverage in supplementary table S2 SupplementaryMaterial online) The reference genomes used were S cerevi-siae v3 (Engel et al 2014) S uvarum (Scannell et al 2011) anda hybrid reference genome created by concatenating the twogenomes Sequence was aligned to the appropriate referencegenome using bwa v062 (Li and Durbin 2009) and mutationswere called using GATK (McKenna et al 2010) and samtools0119 (Li et al 2009) Mutations in evolved clones were filteredin comparison with the ancestor to obtain de novo mutationsAll mutations were first visually inspected using IntegrativeGenomics Viewer (Robinson et al 2011) Subsequently pointmutations in the hybrids were confirmed with Sanger se-quencing (supplementary table S3 Supplementary Materialonline) Copy number variants were visualized usingDNAcopy for S cerevisiae and S uvarum (Seshan andOlshen 2016) LOH events were called based on sequencingcoverage in the hybrids and by identifying homozygous variantcalls in S cerevisiae All break points were called by visual in-spection of sequencing reads and are thus approximate

Fitness AssaysThe pairwise competition experiments were performed in 20ml chemostats (Miller and Dunham 2013) Each competitorstrain was cultured individually until steady state was reachedand then was mixed 5050 with a GFP-tagged ancestor Eachcompetition was conducted in at least two biological repli-cates for approximately 15 generations after mixing Sampleswere collected and analyzed twice daily The proportion ofGFPthorn cells in the population was detected using a BD AccuriC6 flow cytometer (BD Biosciences) The data were plottedwith ln [dark cellsGFPthorn cells] versus generations The rela-tive fitness coefficient was determined from the slope of thelinear region

Strain ConstructionAllele replacements for the PHO84 locus were done followingthe protocol of the Caudy lab with further modifications de-scribed here The native locus was replaced withKluyveromyces lactis URA3 The pho84DURA3 strain wasgrown overnight in 5 ml of C-URA media then inoculatedin a flask of 100 ml yeast extract peptone dextrose (YPD) andgrown to an optical density of 06ndash08 Cells were washed then

aliquoted 275 ml of transformation mix (35 ml 1 M lithiumacetate 240 ml of 50 3500 polyethylene glycol) 10 ml ofsalmon sperm and approximately 3 mg of polymerase chainreaction product were added to the cell pellet It was incu-bated at 37 C (S uvarum) or 42 C (S cerevisiae) for 45 minthen plated to YPD It was replica plated to 5-fluorooroticacid the following day and colonies were tested for the gain ofthe appropriate species allele The GRF167 allele was clonedinto the pIL37 plasmid using Gibson assembly (Gibson et al2009) Correct assembly was verified by Sanger sequencing Allprimers used can be found in supplementary table S3Supplementary Material online

Supplementary MaterialSupplementary data are available at Molecular Biology andEvolution online

AcknowledgmentsWe thank Noah Hanson and Erica Alcantara for technicalassistance We thank Monica Sanchez and three anonymousreviewers for helpful comments on this manuscript JosephSchacherer kindly confirmed the contribution of FL100 to theGRF167 strain background Thanks to Yixian Zheng and DougKoshland for contributing to the initial experimental designcreating yeast strains and purchasing the oligonucleotidesused for the microarrays This work was supported by theNational Science Foundation (grant number 1516330) andthe National Institutes of Health (grant number R01GM094306) MD is a Senior Fellow in the GeneticNetworks program at the Canadian Institute for AdvancedResearch and a Rita Allen Foundation Scholar CSH wassupported in part by National Institutes of Health (grantnumber T32 HG00035) This work was also supported bythe National Institutes of Health (grant number P50GM071508) to the Lewis-Sigler Institute and from theHoward Hughes Medical Institute to Doug Koshland andYixian Zheng Sequencing read data have been deposited atthe NCBI under BioProject accession PRJNA374049Microarray data are deposited in the Gene ExpressionOmnibus (GEO) repository under accession numbersGSE95086 and GSE87401 and in the Princeton MicroarrayDatabase

ReferencesAbbott R Albach D Ansell S Arntzen JW Baird SJ Bierne N Boughman J

Brelsford A Buerkle CA Buggs R et al 2013 Hybridization andspeciation J Evol Biol 26229ndash246

Ainouche ML Jenczewski E 2010 Focus on polyploidy New Phytol1861ndash4

Albertin W Marullo P 2012 Polyploidy in fungi evolution after whole-genome duplication Proc Biol Sci 2792497ndash2509

Almeida P Goncalves C Teixeira S Libkind D Bontrager M Masneuf-Pomarede I Albertin W Durrens P Sherman DJ Marullo P et al2014 A Gondwanan imprint on global diversity and domesticationof wine and cider yeast Saccharomyces uvarum Nat Commun54044

Antunovics Z Nguyen HV Gaillardin C Sipiczki M 2005 Gradual ge-nome stabilisation by progressive reduction of the Saccharomyces

Smukowski Heil et al doi101093molbevmsx098 MBE

14

uvarum genome in an interspecific hybrid with Saccharomyces cer-evisiae FEMS Yeast Res 51141ndash1150

Baker E Wang B Bellora N Peris D Hulfachor AB Koshalek JA AdamsM Libkind D Hittinger CT 2015 The Genome sequence ofSaccharomyces eubayanus and the domestication of lager-brewingyeasts Mol Biol Evol 322818ndash2831

Barbosa R Almeida P Safar SVB Santos RO Morais PB Nielly-Thibault LLeducq JB Landry CR Goncalves P Rosa CA Sampaio JP 2016Evidence of natural hybridization in Brazilian wild lineages ofSaccharomyces cerevisiae Genome Biol Evol 8317ndash329

Barton NH 2001 The role of hybridization in evolution Mol Ecol10551ndash568

Belloch C Orlic S Barrio E Querol A 2008 Fermentative stress adapta-tion of hybrids within the Saccharomyces sensu stricto complex Int JFood Microbiol 122188ndash195

Bellon JR Yang F Day MP Inglis DL Chambers PJ 2015 Designingand creating Saccharomyces interspecific hybrids for improvedindustry relevant phenotypes Appl Microbiol Biotechnol998597ndash8609

Borneman AR Zeppel R Chambers PJ Curtin CD 2014 Insights into theDekkera bruxellensis genomic landscape comparative genomics re-veals variations in ploidy and nutrient utilisation potential amongstwine isolates PLoS Genet 10e1004161

Brewer BJ Payen C Di Rienzi SC Higgins MM Ong G Dunham MJRaghuraman MK 2015 Origin-dependent inverted-repeat amplifi-cation tests of a model for inverted DNA amplification PLoS Genet11e1005699

Brown CJ Todd KM Rosenzweig RF 1998 Multiple duplications of yeasthexose transport genes in response to selection in a glucose-limitedenvironment Mol Biol Evol 15931ndash942

Bullini L 1994 Origin and evolution of animal hybrid species Trends EcolEvol 9422ndash426

Bun-Ya M Nishimura M Harashima S Oshima Y 1991 The PHO84 geneof Saccharomyces cerevisiae encodes an inorganic phosphate trans-porter Mol Cell Biol 113229ndash3238

Burke MK Dunham JP Shahrestani P Thornton KR Rose MR Long AD2010 Genome-wide analysis of a long-term evolution experimentwith Drosophila Nature 467587ndash590

Burke MK Liti G Long AD 2014 Standing genetic variation drives re-peatable experimental evolution in outcrossing populations ofSaccharomyces cerevisiae Mol Biol Evol 313228ndash3239

Cherry JM Hong EL Amundsen C Balakrishnan R Binkley G Chan ETChristie KR Costanzo MC Dwight SS Engel SR et al 2012Saccharomyces genome database the genomics resource of buddingyeast Nucleic Acids Res 40D700ndashD705

Chester M Riley RK Soltis PS Soltis DE 2015 Patterns of chromosomalvariation in natural populations of the neoallotetraploid Tragopogonmirus (Asteraceae) Heredity (Edinb) 114309ndash317

Chou JY Leu JY 2010 Speciation through cytonuclear incompatibilityinsights from yeast and implications for higher eukaryotes Bioessays32401ndash411

Cliften PF Fulton RS Wilson RK Johnston M 2006 After the duplicationgene loss and adaptation in Saccharomyces genomes Genetics172863ndash872

Conte GL Arnegard ME Peichel CL Schluter D 2012 The probability ofgenetic parallelism and convergence in natural populations Proc BiolSci 2795039ndash5047

Cordes FS Bright JN Sansom MS 2002 Proline-induced distortions oftransmembrane helices J Mol Biol 323951ndash960

Crow JF 1998 90 years ago the beginning of hybrid maize Genetics148923ndash928

Dasmahapatra KK Walters JR Briscoe AD Davey JW Whibley A NadeauNJ Zimin AV Hughes DST Ferguson LC Martin SH et al 2012Butterfly genome reveals promiscuous exchange of mimicry adap-tations among species Nature 48794ndash98

Dowling TE Smith GR Brown WM 1989 Reproductive isolation andintrogression between Notropis-Cornutus and Notropis-Chrysocephalus (Family Cyprinidae)mdashcomparison of morphologyallozymes and mitochondrial-DNA Evolution 43620ndash634

Doyle JJ Flagel LE Paterson AH Rapp RA Soltis DE Soltis PS Wendel JF2008 Evolutionary genetics of genome merger and doubling inplants Annu Rev Genet 42443ndash461

Dunham MJ Badrane H Ferea T Adams J Brown PO Rosenzweig FBotstein D 2002 Characteristic genome rearrangements in experi-mental evolution of Saccharomyces cerevisiae Proc Natl Acad Sci U SA 9916144ndash16149

Dunn B Paulish T Stanbery A Piotrowski J Koniges G Kroll E LouisEJ Liti G Sherlock G Rosenzweig F 2013 Recurrent rearrange-ment during adaptive evolution in an interspecific yeast hybridsuggests a model for rapid introgression PLoS Genet9e1003366

Dunn B Richter C Kvitek DJ Pugh T Sherlock G 2012 Analysis of theSaccharomyces cerevisiae pan-genome reveals a pool of copy num-ber variants distributed in diverse yeast strains from differing indus-trial environments Genome Res 22908ndash924

Dunn B Sherlock G 2008 Reconstruction of the genome origins andevolution of the hybrid lager yeast Saccharomyces pastorianusGenome Res 181610ndash1623

Elmer KR Meyer A 2011 Adaptation in the age of ecological genomicsinsights from parallelism and convergence Trends Ecol Evol26298ndash306

Engel SR Dietrich FS Fisk DG Binkley G Balakrishnan R Costanzo MCDwight SS Hitz BC Karra K Nash RS et al 2014 The referencegenome sequence of Saccharomyces cerevisiae then and now G3(Bethesda) 4389ndash398

Fedoroff NV 2012 Presidential address Transposable elements epige-netics and genome evolution Science 338758ndash767

Ferea TL Botstein D Brown PO Rosenzweig RF 1999 Systematicchanges in gene expression patterns following adaptive evolutionin yeast Proc Natl Acad Sci U S A 969721ndash9726

Gibson B Liti G 2015 Saccharomyces pastorianus genomic insightsinspiring innovation for industry Yeast 3217ndash27

Gibson DG Young L Chuang RY Venter JC Hutchison CA Smith HO2009 Enzymatic assembly of DNA molecules up to several hundredkilobases Nat Methods 6343ndash5

Gonzalez SS Barrio E Gafner J Querol A 2006 Natural hybrids fromSaccharomyces cerevisiae Saccharomyces bayanus andSaccharomyces kudriavzevii in wine fermentations FEMS Yeast Res61221ndash1234

Gonzalez SS Barrio E Querol A 2008 Molecular characterization of newnatural hybrids of Saccharomyces cerevisiae and S kudriavzevii inbrewing Appl Environ Microbiol 742314ndash2320

Grant PR Grant BR 1994 Phenotypic and genetic consequences ofhybridization in Darwinrsquos finches Evolution 48297ndash316

Grant PR Grant BR 2002 Unpredictable evolution in a 30-year study ofDarwinrsquos finches Science 296707ndash711

Grant PR Grant BR 2010 Natural selection speciation and Darwinrsquosfinches Proc Calif Acad Sci 61245ndash260

Grant PR Grant BR Petren K 2005 Hybridization in the recent past AmNat 16656ndash67

Greig D 2009 Reproductive isolation in Saccharomyces Heredity (Edinb)10239ndash44

Greig D Leu JY 2009 Natural history of budding yeast Current Biol19R886ndashR890

Greig D Louis EJ Borts RH Travisano M 2002 Hybrid speciation inexperimental populations of yeast Science 2981773ndash1775

Gresham D Desai MM Tucker CM Jenq HT Pai DA Ward A DeSevoCG Botstein D Dunham MJ 2008 The repertoire and dynamics ofevolutionary adaptations to controlled nutrient-limited environ-ments in yeast PLoS Genet 4e1000303

Hajjar R Hodgkin T 2007 The use of wild relatives in crop improvementa survey of developments over the last 20 years Euphytica 1561ndash13

Heiser CB 1954 Variation and subspeciation in the common sunflowerHelianthus annuus Am Midl Nat 51287ndash305

Hittinger CT 2013 Saccharomyces diversity and evolution a buddingmodel genus Trends Genet 29309ndash317

Hoang ML Tan FJ Lai DC Celniker SE Hoskins RA Dunham MJ ZhengY Koshland D 2010 Competitive repair by naturally dispersed

Adaptation in Hybrids doi101093molbevmsx098 MBE

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repetitive DNA during non-allelic homologous recombination PLoSGenet 6e1001228

Hoffmann AA Sgro CM 2011 Climate change and evolutionary adap-tation Nature 470479ndash485

Hoffman CS Winston F 1987 A 10-minute DNA preparation from yeastefficiently releases autonomous plasmids for transformation ofEscherichia coli Gene 57267ndash272

Hou J Friedrich A de Montigny J Schacherer J 2014 Chromosomalrearrangements as a major mechanism in the onset of reproductiveisolation in Saccharomyces cerevisiae Curr Biol 241153ndash1159

Hou J Friedrich A Gounot JS Schacherer J 2015 Comprehensive surveyof condition-specific reproductive isolation reveals genetic incom-patibility in yeast Nat Commun 67214

Huerta-Sanchez E Casey FP 2015 Archaic inheritance supporting high-altitude life in Tibet J Appl Physiol (1985) 1191129ndash1134

Jones FC Grabherr MG Chan YF Russell P Mauceli E Johnson JSwofford R Pirun M Zody MC White S et al 2012 The genomicbasis of adaptive evolution in threespine sticklebacks Nature48455ndash61

Kao KC Sherlock G 2008 Molecular characterization of clonal interfer-ence during adaptive evolution in asexual populations ofSaccharomyces cerevisiae Nat Genet 401499ndash1504

Kawecki TJ Lenski RE Ebert D Hollis B Olivieri I Whitlock MC 2012Experimental evolution Trends Ecol Evol 27547ndash560

Kellis M Patterson N Endrizzi M Birren B Lander ES 2003 Sequencingand comparison of yeast species to identify genes and regulatoryelements Nature 423241ndash254

Kelly B Whiteley A Tallmon D 2010 The Arctic melting pot Nature468891ndash891

Kvitek DJ Sherlock G 2011 Reciprocal sign epistasis between frequentlyexperimentally evolved adaptive mutations causes a rugged fitnesslandscape PLoS Genet 7e1002056

Kvitek DJ Sherlock G 2013 Whole genome whole population sequenc-ing reveals that loss of signaling networks is the major adaptivestrategy in a constant environment PLoS Genet 9e1003972

Landry CR Hartl DL Ranz JM 2007 Genome clashes in hybrids insightsfrom gene expression Heredity (Edinb) 99483ndash493

Lang GI Desai MM 2014 The spectrum of adaptive mutations in ex-perimental evolution Genomics 104412ndash416

Lapunzina P Monk D 2011 The consequences of uniparental disomyand copy number neutral loss-of-heterozygosity during human de-velopment and cancer Biol Cell 103303ndash317

Leducq JB Nielly-Thibault L Charron G Eberlein C Verta JP Samani PSylvester K Hittinger CT Bell G Landry CR 2016 Speciation drivenby hybridization and chromosomal plasticity in a wild yeast NatMicrobiol 115003

Lee HY Chou JY Cheong L Chang NH Yang SY Leu JY 2008Incompatibility of nuclear and mitochondrial genomes causes hy-brid sterility between two yeast species Cell 1351065ndash1073

Li H Durbin R 2009 Fast and accurate short read alignment withBurrows-Wheeler transform Bioinformatics 251754ndash1760

Li H Handsaker B Wysoker A Fennell T Ruan J Homer N Marth GAbecasis G Durbin R 2009 The Sequence AlignmentMap formatand SAMtools Bioinformatics 252078ndash2079

Liti G 2015 The fascinating and secret wild life of the budding yeast Scerevisiae Elife 4e05835

Liti G Barton DB Louis EJ 2006 Sequence diversity reproductiveisolation and species concepts in Saccharomyces Genetics174839ndash850

Lloyd AH Ranoux M Vautrin S Glover N Fourment J Charif D ChouletF Lassalle G Marande W Tran J et al 2014 Meiotic gene evolutioncan you teach a new dog new tricks Mol Biol Evol 311724ndash1727

Losos JB Jackman TR Larson A de Queiroz K Rodriguez-Schettino L1998 Contingency and determinism in replicated adaptive radia-tions of island lizards Science 2792115ndash2118

Louis VL Despons L Friedrich A Martin T Durrens P Casaregola SNeuveglise C Fairhead C Marck C Cruz JA et al 2012 Pichia sorbi-tophila an interspecies yeast hybrid reveals early steps of genomeresolution after polyploidization G3 (Bethesda) 2299ndash311

Mallet J 2007 Hybrid speciation Nature 446279ndash283Marcet-Houben M Gabaldon T 2015 Beyond the whole-genome du-

plication phylogenetic evidence for an ancient interspecies hybrid-ization in the bakerrsquos yeast lineage PLoS Biol 13e1002220

Marinoni G Manuel M Petersen RF Hvidtfeldt J Sulo P Piskur J 1999Horizontal transfer of genetic material among Saccharomyces yeastsJ Bacteriol 1816488ndash6496

Martin A Orgogozo V 2013 The loci of repeated evolution a catalog ofgenetic hotspots of phenotypic variation Evolution 671235ndash1250

Masly JP Jones CD Noor MAF Locke J Orr HA 2006 Gene transpositionas a cause of hybrid sterility in Drosophila Science 3131448ndash1450

Mason AS Batley J 2015 Creating new interspecific hybrid and polyploidcrops Trends Biotechnol 33436ndash441

Mavarez J Salazar CA Bermingham E Salcedo C Jiggins CD Linares M2006 Speciation by hybridization in Heliconius butterflies Nature441868ndash871

McKenna A Hanna M Banks E Sivachenko A Cibulskis K Kernytsky AGarimella K Altshuler D Gabriel S Daly M et al 2010 The genomeanalysis toolkit a MapReduce framework for analyzing next-generation DNA sequencing data Genome Res 201297ndash1303

Meiklejohn CD Holmbeck MA Siddiq MA Abt DN Rand DMMontooth KL 2013 An Incompatibility between a mitochondrialtRNA and its nuclear-encoded tRNA synthetase compromises de-velopment and fitness in Drosophila PLoS Genet 9e1003238

Meyer A Salzburger W Schartl M 2006 Hybrid origin of a swordtailspecies (Teleostei Xiphophorus clemenciae) driven by sexual selec-tion Mol Ecol 15721ndash730

Michalak P 2009 Epigenetic transposon and small RNA determinants ofhybrid dysfunctions Heredity (Edinb) 10245ndash50

Miller AW Dunham MJ 2013 Design and use of multiplexed chemostatarrays J Vis Exp 72e50262

Monod J 1949 The growth of bacterial cultures Annu Rev Microbiol3371ndash394

Mouillon JM Persson BL 2006 New aspects on phosphate sensing andsignalling in Saccharomyces cerevisiae FEMS Yeast Res 6171ndash176

Muhlfeld CC Kovach RP Jones LA Al-Chokhachy R Boyer MC Leary RFLowe WH Luikart G Allendorf FW 2014 Invasive hybridization in athreatened species is accelerated by climate change Nat ClimChange 4620ndash624

Muller LAH McCusker JH 2009 A multispecies-based taxonomic mi-croarray reveals interspecies hybridization and introgression inSaccharomyces cerevisiae FEMS Yeast Res 9143ndash152

Nagalakshmi U Wang Z Waern K Shou C Raha D Gerstein M SnyderM 2008 The transcriptional landscape of the yeast genome definedby RNA sequencing Science 3201344ndash1349

Nakao Y Kanamori T Itoh T Kodama Y Rainieri S Nakamura NShimonaga T Hattori M Ashikari T 2009 Genome sequence ofthe lager brewing yeast an interspecies hybrid DNA Res 16115ndash129

Nolte AW Freyhof J Stemshorn KC Tautz D 2005 An invasive lineage ofsculpins Cottus sp (Pisces Teleostei) in the Rhine with new habitatadaptations has originated from hybridization between old phylo-geographic groups Proc Biol Sci 2722379ndash2387

Novick A Szilard L 1950a Description of the chemostat Science112715ndash716

Novick A Szilard L 1950b Experiments with the Chemostat on spon-taneous mutations of bacteria Proc Natl Acad Sci U S A 36708ndash719

Ouyang YD Liu YG Zhang QF 2010 Hybrid sterility in plant storiesfrom rice Curr Opin Plant Biol 13186ndash192

Payen C Di Rienzi SC Ong GT Pogachar JL Sanchez JC SunshineAB Raghuraman MK Brewer BJ Dunham MJ 2014 The dynam-ics of diverse segmental amplifications in populations ofSaccharomyces cerevisiae adapting to strong selection G3(Bethesda) 4399ndash409

Payen C Sunshine AB Ong GT Pogachar JL Zhao W Dunham MJ 2016High-throughput identification of adaptive mutations in experimen-tally evolved yeast populations PLoS Genet 12e1006339

Perez-Prat E van Lookeren Campagne MM 2002 Hybrid seed produc-tion and the challenge of propagating male-sterile plants TrendsPlant Sci 7199ndash203

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Peris D Langdon QK Moriarty RV Sylvester K Bontrager M Charron GLeducq JB Landry CR Libkind D Hittinger CT 2016 Complex ances-tries of lager-brewing hybrids were shaped by standing variation inthe wild yeast Saccharomyces eubayanus PLoS Genet 12e1006155

Perlstein EO Ruderfer DM Roberts DC Schreiber SL Kruglyak L 2007Genetic basis of individual differences in the response to small-molecule drugs in yeast Nat Genet 39496ndash502

Piatkowska EM Naseeb S Knight D Delneri D 2013 Chimeric proteincomplexes in hybrid species generate novel phenotypes PLoS Genet9e1003836

Piotrowski JS Nagarajan S Kroll E Stanbery A Chiotti KE Kruckeberg ALDunn B Sherlock G Rosenzweig F 2012 Different selective pressureslead to different genomic outcomes as newly-formed hybrid yeastsevolve BMC Evol Biol 1246

Pryszcz LP Nemeth T Gacser A Gabaldon T 2014 Genome comparisonof Candida orthopsilosis clinical strains reveals the existence of hy-brids between two distinct subspecies Genome Biol Evol61069ndash1078

Racimo F Sankararaman S Nielsen R Huerta-Sanchez E 2015 Evidencefor archaic adaptive introgression in humans Nat Rev Genet16359ndash371

Rieseberg LH 1991 Homoploid reticulate evolution in Helianthus(Asteraceae)mdashevidence from ribosomal genes Am J Bot781218ndash1237

Rieseberg LH 1997 Hybrid origins of plant species Annu Rev EcolSystemat 28359ndash389

Robinson JT Thorvaldsdottir H Winckler W Guttman M Lander ESGetz G Mesirov JP 2011 Integrative genomics viewer NatBiotechnol 2924ndash26

Rosenthal DM Schwarzbach AE Donovan LA Raymond O RiesebergLH 2002 Phenotypic differentiation between three ancient hybridtaxa and their parental species Int J Plant Sci 163387ndash398

Ruderfer DM Pratt SC Seidel HS Kruglyak L 2006 Population genomicanalysis of outcrossing and recombination in yeast Nat Genet381077ndash1081

Rundle HD Nagel L Boughman JW Schluter D 2000 Natural selectionand parallel speciation in sympatric sticklebacks Science287306ndash308

Ryland GL Doyle MA Goode D Boyle SE Choong DY Rowley SM Li JBowtell DD Tothill RW Campbell IG et al 2015 Australian OvarianCancer Study Group Loss of heterozygosity what is it good for BMCMed Genomics 845

Salverda MLM Dellus E Gorter FA Debets AJM van der Oost J HoekstraRF Tawfik DS de Visser JAGM 2011 Initial mutations direct alter-native pathways of protein evolution PLoS Genet 7e1001321

Sanchez MR Miller AW Liachko I Sunshine AB Lynch B Huang MAlcantara E DeSevo CG Pai DA Tucker CM et al 2017 Differentialparalog divergence modulates genome evolution across yeast spe-cies PLoS Genet 13(2)e1006585

Scannell DR Zill OA Rokas A Payen C Dunham MJ Eisen MB Rine JJohnston M Hittinger CT 2011 The awesome power of yeast evo-lutionary genetics new genome sequences and strain resources forthe Saccharomyces sensu stricto genus G3 (Bethesda) 111ndash25

Schroder MS Martinez de San Vicente K Prandini TH Hammel SHiggins DG Bagagli E Wolfe KH Butler G 2016 Multiple originsof the pathogenic yeast Candida orthopsilosis by separate hybridiza-tions between two parental species PLoS Genet 12e1006404

Schumer M Rosenthal GG Andolfatto P 2014 How common is homo-ploid hybrid speciation Evolution 681553ndash1560

Schwarzbach AE Donovan LA Rieseberg LH 2001 Transgressivecharacter expression in a hybrid sunflower species Am J Bot88270ndash277

Selmecki AM Maruvka YE Richmond PA Guillet M Shoresh NSorenson AL De S Kishony R Michor F Dowell R et al 2015Polyploidy can drive rapid adaptation in yeast Nature 519349ndash52

Seshan VE Olshen A 2016 DNAcopy DNA copy number data analysisVersion R package version 1460

Soltis DE Visger CJ Soltis PS 2014 The polyploidy revolution then andnow Stebbins revisited Am J Bot 1011057ndash1078

Soltis PS 2013 Hybridization speciation and novelty J Evol Biol26291ndash293

Soltis PS Soltis DE 2009 The role of hybridization in plant speciationAnnu Rev Plant Biol 60561ndash588

Sunshine AB Payen C Ong GT Liachko I Tan KM Dunham MJ 2015The fitness consequences of aneuploidy are driven by condition-dependent gene effects PLoS Biol 13e1002155

Tamai Y Momma T Yoshimoto H Kaneko Y 1998 Co-existence of twotypes of chromosome in the bottom fermenting yeastSaccharomyces pastorianus Yeast 14923ndash933

Taylor DJ Hebert PDN 1993 Habitat-dependent hybrid parentage anddifferential introgression between neighboringly sympatric Daphniaspecies Proc Natl Acad Sci U S A 907079ndash7083

Thiagalingam S Laken S Willson JK Markowitz SD Kinzler KWVogelstein B Lengauer C 2001 Mechanisms underlying losses ofheterozygosity in human colorectal cancers Proc Natl Acad Sci US A 982698ndash2702

Tuna M Knuutila S Mills GB 2009 Uniparental disomy in cancer TrendsMol Med 15120ndash128

Walther A Hesselbart A Wendland J 2014 Genome sequence ofSaccharomyces carlsbergensis the worldrsquos first pure culture lageryeast G3 (Bethesda) 4783ndash793

Wayne RK 1993 Molecular evolution of the dog family Trends Genet9218ndash224

Wenger JW Schwartz K Sherlock G 2010 Bulk segregant analysis byhigh-throughput sequencing reveals a novel xylose utilization genefrom Saccharomyces cerevisiae PLoS Genet 6e1000942

Wessinger CA Rausher MD 2014 Predictability and irreversibility ofgenetic changes associated with flower color evolution inPenstemon barbatus Evolution 681058ndash1070

Wolfe KH 2015 Origin of the yeast whole-genome duplication PLoSBiol 13e1002221

Woods R Schneider D Winkworth CL Riley MA Lenski RE 2006 Testsof parallel molecular evolution in a long-term experiment withEscherichia coli Proc Natl Acad Sci U S A 1039107ndash9112

Wykoff DD OrsquoShea EK 2001 Phosphate transport and sensing inSaccharomyces cerevisiae Genetics 1591491ndash1499

Xiong Z Gaeta RT Pires JC 2011 Homoeologous shuffling andchromosome compensation maintain genome balance inresynthesized allopolyploid Brassica napus Proc Natl Acad SciU S A 1087908ndash7913

Yassour M Kapian T Fraser HB Levin JZ Pfiffner J Adiconis X Schroth GLuo SJ Khrebtukova I Gnirke A et al 2009 Ab initio construction ofa eukaryotic transcriptome by massively parallel mRNA sequencingProc Natl Acad Sci U S A 1063264ndash3269

Zhu YO Sherlock G Petrov DA 2016 Whole genome analysis of 132clinical Saccharomyces cerevisiae strains reveals extensive ploidy var-iation G3 (Bethesda) doi101534g3116029397

Adaptation in Hybrids doi101093molbevmsx098 MBE

17

  • msx098-TF1
  • msx098-TF2
Page 15: Loss of Heterozygosity Drives Adaptation in Hybrid Yeastdunham.gs.washington.edu/SmukowskiHeil.pdf · 2017-05-20 · Loss of Heterozygosity Drives Adaptation in Hybrid Yeast Caiti

uvarum genome in an interspecific hybrid with Saccharomyces cer-evisiae FEMS Yeast Res 51141ndash1150

Baker E Wang B Bellora N Peris D Hulfachor AB Koshalek JA AdamsM Libkind D Hittinger CT 2015 The Genome sequence ofSaccharomyces eubayanus and the domestication of lager-brewingyeasts Mol Biol Evol 322818ndash2831

Barbosa R Almeida P Safar SVB Santos RO Morais PB Nielly-Thibault LLeducq JB Landry CR Goncalves P Rosa CA Sampaio JP 2016Evidence of natural hybridization in Brazilian wild lineages ofSaccharomyces cerevisiae Genome Biol Evol 8317ndash329

Barton NH 2001 The role of hybridization in evolution Mol Ecol10551ndash568

Belloch C Orlic S Barrio E Querol A 2008 Fermentative stress adapta-tion of hybrids within the Saccharomyces sensu stricto complex Int JFood Microbiol 122188ndash195

Bellon JR Yang F Day MP Inglis DL Chambers PJ 2015 Designingand creating Saccharomyces interspecific hybrids for improvedindustry relevant phenotypes Appl Microbiol Biotechnol998597ndash8609

Borneman AR Zeppel R Chambers PJ Curtin CD 2014 Insights into theDekkera bruxellensis genomic landscape comparative genomics re-veals variations in ploidy and nutrient utilisation potential amongstwine isolates PLoS Genet 10e1004161

Brewer BJ Payen C Di Rienzi SC Higgins MM Ong G Dunham MJRaghuraman MK 2015 Origin-dependent inverted-repeat amplifi-cation tests of a model for inverted DNA amplification PLoS Genet11e1005699

Brown CJ Todd KM Rosenzweig RF 1998 Multiple duplications of yeasthexose transport genes in response to selection in a glucose-limitedenvironment Mol Biol Evol 15931ndash942

Bullini L 1994 Origin and evolution of animal hybrid species Trends EcolEvol 9422ndash426

Bun-Ya M Nishimura M Harashima S Oshima Y 1991 The PHO84 geneof Saccharomyces cerevisiae encodes an inorganic phosphate trans-porter Mol Cell Biol 113229ndash3238

Burke MK Dunham JP Shahrestani P Thornton KR Rose MR Long AD2010 Genome-wide analysis of a long-term evolution experimentwith Drosophila Nature 467587ndash590

Burke MK Liti G Long AD 2014 Standing genetic variation drives re-peatable experimental evolution in outcrossing populations ofSaccharomyces cerevisiae Mol Biol Evol 313228ndash3239

Cherry JM Hong EL Amundsen C Balakrishnan R Binkley G Chan ETChristie KR Costanzo MC Dwight SS Engel SR et al 2012Saccharomyces genome database the genomics resource of buddingyeast Nucleic Acids Res 40D700ndashD705

Chester M Riley RK Soltis PS Soltis DE 2015 Patterns of chromosomalvariation in natural populations of the neoallotetraploid Tragopogonmirus (Asteraceae) Heredity (Edinb) 114309ndash317

Chou JY Leu JY 2010 Speciation through cytonuclear incompatibilityinsights from yeast and implications for higher eukaryotes Bioessays32401ndash411

Cliften PF Fulton RS Wilson RK Johnston M 2006 After the duplicationgene loss and adaptation in Saccharomyces genomes Genetics172863ndash872

Conte GL Arnegard ME Peichel CL Schluter D 2012 The probability ofgenetic parallelism and convergence in natural populations Proc BiolSci 2795039ndash5047

Cordes FS Bright JN Sansom MS 2002 Proline-induced distortions oftransmembrane helices J Mol Biol 323951ndash960

Crow JF 1998 90 years ago the beginning of hybrid maize Genetics148923ndash928

Dasmahapatra KK Walters JR Briscoe AD Davey JW Whibley A NadeauNJ Zimin AV Hughes DST Ferguson LC Martin SH et al 2012Butterfly genome reveals promiscuous exchange of mimicry adap-tations among species Nature 48794ndash98

Dowling TE Smith GR Brown WM 1989 Reproductive isolation andintrogression between Notropis-Cornutus and Notropis-Chrysocephalus (Family Cyprinidae)mdashcomparison of morphologyallozymes and mitochondrial-DNA Evolution 43620ndash634

Doyle JJ Flagel LE Paterson AH Rapp RA Soltis DE Soltis PS Wendel JF2008 Evolutionary genetics of genome merger and doubling inplants Annu Rev Genet 42443ndash461

Dunham MJ Badrane H Ferea T Adams J Brown PO Rosenzweig FBotstein D 2002 Characteristic genome rearrangements in experi-mental evolution of Saccharomyces cerevisiae Proc Natl Acad Sci U SA 9916144ndash16149

Dunn B Paulish T Stanbery A Piotrowski J Koniges G Kroll E LouisEJ Liti G Sherlock G Rosenzweig F 2013 Recurrent rearrange-ment during adaptive evolution in an interspecific yeast hybridsuggests a model for rapid introgression PLoS Genet9e1003366

Dunn B Richter C Kvitek DJ Pugh T Sherlock G 2012 Analysis of theSaccharomyces cerevisiae pan-genome reveals a pool of copy num-ber variants distributed in diverse yeast strains from differing indus-trial environments Genome Res 22908ndash924

Dunn B Sherlock G 2008 Reconstruction of the genome origins andevolution of the hybrid lager yeast Saccharomyces pastorianusGenome Res 181610ndash1623

Elmer KR Meyer A 2011 Adaptation in the age of ecological genomicsinsights from parallelism and convergence Trends Ecol Evol26298ndash306

Engel SR Dietrich FS Fisk DG Binkley G Balakrishnan R Costanzo MCDwight SS Hitz BC Karra K Nash RS et al 2014 The referencegenome sequence of Saccharomyces cerevisiae then and now G3(Bethesda) 4389ndash398

Fedoroff NV 2012 Presidential address Transposable elements epige-netics and genome evolution Science 338758ndash767

Ferea TL Botstein D Brown PO Rosenzweig RF 1999 Systematicchanges in gene expression patterns following adaptive evolutionin yeast Proc Natl Acad Sci U S A 969721ndash9726

Gibson B Liti G 2015 Saccharomyces pastorianus genomic insightsinspiring innovation for industry Yeast 3217ndash27

Gibson DG Young L Chuang RY Venter JC Hutchison CA Smith HO2009 Enzymatic assembly of DNA molecules up to several hundredkilobases Nat Methods 6343ndash5

Gonzalez SS Barrio E Gafner J Querol A 2006 Natural hybrids fromSaccharomyces cerevisiae Saccharomyces bayanus andSaccharomyces kudriavzevii in wine fermentations FEMS Yeast Res61221ndash1234

Gonzalez SS Barrio E Querol A 2008 Molecular characterization of newnatural hybrids of Saccharomyces cerevisiae and S kudriavzevii inbrewing Appl Environ Microbiol 742314ndash2320

Grant PR Grant BR 1994 Phenotypic and genetic consequences ofhybridization in Darwinrsquos finches Evolution 48297ndash316

Grant PR Grant BR 2002 Unpredictable evolution in a 30-year study ofDarwinrsquos finches Science 296707ndash711

Grant PR Grant BR 2010 Natural selection speciation and Darwinrsquosfinches Proc Calif Acad Sci 61245ndash260

Grant PR Grant BR Petren K 2005 Hybridization in the recent past AmNat 16656ndash67

Greig D 2009 Reproductive isolation in Saccharomyces Heredity (Edinb)10239ndash44

Greig D Leu JY 2009 Natural history of budding yeast Current Biol19R886ndashR890

Greig D Louis EJ Borts RH Travisano M 2002 Hybrid speciation inexperimental populations of yeast Science 2981773ndash1775

Gresham D Desai MM Tucker CM Jenq HT Pai DA Ward A DeSevoCG Botstein D Dunham MJ 2008 The repertoire and dynamics ofevolutionary adaptations to controlled nutrient-limited environ-ments in yeast PLoS Genet 4e1000303

Hajjar R Hodgkin T 2007 The use of wild relatives in crop improvementa survey of developments over the last 20 years Euphytica 1561ndash13

Heiser CB 1954 Variation and subspeciation in the common sunflowerHelianthus annuus Am Midl Nat 51287ndash305

Hittinger CT 2013 Saccharomyces diversity and evolution a buddingmodel genus Trends Genet 29309ndash317

Hoang ML Tan FJ Lai DC Celniker SE Hoskins RA Dunham MJ ZhengY Koshland D 2010 Competitive repair by naturally dispersed

Adaptation in Hybrids doi101093molbevmsx098 MBE

15

repetitive DNA during non-allelic homologous recombination PLoSGenet 6e1001228

Hoffmann AA Sgro CM 2011 Climate change and evolutionary adap-tation Nature 470479ndash485

Hoffman CS Winston F 1987 A 10-minute DNA preparation from yeastefficiently releases autonomous plasmids for transformation ofEscherichia coli Gene 57267ndash272

Hou J Friedrich A de Montigny J Schacherer J 2014 Chromosomalrearrangements as a major mechanism in the onset of reproductiveisolation in Saccharomyces cerevisiae Curr Biol 241153ndash1159

Hou J Friedrich A Gounot JS Schacherer J 2015 Comprehensive surveyof condition-specific reproductive isolation reveals genetic incom-patibility in yeast Nat Commun 67214

Huerta-Sanchez E Casey FP 2015 Archaic inheritance supporting high-altitude life in Tibet J Appl Physiol (1985) 1191129ndash1134

Jones FC Grabherr MG Chan YF Russell P Mauceli E Johnson JSwofford R Pirun M Zody MC White S et al 2012 The genomicbasis of adaptive evolution in threespine sticklebacks Nature48455ndash61

Kao KC Sherlock G 2008 Molecular characterization of clonal interfer-ence during adaptive evolution in asexual populations ofSaccharomyces cerevisiae Nat Genet 401499ndash1504

Kawecki TJ Lenski RE Ebert D Hollis B Olivieri I Whitlock MC 2012Experimental evolution Trends Ecol Evol 27547ndash560

Kellis M Patterson N Endrizzi M Birren B Lander ES 2003 Sequencingand comparison of yeast species to identify genes and regulatoryelements Nature 423241ndash254

Kelly B Whiteley A Tallmon D 2010 The Arctic melting pot Nature468891ndash891

Kvitek DJ Sherlock G 2011 Reciprocal sign epistasis between frequentlyexperimentally evolved adaptive mutations causes a rugged fitnesslandscape PLoS Genet 7e1002056

Kvitek DJ Sherlock G 2013 Whole genome whole population sequenc-ing reveals that loss of signaling networks is the major adaptivestrategy in a constant environment PLoS Genet 9e1003972

Landry CR Hartl DL Ranz JM 2007 Genome clashes in hybrids insightsfrom gene expression Heredity (Edinb) 99483ndash493

Lang GI Desai MM 2014 The spectrum of adaptive mutations in ex-perimental evolution Genomics 104412ndash416

Lapunzina P Monk D 2011 The consequences of uniparental disomyand copy number neutral loss-of-heterozygosity during human de-velopment and cancer Biol Cell 103303ndash317

Leducq JB Nielly-Thibault L Charron G Eberlein C Verta JP Samani PSylvester K Hittinger CT Bell G Landry CR 2016 Speciation drivenby hybridization and chromosomal plasticity in a wild yeast NatMicrobiol 115003

Lee HY Chou JY Cheong L Chang NH Yang SY Leu JY 2008Incompatibility of nuclear and mitochondrial genomes causes hy-brid sterility between two yeast species Cell 1351065ndash1073

Li H Durbin R 2009 Fast and accurate short read alignment withBurrows-Wheeler transform Bioinformatics 251754ndash1760

Li H Handsaker B Wysoker A Fennell T Ruan J Homer N Marth GAbecasis G Durbin R 2009 The Sequence AlignmentMap formatand SAMtools Bioinformatics 252078ndash2079

Liti G 2015 The fascinating and secret wild life of the budding yeast Scerevisiae Elife 4e05835

Liti G Barton DB Louis EJ 2006 Sequence diversity reproductiveisolation and species concepts in Saccharomyces Genetics174839ndash850

Lloyd AH Ranoux M Vautrin S Glover N Fourment J Charif D ChouletF Lassalle G Marande W Tran J et al 2014 Meiotic gene evolutioncan you teach a new dog new tricks Mol Biol Evol 311724ndash1727

Losos JB Jackman TR Larson A de Queiroz K Rodriguez-Schettino L1998 Contingency and determinism in replicated adaptive radia-tions of island lizards Science 2792115ndash2118

Louis VL Despons L Friedrich A Martin T Durrens P Casaregola SNeuveglise C Fairhead C Marck C Cruz JA et al 2012 Pichia sorbi-tophila an interspecies yeast hybrid reveals early steps of genomeresolution after polyploidization G3 (Bethesda) 2299ndash311

Mallet J 2007 Hybrid speciation Nature 446279ndash283Marcet-Houben M Gabaldon T 2015 Beyond the whole-genome du-

plication phylogenetic evidence for an ancient interspecies hybrid-ization in the bakerrsquos yeast lineage PLoS Biol 13e1002220

Marinoni G Manuel M Petersen RF Hvidtfeldt J Sulo P Piskur J 1999Horizontal transfer of genetic material among Saccharomyces yeastsJ Bacteriol 1816488ndash6496

Martin A Orgogozo V 2013 The loci of repeated evolution a catalog ofgenetic hotspots of phenotypic variation Evolution 671235ndash1250

Masly JP Jones CD Noor MAF Locke J Orr HA 2006 Gene transpositionas a cause of hybrid sterility in Drosophila Science 3131448ndash1450

Mason AS Batley J 2015 Creating new interspecific hybrid and polyploidcrops Trends Biotechnol 33436ndash441

Mavarez J Salazar CA Bermingham E Salcedo C Jiggins CD Linares M2006 Speciation by hybridization in Heliconius butterflies Nature441868ndash871

McKenna A Hanna M Banks E Sivachenko A Cibulskis K Kernytsky AGarimella K Altshuler D Gabriel S Daly M et al 2010 The genomeanalysis toolkit a MapReduce framework for analyzing next-generation DNA sequencing data Genome Res 201297ndash1303

Meiklejohn CD Holmbeck MA Siddiq MA Abt DN Rand DMMontooth KL 2013 An Incompatibility between a mitochondrialtRNA and its nuclear-encoded tRNA synthetase compromises de-velopment and fitness in Drosophila PLoS Genet 9e1003238

Meyer A Salzburger W Schartl M 2006 Hybrid origin of a swordtailspecies (Teleostei Xiphophorus clemenciae) driven by sexual selec-tion Mol Ecol 15721ndash730

Michalak P 2009 Epigenetic transposon and small RNA determinants ofhybrid dysfunctions Heredity (Edinb) 10245ndash50

Miller AW Dunham MJ 2013 Design and use of multiplexed chemostatarrays J Vis Exp 72e50262

Monod J 1949 The growth of bacterial cultures Annu Rev Microbiol3371ndash394

Mouillon JM Persson BL 2006 New aspects on phosphate sensing andsignalling in Saccharomyces cerevisiae FEMS Yeast Res 6171ndash176

Muhlfeld CC Kovach RP Jones LA Al-Chokhachy R Boyer MC Leary RFLowe WH Luikart G Allendorf FW 2014 Invasive hybridization in athreatened species is accelerated by climate change Nat ClimChange 4620ndash624

Muller LAH McCusker JH 2009 A multispecies-based taxonomic mi-croarray reveals interspecies hybridization and introgression inSaccharomyces cerevisiae FEMS Yeast Res 9143ndash152

Nagalakshmi U Wang Z Waern K Shou C Raha D Gerstein M SnyderM 2008 The transcriptional landscape of the yeast genome definedby RNA sequencing Science 3201344ndash1349

Nakao Y Kanamori T Itoh T Kodama Y Rainieri S Nakamura NShimonaga T Hattori M Ashikari T 2009 Genome sequence ofthe lager brewing yeast an interspecies hybrid DNA Res 16115ndash129

Nolte AW Freyhof J Stemshorn KC Tautz D 2005 An invasive lineage ofsculpins Cottus sp (Pisces Teleostei) in the Rhine with new habitatadaptations has originated from hybridization between old phylo-geographic groups Proc Biol Sci 2722379ndash2387

Novick A Szilard L 1950a Description of the chemostat Science112715ndash716

Novick A Szilard L 1950b Experiments with the Chemostat on spon-taneous mutations of bacteria Proc Natl Acad Sci U S A 36708ndash719

Ouyang YD Liu YG Zhang QF 2010 Hybrid sterility in plant storiesfrom rice Curr Opin Plant Biol 13186ndash192

Payen C Di Rienzi SC Ong GT Pogachar JL Sanchez JC SunshineAB Raghuraman MK Brewer BJ Dunham MJ 2014 The dynam-ics of diverse segmental amplifications in populations ofSaccharomyces cerevisiae adapting to strong selection G3(Bethesda) 4399ndash409

Payen C Sunshine AB Ong GT Pogachar JL Zhao W Dunham MJ 2016High-throughput identification of adaptive mutations in experimen-tally evolved yeast populations PLoS Genet 12e1006339

Perez-Prat E van Lookeren Campagne MM 2002 Hybrid seed produc-tion and the challenge of propagating male-sterile plants TrendsPlant Sci 7199ndash203

Smukowski Heil et al doi101093molbevmsx098 MBE

16

Peris D Langdon QK Moriarty RV Sylvester K Bontrager M Charron GLeducq JB Landry CR Libkind D Hittinger CT 2016 Complex ances-tries of lager-brewing hybrids were shaped by standing variation inthe wild yeast Saccharomyces eubayanus PLoS Genet 12e1006155

Perlstein EO Ruderfer DM Roberts DC Schreiber SL Kruglyak L 2007Genetic basis of individual differences in the response to small-molecule drugs in yeast Nat Genet 39496ndash502

Piatkowska EM Naseeb S Knight D Delneri D 2013 Chimeric proteincomplexes in hybrid species generate novel phenotypes PLoS Genet9e1003836

Piotrowski JS Nagarajan S Kroll E Stanbery A Chiotti KE Kruckeberg ALDunn B Sherlock G Rosenzweig F 2012 Different selective pressureslead to different genomic outcomes as newly-formed hybrid yeastsevolve BMC Evol Biol 1246

Pryszcz LP Nemeth T Gacser A Gabaldon T 2014 Genome comparisonof Candida orthopsilosis clinical strains reveals the existence of hy-brids between two distinct subspecies Genome Biol Evol61069ndash1078

Racimo F Sankararaman S Nielsen R Huerta-Sanchez E 2015 Evidencefor archaic adaptive introgression in humans Nat Rev Genet16359ndash371

Rieseberg LH 1991 Homoploid reticulate evolution in Helianthus(Asteraceae)mdashevidence from ribosomal genes Am J Bot781218ndash1237

Rieseberg LH 1997 Hybrid origins of plant species Annu Rev EcolSystemat 28359ndash389

Robinson JT Thorvaldsdottir H Winckler W Guttman M Lander ESGetz G Mesirov JP 2011 Integrative genomics viewer NatBiotechnol 2924ndash26

Rosenthal DM Schwarzbach AE Donovan LA Raymond O RiesebergLH 2002 Phenotypic differentiation between three ancient hybridtaxa and their parental species Int J Plant Sci 163387ndash398

Ruderfer DM Pratt SC Seidel HS Kruglyak L 2006 Population genomicanalysis of outcrossing and recombination in yeast Nat Genet381077ndash1081

Rundle HD Nagel L Boughman JW Schluter D 2000 Natural selectionand parallel speciation in sympatric sticklebacks Science287306ndash308

Ryland GL Doyle MA Goode D Boyle SE Choong DY Rowley SM Li JBowtell DD Tothill RW Campbell IG et al 2015 Australian OvarianCancer Study Group Loss of heterozygosity what is it good for BMCMed Genomics 845

Salverda MLM Dellus E Gorter FA Debets AJM van der Oost J HoekstraRF Tawfik DS de Visser JAGM 2011 Initial mutations direct alter-native pathways of protein evolution PLoS Genet 7e1001321

Sanchez MR Miller AW Liachko I Sunshine AB Lynch B Huang MAlcantara E DeSevo CG Pai DA Tucker CM et al 2017 Differentialparalog divergence modulates genome evolution across yeast spe-cies PLoS Genet 13(2)e1006585

Scannell DR Zill OA Rokas A Payen C Dunham MJ Eisen MB Rine JJohnston M Hittinger CT 2011 The awesome power of yeast evo-lutionary genetics new genome sequences and strain resources forthe Saccharomyces sensu stricto genus G3 (Bethesda) 111ndash25

Schroder MS Martinez de San Vicente K Prandini TH Hammel SHiggins DG Bagagli E Wolfe KH Butler G 2016 Multiple originsof the pathogenic yeast Candida orthopsilosis by separate hybridiza-tions between two parental species PLoS Genet 12e1006404

Schumer M Rosenthal GG Andolfatto P 2014 How common is homo-ploid hybrid speciation Evolution 681553ndash1560

Schwarzbach AE Donovan LA Rieseberg LH 2001 Transgressivecharacter expression in a hybrid sunflower species Am J Bot88270ndash277

Selmecki AM Maruvka YE Richmond PA Guillet M Shoresh NSorenson AL De S Kishony R Michor F Dowell R et al 2015Polyploidy can drive rapid adaptation in yeast Nature 519349ndash52

Seshan VE Olshen A 2016 DNAcopy DNA copy number data analysisVersion R package version 1460

Soltis DE Visger CJ Soltis PS 2014 The polyploidy revolution then andnow Stebbins revisited Am J Bot 1011057ndash1078

Soltis PS 2013 Hybridization speciation and novelty J Evol Biol26291ndash293

Soltis PS Soltis DE 2009 The role of hybridization in plant speciationAnnu Rev Plant Biol 60561ndash588

Sunshine AB Payen C Ong GT Liachko I Tan KM Dunham MJ 2015The fitness consequences of aneuploidy are driven by condition-dependent gene effects PLoS Biol 13e1002155

Tamai Y Momma T Yoshimoto H Kaneko Y 1998 Co-existence of twotypes of chromosome in the bottom fermenting yeastSaccharomyces pastorianus Yeast 14923ndash933

Taylor DJ Hebert PDN 1993 Habitat-dependent hybrid parentage anddifferential introgression between neighboringly sympatric Daphniaspecies Proc Natl Acad Sci U S A 907079ndash7083

Thiagalingam S Laken S Willson JK Markowitz SD Kinzler KWVogelstein B Lengauer C 2001 Mechanisms underlying losses ofheterozygosity in human colorectal cancers Proc Natl Acad Sci US A 982698ndash2702

Tuna M Knuutila S Mills GB 2009 Uniparental disomy in cancer TrendsMol Med 15120ndash128

Walther A Hesselbart A Wendland J 2014 Genome sequence ofSaccharomyces carlsbergensis the worldrsquos first pure culture lageryeast G3 (Bethesda) 4783ndash793

Wayne RK 1993 Molecular evolution of the dog family Trends Genet9218ndash224

Wenger JW Schwartz K Sherlock G 2010 Bulk segregant analysis byhigh-throughput sequencing reveals a novel xylose utilization genefrom Saccharomyces cerevisiae PLoS Genet 6e1000942

Wessinger CA Rausher MD 2014 Predictability and irreversibility ofgenetic changes associated with flower color evolution inPenstemon barbatus Evolution 681058ndash1070

Wolfe KH 2015 Origin of the yeast whole-genome duplication PLoSBiol 13e1002221

Woods R Schneider D Winkworth CL Riley MA Lenski RE 2006 Testsof parallel molecular evolution in a long-term experiment withEscherichia coli Proc Natl Acad Sci U S A 1039107ndash9112

Wykoff DD OrsquoShea EK 2001 Phosphate transport and sensing inSaccharomyces cerevisiae Genetics 1591491ndash1499

Xiong Z Gaeta RT Pires JC 2011 Homoeologous shuffling andchromosome compensation maintain genome balance inresynthesized allopolyploid Brassica napus Proc Natl Acad SciU S A 1087908ndash7913

Yassour M Kapian T Fraser HB Levin JZ Pfiffner J Adiconis X Schroth GLuo SJ Khrebtukova I Gnirke A et al 2009 Ab initio construction ofa eukaryotic transcriptome by massively parallel mRNA sequencingProc Natl Acad Sci U S A 1063264ndash3269

Zhu YO Sherlock G Petrov DA 2016 Whole genome analysis of 132clinical Saccharomyces cerevisiae strains reveals extensive ploidy var-iation G3 (Bethesda) doi101534g3116029397

Adaptation in Hybrids doi101093molbevmsx098 MBE

17

  • msx098-TF1
  • msx098-TF2
Page 16: Loss of Heterozygosity Drives Adaptation in Hybrid Yeastdunham.gs.washington.edu/SmukowskiHeil.pdf · 2017-05-20 · Loss of Heterozygosity Drives Adaptation in Hybrid Yeast Caiti

repetitive DNA during non-allelic homologous recombination PLoSGenet 6e1001228

Hoffmann AA Sgro CM 2011 Climate change and evolutionary adap-tation Nature 470479ndash485

Hoffman CS Winston F 1987 A 10-minute DNA preparation from yeastefficiently releases autonomous plasmids for transformation ofEscherichia coli Gene 57267ndash272

Hou J Friedrich A de Montigny J Schacherer J 2014 Chromosomalrearrangements as a major mechanism in the onset of reproductiveisolation in Saccharomyces cerevisiae Curr Biol 241153ndash1159

Hou J Friedrich A Gounot JS Schacherer J 2015 Comprehensive surveyof condition-specific reproductive isolation reveals genetic incom-patibility in yeast Nat Commun 67214

Huerta-Sanchez E Casey FP 2015 Archaic inheritance supporting high-altitude life in Tibet J Appl Physiol (1985) 1191129ndash1134

Jones FC Grabherr MG Chan YF Russell P Mauceli E Johnson JSwofford R Pirun M Zody MC White S et al 2012 The genomicbasis of adaptive evolution in threespine sticklebacks Nature48455ndash61

Kao KC Sherlock G 2008 Molecular characterization of clonal interfer-ence during adaptive evolution in asexual populations ofSaccharomyces cerevisiae Nat Genet 401499ndash1504

Kawecki TJ Lenski RE Ebert D Hollis B Olivieri I Whitlock MC 2012Experimental evolution Trends Ecol Evol 27547ndash560

Kellis M Patterson N Endrizzi M Birren B Lander ES 2003 Sequencingand comparison of yeast species to identify genes and regulatoryelements Nature 423241ndash254

Kelly B Whiteley A Tallmon D 2010 The Arctic melting pot Nature468891ndash891

Kvitek DJ Sherlock G 2011 Reciprocal sign epistasis between frequentlyexperimentally evolved adaptive mutations causes a rugged fitnesslandscape PLoS Genet 7e1002056

Kvitek DJ Sherlock G 2013 Whole genome whole population sequenc-ing reveals that loss of signaling networks is the major adaptivestrategy in a constant environment PLoS Genet 9e1003972

Landry CR Hartl DL Ranz JM 2007 Genome clashes in hybrids insightsfrom gene expression Heredity (Edinb) 99483ndash493

Lang GI Desai MM 2014 The spectrum of adaptive mutations in ex-perimental evolution Genomics 104412ndash416

Lapunzina P Monk D 2011 The consequences of uniparental disomyand copy number neutral loss-of-heterozygosity during human de-velopment and cancer Biol Cell 103303ndash317

Leducq JB Nielly-Thibault L Charron G Eberlein C Verta JP Samani PSylvester K Hittinger CT Bell G Landry CR 2016 Speciation drivenby hybridization and chromosomal plasticity in a wild yeast NatMicrobiol 115003

Lee HY Chou JY Cheong L Chang NH Yang SY Leu JY 2008Incompatibility of nuclear and mitochondrial genomes causes hy-brid sterility between two yeast species Cell 1351065ndash1073

Li H Durbin R 2009 Fast and accurate short read alignment withBurrows-Wheeler transform Bioinformatics 251754ndash1760

Li H Handsaker B Wysoker A Fennell T Ruan J Homer N Marth GAbecasis G Durbin R 2009 The Sequence AlignmentMap formatand SAMtools Bioinformatics 252078ndash2079

Liti G 2015 The fascinating and secret wild life of the budding yeast Scerevisiae Elife 4e05835

Liti G Barton DB Louis EJ 2006 Sequence diversity reproductiveisolation and species concepts in Saccharomyces Genetics174839ndash850

Lloyd AH Ranoux M Vautrin S Glover N Fourment J Charif D ChouletF Lassalle G Marande W Tran J et al 2014 Meiotic gene evolutioncan you teach a new dog new tricks Mol Biol Evol 311724ndash1727

Losos JB Jackman TR Larson A de Queiroz K Rodriguez-Schettino L1998 Contingency and determinism in replicated adaptive radia-tions of island lizards Science 2792115ndash2118

Louis VL Despons L Friedrich A Martin T Durrens P Casaregola SNeuveglise C Fairhead C Marck C Cruz JA et al 2012 Pichia sorbi-tophila an interspecies yeast hybrid reveals early steps of genomeresolution after polyploidization G3 (Bethesda) 2299ndash311

Mallet J 2007 Hybrid speciation Nature 446279ndash283Marcet-Houben M Gabaldon T 2015 Beyond the whole-genome du-

plication phylogenetic evidence for an ancient interspecies hybrid-ization in the bakerrsquos yeast lineage PLoS Biol 13e1002220

Marinoni G Manuel M Petersen RF Hvidtfeldt J Sulo P Piskur J 1999Horizontal transfer of genetic material among Saccharomyces yeastsJ Bacteriol 1816488ndash6496

Martin A Orgogozo V 2013 The loci of repeated evolution a catalog ofgenetic hotspots of phenotypic variation Evolution 671235ndash1250

Masly JP Jones CD Noor MAF Locke J Orr HA 2006 Gene transpositionas a cause of hybrid sterility in Drosophila Science 3131448ndash1450

Mason AS Batley J 2015 Creating new interspecific hybrid and polyploidcrops Trends Biotechnol 33436ndash441

Mavarez J Salazar CA Bermingham E Salcedo C Jiggins CD Linares M2006 Speciation by hybridization in Heliconius butterflies Nature441868ndash871

McKenna A Hanna M Banks E Sivachenko A Cibulskis K Kernytsky AGarimella K Altshuler D Gabriel S Daly M et al 2010 The genomeanalysis toolkit a MapReduce framework for analyzing next-generation DNA sequencing data Genome Res 201297ndash1303

Meiklejohn CD Holmbeck MA Siddiq MA Abt DN Rand DMMontooth KL 2013 An Incompatibility between a mitochondrialtRNA and its nuclear-encoded tRNA synthetase compromises de-velopment and fitness in Drosophila PLoS Genet 9e1003238

Meyer A Salzburger W Schartl M 2006 Hybrid origin of a swordtailspecies (Teleostei Xiphophorus clemenciae) driven by sexual selec-tion Mol Ecol 15721ndash730

Michalak P 2009 Epigenetic transposon and small RNA determinants ofhybrid dysfunctions Heredity (Edinb) 10245ndash50

Miller AW Dunham MJ 2013 Design and use of multiplexed chemostatarrays J Vis Exp 72e50262

Monod J 1949 The growth of bacterial cultures Annu Rev Microbiol3371ndash394

Mouillon JM Persson BL 2006 New aspects on phosphate sensing andsignalling in Saccharomyces cerevisiae FEMS Yeast Res 6171ndash176

Muhlfeld CC Kovach RP Jones LA Al-Chokhachy R Boyer MC Leary RFLowe WH Luikart G Allendorf FW 2014 Invasive hybridization in athreatened species is accelerated by climate change Nat ClimChange 4620ndash624

Muller LAH McCusker JH 2009 A multispecies-based taxonomic mi-croarray reveals interspecies hybridization and introgression inSaccharomyces cerevisiae FEMS Yeast Res 9143ndash152

Nagalakshmi U Wang Z Waern K Shou C Raha D Gerstein M SnyderM 2008 The transcriptional landscape of the yeast genome definedby RNA sequencing Science 3201344ndash1349

Nakao Y Kanamori T Itoh T Kodama Y Rainieri S Nakamura NShimonaga T Hattori M Ashikari T 2009 Genome sequence ofthe lager brewing yeast an interspecies hybrid DNA Res 16115ndash129

Nolte AW Freyhof J Stemshorn KC Tautz D 2005 An invasive lineage ofsculpins Cottus sp (Pisces Teleostei) in the Rhine with new habitatadaptations has originated from hybridization between old phylo-geographic groups Proc Biol Sci 2722379ndash2387

Novick A Szilard L 1950a Description of the chemostat Science112715ndash716

Novick A Szilard L 1950b Experiments with the Chemostat on spon-taneous mutations of bacteria Proc Natl Acad Sci U S A 36708ndash719

Ouyang YD Liu YG Zhang QF 2010 Hybrid sterility in plant storiesfrom rice Curr Opin Plant Biol 13186ndash192

Payen C Di Rienzi SC Ong GT Pogachar JL Sanchez JC SunshineAB Raghuraman MK Brewer BJ Dunham MJ 2014 The dynam-ics of diverse segmental amplifications in populations ofSaccharomyces cerevisiae adapting to strong selection G3(Bethesda) 4399ndash409

Payen C Sunshine AB Ong GT Pogachar JL Zhao W Dunham MJ 2016High-throughput identification of adaptive mutations in experimen-tally evolved yeast populations PLoS Genet 12e1006339

Perez-Prat E van Lookeren Campagne MM 2002 Hybrid seed produc-tion and the challenge of propagating male-sterile plants TrendsPlant Sci 7199ndash203

Smukowski Heil et al doi101093molbevmsx098 MBE

16

Peris D Langdon QK Moriarty RV Sylvester K Bontrager M Charron GLeducq JB Landry CR Libkind D Hittinger CT 2016 Complex ances-tries of lager-brewing hybrids were shaped by standing variation inthe wild yeast Saccharomyces eubayanus PLoS Genet 12e1006155

Perlstein EO Ruderfer DM Roberts DC Schreiber SL Kruglyak L 2007Genetic basis of individual differences in the response to small-molecule drugs in yeast Nat Genet 39496ndash502

Piatkowska EM Naseeb S Knight D Delneri D 2013 Chimeric proteincomplexes in hybrid species generate novel phenotypes PLoS Genet9e1003836

Piotrowski JS Nagarajan S Kroll E Stanbery A Chiotti KE Kruckeberg ALDunn B Sherlock G Rosenzweig F 2012 Different selective pressureslead to different genomic outcomes as newly-formed hybrid yeastsevolve BMC Evol Biol 1246

Pryszcz LP Nemeth T Gacser A Gabaldon T 2014 Genome comparisonof Candida orthopsilosis clinical strains reveals the existence of hy-brids between two distinct subspecies Genome Biol Evol61069ndash1078

Racimo F Sankararaman S Nielsen R Huerta-Sanchez E 2015 Evidencefor archaic adaptive introgression in humans Nat Rev Genet16359ndash371

Rieseberg LH 1991 Homoploid reticulate evolution in Helianthus(Asteraceae)mdashevidence from ribosomal genes Am J Bot781218ndash1237

Rieseberg LH 1997 Hybrid origins of plant species Annu Rev EcolSystemat 28359ndash389

Robinson JT Thorvaldsdottir H Winckler W Guttman M Lander ESGetz G Mesirov JP 2011 Integrative genomics viewer NatBiotechnol 2924ndash26

Rosenthal DM Schwarzbach AE Donovan LA Raymond O RiesebergLH 2002 Phenotypic differentiation between three ancient hybridtaxa and their parental species Int J Plant Sci 163387ndash398

Ruderfer DM Pratt SC Seidel HS Kruglyak L 2006 Population genomicanalysis of outcrossing and recombination in yeast Nat Genet381077ndash1081

Rundle HD Nagel L Boughman JW Schluter D 2000 Natural selectionand parallel speciation in sympatric sticklebacks Science287306ndash308

Ryland GL Doyle MA Goode D Boyle SE Choong DY Rowley SM Li JBowtell DD Tothill RW Campbell IG et al 2015 Australian OvarianCancer Study Group Loss of heterozygosity what is it good for BMCMed Genomics 845

Salverda MLM Dellus E Gorter FA Debets AJM van der Oost J HoekstraRF Tawfik DS de Visser JAGM 2011 Initial mutations direct alter-native pathways of protein evolution PLoS Genet 7e1001321

Sanchez MR Miller AW Liachko I Sunshine AB Lynch B Huang MAlcantara E DeSevo CG Pai DA Tucker CM et al 2017 Differentialparalog divergence modulates genome evolution across yeast spe-cies PLoS Genet 13(2)e1006585

Scannell DR Zill OA Rokas A Payen C Dunham MJ Eisen MB Rine JJohnston M Hittinger CT 2011 The awesome power of yeast evo-lutionary genetics new genome sequences and strain resources forthe Saccharomyces sensu stricto genus G3 (Bethesda) 111ndash25

Schroder MS Martinez de San Vicente K Prandini TH Hammel SHiggins DG Bagagli E Wolfe KH Butler G 2016 Multiple originsof the pathogenic yeast Candida orthopsilosis by separate hybridiza-tions between two parental species PLoS Genet 12e1006404

Schumer M Rosenthal GG Andolfatto P 2014 How common is homo-ploid hybrid speciation Evolution 681553ndash1560

Schwarzbach AE Donovan LA Rieseberg LH 2001 Transgressivecharacter expression in a hybrid sunflower species Am J Bot88270ndash277

Selmecki AM Maruvka YE Richmond PA Guillet M Shoresh NSorenson AL De S Kishony R Michor F Dowell R et al 2015Polyploidy can drive rapid adaptation in yeast Nature 519349ndash52

Seshan VE Olshen A 2016 DNAcopy DNA copy number data analysisVersion R package version 1460

Soltis DE Visger CJ Soltis PS 2014 The polyploidy revolution then andnow Stebbins revisited Am J Bot 1011057ndash1078

Soltis PS 2013 Hybridization speciation and novelty J Evol Biol26291ndash293

Soltis PS Soltis DE 2009 The role of hybridization in plant speciationAnnu Rev Plant Biol 60561ndash588

Sunshine AB Payen C Ong GT Liachko I Tan KM Dunham MJ 2015The fitness consequences of aneuploidy are driven by condition-dependent gene effects PLoS Biol 13e1002155

Tamai Y Momma T Yoshimoto H Kaneko Y 1998 Co-existence of twotypes of chromosome in the bottom fermenting yeastSaccharomyces pastorianus Yeast 14923ndash933

Taylor DJ Hebert PDN 1993 Habitat-dependent hybrid parentage anddifferential introgression between neighboringly sympatric Daphniaspecies Proc Natl Acad Sci U S A 907079ndash7083

Thiagalingam S Laken S Willson JK Markowitz SD Kinzler KWVogelstein B Lengauer C 2001 Mechanisms underlying losses ofheterozygosity in human colorectal cancers Proc Natl Acad Sci US A 982698ndash2702

Tuna M Knuutila S Mills GB 2009 Uniparental disomy in cancer TrendsMol Med 15120ndash128

Walther A Hesselbart A Wendland J 2014 Genome sequence ofSaccharomyces carlsbergensis the worldrsquos first pure culture lageryeast G3 (Bethesda) 4783ndash793

Wayne RK 1993 Molecular evolution of the dog family Trends Genet9218ndash224

Wenger JW Schwartz K Sherlock G 2010 Bulk segregant analysis byhigh-throughput sequencing reveals a novel xylose utilization genefrom Saccharomyces cerevisiae PLoS Genet 6e1000942

Wessinger CA Rausher MD 2014 Predictability and irreversibility ofgenetic changes associated with flower color evolution inPenstemon barbatus Evolution 681058ndash1070

Wolfe KH 2015 Origin of the yeast whole-genome duplication PLoSBiol 13e1002221

Woods R Schneider D Winkworth CL Riley MA Lenski RE 2006 Testsof parallel molecular evolution in a long-term experiment withEscherichia coli Proc Natl Acad Sci U S A 1039107ndash9112

Wykoff DD OrsquoShea EK 2001 Phosphate transport and sensing inSaccharomyces cerevisiae Genetics 1591491ndash1499

Xiong Z Gaeta RT Pires JC 2011 Homoeologous shuffling andchromosome compensation maintain genome balance inresynthesized allopolyploid Brassica napus Proc Natl Acad SciU S A 1087908ndash7913

Yassour M Kapian T Fraser HB Levin JZ Pfiffner J Adiconis X Schroth GLuo SJ Khrebtukova I Gnirke A et al 2009 Ab initio construction ofa eukaryotic transcriptome by massively parallel mRNA sequencingProc Natl Acad Sci U S A 1063264ndash3269

Zhu YO Sherlock G Petrov DA 2016 Whole genome analysis of 132clinical Saccharomyces cerevisiae strains reveals extensive ploidy var-iation G3 (Bethesda) doi101534g3116029397

Adaptation in Hybrids doi101093molbevmsx098 MBE

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  • msx098-TF1
  • msx098-TF2
Page 17: Loss of Heterozygosity Drives Adaptation in Hybrid Yeastdunham.gs.washington.edu/SmukowskiHeil.pdf · 2017-05-20 · Loss of Heterozygosity Drives Adaptation in Hybrid Yeast Caiti

Peris D Langdon QK Moriarty RV Sylvester K Bontrager M Charron GLeducq JB Landry CR Libkind D Hittinger CT 2016 Complex ances-tries of lager-brewing hybrids were shaped by standing variation inthe wild yeast Saccharomyces eubayanus PLoS Genet 12e1006155

Perlstein EO Ruderfer DM Roberts DC Schreiber SL Kruglyak L 2007Genetic basis of individual differences in the response to small-molecule drugs in yeast Nat Genet 39496ndash502

Piatkowska EM Naseeb S Knight D Delneri D 2013 Chimeric proteincomplexes in hybrid species generate novel phenotypes PLoS Genet9e1003836

Piotrowski JS Nagarajan S Kroll E Stanbery A Chiotti KE Kruckeberg ALDunn B Sherlock G Rosenzweig F 2012 Different selective pressureslead to different genomic outcomes as newly-formed hybrid yeastsevolve BMC Evol Biol 1246

Pryszcz LP Nemeth T Gacser A Gabaldon T 2014 Genome comparisonof Candida orthopsilosis clinical strains reveals the existence of hy-brids between two distinct subspecies Genome Biol Evol61069ndash1078

Racimo F Sankararaman S Nielsen R Huerta-Sanchez E 2015 Evidencefor archaic adaptive introgression in humans Nat Rev Genet16359ndash371

Rieseberg LH 1991 Homoploid reticulate evolution in Helianthus(Asteraceae)mdashevidence from ribosomal genes Am J Bot781218ndash1237

Rieseberg LH 1997 Hybrid origins of plant species Annu Rev EcolSystemat 28359ndash389

Robinson JT Thorvaldsdottir H Winckler W Guttman M Lander ESGetz G Mesirov JP 2011 Integrative genomics viewer NatBiotechnol 2924ndash26

Rosenthal DM Schwarzbach AE Donovan LA Raymond O RiesebergLH 2002 Phenotypic differentiation between three ancient hybridtaxa and their parental species Int J Plant Sci 163387ndash398

Ruderfer DM Pratt SC Seidel HS Kruglyak L 2006 Population genomicanalysis of outcrossing and recombination in yeast Nat Genet381077ndash1081

Rundle HD Nagel L Boughman JW Schluter D 2000 Natural selectionand parallel speciation in sympatric sticklebacks Science287306ndash308

Ryland GL Doyle MA Goode D Boyle SE Choong DY Rowley SM Li JBowtell DD Tothill RW Campbell IG et al 2015 Australian OvarianCancer Study Group Loss of heterozygosity what is it good for BMCMed Genomics 845

Salverda MLM Dellus E Gorter FA Debets AJM van der Oost J HoekstraRF Tawfik DS de Visser JAGM 2011 Initial mutations direct alter-native pathways of protein evolution PLoS Genet 7e1001321

Sanchez MR Miller AW Liachko I Sunshine AB Lynch B Huang MAlcantara E DeSevo CG Pai DA Tucker CM et al 2017 Differentialparalog divergence modulates genome evolution across yeast spe-cies PLoS Genet 13(2)e1006585

Scannell DR Zill OA Rokas A Payen C Dunham MJ Eisen MB Rine JJohnston M Hittinger CT 2011 The awesome power of yeast evo-lutionary genetics new genome sequences and strain resources forthe Saccharomyces sensu stricto genus G3 (Bethesda) 111ndash25

Schroder MS Martinez de San Vicente K Prandini TH Hammel SHiggins DG Bagagli E Wolfe KH Butler G 2016 Multiple originsof the pathogenic yeast Candida orthopsilosis by separate hybridiza-tions between two parental species PLoS Genet 12e1006404

Schumer M Rosenthal GG Andolfatto P 2014 How common is homo-ploid hybrid speciation Evolution 681553ndash1560

Schwarzbach AE Donovan LA Rieseberg LH 2001 Transgressivecharacter expression in a hybrid sunflower species Am J Bot88270ndash277

Selmecki AM Maruvka YE Richmond PA Guillet M Shoresh NSorenson AL De S Kishony R Michor F Dowell R et al 2015Polyploidy can drive rapid adaptation in yeast Nature 519349ndash52

Seshan VE Olshen A 2016 DNAcopy DNA copy number data analysisVersion R package version 1460

Soltis DE Visger CJ Soltis PS 2014 The polyploidy revolution then andnow Stebbins revisited Am J Bot 1011057ndash1078

Soltis PS 2013 Hybridization speciation and novelty J Evol Biol26291ndash293

Soltis PS Soltis DE 2009 The role of hybridization in plant speciationAnnu Rev Plant Biol 60561ndash588

Sunshine AB Payen C Ong GT Liachko I Tan KM Dunham MJ 2015The fitness consequences of aneuploidy are driven by condition-dependent gene effects PLoS Biol 13e1002155

Tamai Y Momma T Yoshimoto H Kaneko Y 1998 Co-existence of twotypes of chromosome in the bottom fermenting yeastSaccharomyces pastorianus Yeast 14923ndash933

Taylor DJ Hebert PDN 1993 Habitat-dependent hybrid parentage anddifferential introgression between neighboringly sympatric Daphniaspecies Proc Natl Acad Sci U S A 907079ndash7083

Thiagalingam S Laken S Willson JK Markowitz SD Kinzler KWVogelstein B Lengauer C 2001 Mechanisms underlying losses ofheterozygosity in human colorectal cancers Proc Natl Acad Sci US A 982698ndash2702

Tuna M Knuutila S Mills GB 2009 Uniparental disomy in cancer TrendsMol Med 15120ndash128

Walther A Hesselbart A Wendland J 2014 Genome sequence ofSaccharomyces carlsbergensis the worldrsquos first pure culture lageryeast G3 (Bethesda) 4783ndash793

Wayne RK 1993 Molecular evolution of the dog family Trends Genet9218ndash224

Wenger JW Schwartz K Sherlock G 2010 Bulk segregant analysis byhigh-throughput sequencing reveals a novel xylose utilization genefrom Saccharomyces cerevisiae PLoS Genet 6e1000942

Wessinger CA Rausher MD 2014 Predictability and irreversibility ofgenetic changes associated with flower color evolution inPenstemon barbatus Evolution 681058ndash1070

Wolfe KH 2015 Origin of the yeast whole-genome duplication PLoSBiol 13e1002221

Woods R Schneider D Winkworth CL Riley MA Lenski RE 2006 Testsof parallel molecular evolution in a long-term experiment withEscherichia coli Proc Natl Acad Sci U S A 1039107ndash9112

Wykoff DD OrsquoShea EK 2001 Phosphate transport and sensing inSaccharomyces cerevisiae Genetics 1591491ndash1499

Xiong Z Gaeta RT Pires JC 2011 Homoeologous shuffling andchromosome compensation maintain genome balance inresynthesized allopolyploid Brassica napus Proc Natl Acad SciU S A 1087908ndash7913

Yassour M Kapian T Fraser HB Levin JZ Pfiffner J Adiconis X Schroth GLuo SJ Khrebtukova I Gnirke A et al 2009 Ab initio construction ofa eukaryotic transcriptome by massively parallel mRNA sequencingProc Natl Acad Sci U S A 1063264ndash3269

Zhu YO Sherlock G Petrov DA 2016 Whole genome analysis of 132clinical Saccharomyces cerevisiae strains reveals extensive ploidy var-iation G3 (Bethesda) doi101534g3116029397

Adaptation in Hybrids doi101093molbevmsx098 MBE

17

  • msx098-TF1
  • msx098-TF2

Thermal adaptation rather than demographic history drives

Application Note, Copy-neutral Loss of Heterozygosity in Cancertools.thermofisher.com/content/sfs/brochures/copy... · 2017-01-14 · Copy-neutral loss of heterozygosity (LOH)representsoneexampleofagenom

LOSS OF HETEROZYGOSITY

Lab 4: Inbreeding and Kinship. Inbreeding Reduces heterozygosity Does not change allele frequencies

Compound Heterozygosity of the Functionally Nulldspace.lib.niigata-u.ac.jp/dspace/bitstream/10191/41920/2/h27nmk692.pdf · —Original— Compound Heterozygosity of the Functionally

HETEROZYGOSITY IN PIN-THRUM PLANTS OR WITH

Acyl-CoA Dehydrogenase Drives Heat Adaptation by ...genetics.wustl.edu/bio5491/files/2016/01/Acyl-CoA-Dehydrogenase... · Acyl-CoA Dehydrogenase Drives Heat Adaptation by Sequestering

Thermal adaptation rather than demographic history drives ... · 5/4/2020  · 1 Thermal adaptation rather than demographic history drives genetic structure inferred by copy number

2 CAR0RYIN0G T7HE FecX /ROA IN HETEROZYGOSITY INCREASE ...€¦ · (FecX /ROA) was not decribed before that date. FEMALES2 CAR0RYIN0G T7HE FecX /ROA IN HETEROZYGOSITY INCREASE PROLIFICACY

Compound heterozygosity for hemoglobin S and hemoglobin E

High genome heterozygosity and endemic genetic ...xulab.org/files/pdf/ZhengW-2013-NC.pdf · High genome heterozygosity and endemic genetic recombination in the wheat stripe rust fungus

Title page Individual multi-locus heterozygosity is associated with

Heterozygosity at individual amino acid sites: Extremely high levels

Loss of heterozygosity at D8S262: an early genetic event … of heterozygosity at D8S262: an early genetic event of hepatocarcinogenesis Qiao Zhu1†, Li Gong1*†, Xiaoyan Liu1, Jun

Heterozygosity predicts clutch and egg size but not …udel.edu/~istew/Wetzel et al. 2012 Heterozygosity and egg size.pdf · Heterozygosity predicts clutch and egg size but not plasticity

Program SLED 2018 - IEEEPermanent-Magnet Flux Adaptation for Sensorless Synchronous Motor Drives Sensorless Control of Induction Motor Drives Using Additional Windings on the Stator

Constant Torque Drives - AC Drives | DC Drives | Variable

Decrease of Heterozygosity Under Inbreeding …hussinju/l2010/lecture4-inbreeding...increase diversity, or at least the variance, among populations). Decrease of Heterozygosity Under

STUDI TUMORIGENESIS DAN LOSS OF HETEROZYGOSITY …digilib.batan.go.id/e-prosiding/File Prosiding/Lingkungan/P3TM-Juli... · studi tumorigenesis dan loss of heterozygosity (loh) pada

ELECTRiCaL MaiNTENaNCE · Motor Drives Motor Drives: Identification Motor Drives: Open & Closed Loop Systems Motor Drives: Variable Speed AC Drives Motor Drives: Servo & Stepper Motors

Local adaptation drives thermal tolerance among parasite

Acyl-CoA Dehydrogenase Drives Heat Adaptation by ...web.mit.edu/horvitz/www/assets/publications/ma2015.pdf · Acyl-CoA Dehydrogenase Drives Heat Adaptation by ... normal egg laying

ABB and Baldor Drives - СоюзИмпортBaldor DC Drives Analog AC Drives AC Vector Drives AC Inverter Drives AC Micro Drives AC Drives Selection Chart Baldor ACB & ACS Drives

Loss of Heterozygosity Analysis at selected Single ... Journal of American Science, 2(4), 2006, Mitchell, et al, Loss of Heterozygosity Analysis Loss of Heterozygosity Analysis at

Noise and adaptation in multistable perception: Noise ... · Noise and adaptation in multistable perception: Noise drives when to switch, adaptation determines percept choice

Predation drives local adaptation of phenotypic plasticityeprints.whiterose.ac.uk/125047/8/Beckerman_Revision2_Accepted.pdf · Our findings are evidence for local adaptation of plasticity,

HT200/HTD Synchronous Belt Drives V-Drives … Synchronous Drives HTR Synchronous Drives HTRC Synchronous Drives HT200/HTD Synchronous Belt Drives Specification.. HT.TAPER-LOCK. MPB.&.BTS

Fatal CTLA‐4 heterozygosity with autoimmunity and ... · Fatal CTLA-4 heterozygosity with autoimmunity and recurrent infections: ... IgE and IgD were ... Fatal CTLA‐4 heterozygosity

GENERAL ELECTRICAL DRIVES What is electrical drives? Components of electrical drives Advantages of electrical drives DC drives Vs AC drives Torque equations

Environmental change drives accelerated adaptation through ...€¦ · RESEARCHARTICLE Environmental change drives accelerated adaptation through stimulated copy number variation

ADL300 Traction Elevator Drives E-Catalog · Elevator Drives E ADL. Elevator Drives. Elevator Drives. Elevator Drives E. Elevator Drives. Elevator Drives

Inferring Heterozygosity from Ancient and Low Coverage Genomes · Inferring Heterozygosity from Ancient and Low Coverage Genomes Athanasios Kousathanas,*,† Christoph Leuenberger,‡

Analyzing Loss of Heterozygosity in in Partek Genomics ... · PDF fileAnalyzing loss of heterozygosity 1 Analyzing Loss of Heterozygosity in in Partek ® Genomics ... gene copy number,

Postnatal mechanical loading drives adaptation of tissues … · 2020-04-23 · 1 1 Post natal mechanical loading drives adaptation of tissues primarily through modulation 2 of the

Program SLED 2018 - IEEEsite.ieee.org/sled2018/files/2018/09/SLED-2018-program.pdf · Permanent-Magnet Flux Adaptation for Sensorless Synchronous Motor Drives Sensorless Control of