Goal: Establish Aquilegia as an evolutionary model system

70 taxa (diverse in floral morphology & ecology) recent & rapid radiation (little genetic variation & successful hybrid formation) diploid (n=7), small genome (320-400Mbp) basal lineage in the eu-dicots

University of California Santa BarbaraEcology, Evolution & Marine Biology

Hodges Lab

Reproductive isolation & speciation in Columbines (Aquilegia)

Requires: Development of molecular resources(J. Borevitz, University of Chicago, E. Kramer, Harvard University, J. Tomkins, Clemson University, M. Nordborg, University of Southern California)

EST database (TIGR) (microarrays for both expression & genotyping studies) physical map (CUGI) transformation system to manipulate candidate gene expression

AFLP-based Phylogeny of North American Columbines

(Whitall and Hodges, in prep)

Diversity in pollination syndromes

Bee-pollinated flowers

Hummingbird-pollinated flowers

Hawkmoth-pollinated flowers

Shifts from blue/red to white/yellow

=

Shift to hawkmoth pollination (?)

*

**

*

0.1

SHTR

FOEX

FOFL

PUCOOC

COCOCOAL

COOCSp. nov.

EL BAMI

SCDE

CHHICH

PI CHAP

LOLO

SKCA

BRLA

JOSA

VUL

Bob SkowronBob Skowron

Bob SkowronBob Skowron

triternata

shockleyi

formosa (East)

formosa (West)

eximia

flavescens

pubescens

coerulea var. ochroleuca (UT)

coerulea var. alpina

coerulea var. coerulea

coerulea var. ochroleuca (CO)

sp. nov.

elegantula

micrantha

barnebyi

scopulorum

desertorum

pinetorum

chrysantha (NM)

hinckleyana

chrysantha (CHI)

chaplinei

longissima (AZ)

skinneri

longissima (TX)

brevistyla

canadensis

laramiensis

saximontana

jonesii

vulgaris

triternata

shockleyi

formosa (East)

formosa (West)

eximia

flavescens

pubescens

coerulea var. ochroleuca (UT)

coerulea var. alpina

coerulea var. coerulea

coerulea var. ochroleuca (CO)

sp. nov.

elegantula

micrantha

barnebyi

scopulorum

desertorum

pinetorum

chrysantha (NM)

hinckleyana

chrysantha (CHI)

chaplinei

longissima (AZ)

skinneri

longissima (TX)

brevistyla

canadensis

laramiensis

saximontana

jonesii

vulgaris

TR - A. triternata

SH - A. shockleyi

FO - A. formosa (East)

FO - A. formosa (West)

EX - A. eximia

FL - A. flavescens

PU - A. pubescens

COOC - A. coer. ochrol.

COAP - A. coer. alpina

COCO - A. coer. coerulea

COOC - A. coer. ochrol.

A. sp. nov.

EL - A. elegantula

MI - A. micrantha

BA - A. barnebyi

SC - A. scopulorum

DE - A. desertorum

PI – A. pinetorum

CH - A. chrysantha (NM)

HI - A. hinckleyana

CH - A. chrysantha (CHI)

CHAP - A. chaplinei

LO - A. longissima (AZ)

SK - A. skinneri

LO - A. longissima (TX)

BR - A. brevistyla

CA - A. canadensis

LA - A. laramiensis

SA - A. saximontana

JO - A. jonesii

VUL - A. vulgaris

Phylogenetic Mapping of Floral Anthocyanins

Which genes drive anthocyanin loss?

Relative importance of structural versus regulatory mutations?

Are the molecular mechanisms of a convergent phenotype convergent themselves?

6 independent losses of floral anthocyanins...

A+

A-

A+/A-

Coumaroyl CoA + Malonyl CoA

Chalcones

Flavanones

3-OH Flavonols

Leucanthocyanidins

Anthocyanins

Early

Late

Anthocyanidins

The Anthocyanin Biosynthetic Pathway (ABP)

CHS

CHI

F3H

DFR

ANS

UF3GT

6 core lociIsoflavenoids

Aurones

Phlobaphenes

Flavones

Flavonols

Flavan-3-ols

Condensed Tannins

Many side branches

3 classes of regulators

• bHLH (myc)

• myb

• WD 40

Petunia, Zea, Arabidopsis, Ipomoea, Antirrhynum...

Convergence in ABP Gene Expression in A- species?

....|....| ....|....| ....|....| ....|....| ....|....| 55 65 75 85 95 Callistephus chinensis SDQ~~GPQVP IIDLHDI~~N SNDPKTRDKC TYELRKAATE WGVMHLVNHG Ipomoea purpurea IAG~~GPQVP TVDLKGI~~N SEDLEVREKC REELRKAAVD WGVMHLVNHG Arabidopsis thaliana KED~~GPQVP TIDLKNI~~E SDDEKIRENC IEELKKASLD WGVMHLINHG Vitis labrusca x Vitis vinifera KDE~~GPQVP TIDLKDI~~E SEDEVVRERC REELKKAAME WGVMHLVNHG Citrus sinensis KDE~~GPQVP TIDLKEI~~D SEDRVEREKC REELKKAAMD WGVMHLVNHG Malus sp. NNE~~GPQVP TIDLKEI~~E SDNEKVRAKC REKLKKAAVD WGVMHLVNHG Daucus carota SDE~~GPQVP TVDIADI~~L SDDKAVREKC YERIKDAAVD WGVMHLVNHG Anthurium andraeanum AED~~GPQIP TVDVAGF~~S SGDEAARRAC AEAVRRAATD WGVMHVVNHG Allium cepa SNS~~GPQIP IIDLD~~~~~ S~~~~~RD~C IEKVTKAAKE WGVMHIVNHG Zea mays ANDHTAPRIP VVDISPFLDS SSQQQQRDEC VEAVRAAAAD WGVMHIAGHG Oryza sativa SDDDATARIP VVDISAF~DN DGD~~GRHAC VEAVRAAAEE WGVMHIAGHG Aquilegia formosa ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~**** *****LVNHG ....|....| ....|....| ....|....| ....|....| ....|....|

• Degenerate primers from multiple aa alignments for 6 loci

• Clone 6 loci from A. formosa (A+)

• Optimise RT-PCR (primers, temperatures, cycle number)

• Find the right floral tissue and developmental stage!

CHS

ACT

CHI

F3H

UF3GT

DFR

ANS

GB YB PrAOB PoA GB YB PrAOB PoA GB YB PrAOB PoA

1 2 3 AN1 2 3

GB YB PrAOB PoA

A. canadensis as an A+ Reference Species

5 stages, 3 individuals, 2 tissues

• Most consistent expression in old bud and pre-anthesis flowers

• Little variation between individuals and tissues

• No expression of DFR and ANS in anthers (A- tissue of an A+ species)

GB YB PrAOB PoA PrA

CA (A+)

FO (A+)

CH (A-)

CA (A+)

FO (A+)

CH (A-)

CA (A+)

FO (A+)

CH (A-)

CA (A+)

FO (A+)

CH (A-)

CA (A+)

FO (A+)

CH (A-)

CA (A+)

FO (A+)

CH (A-)

CA (A+)

FO (A+)

CH (A-)

CHS

CHI

F3H

DFR

ANS

UF3GT

ACT

Petals Anthers

A. canadensis (A+)

A. formosa (A+)

A. chrysantha (A-)

• Different kinetics in A. chrysantha

• DFR and ANS missing in A. chrysantha

• Select OB+PrA for multispecies comparison

Comparing kinetics in three species...

LA CA LO PI CH SC MI BA CO CO SP PU FL FO FLA OW OC NV PU

CHS

ACT

CHI

F3H

UF3GT

DFR

ANS

X X

X X

X

X

X

X

XX

X X

X

31 1 21 22 33 4 4 1A+ A+ A+ A+Pattern

ABP gene expression in 14+2 species - Results

Species

ABP gene expression in 16 species - Summary

• 2 main patterns among A- species

1. all genes expressed comparable to A+ species or

2. reduction/lack of expression in the second half of the ABP

• Hypotheses for Pattern 1 - structural mutation in one or more ABP loci or side branches of the ABP „took over“ Pattern 2 - deficiency in regulation of expression

• Convergence within the A. chrysantha clade, the A. micrantha clade and the A. coerulea clade

• CHS and CHI always expressed – pleiotropy!

• Amplification of ABP loci worked from DNA

5 possible scenarios leading to anthocyanin loss:

ABP Gene

ABP Gene

ABP Gene

ABP Gene

ABP Gene

R

R

R

R

X

X

X

A- 1

A- 2

A- 3

A- 4 & 5

A+

A+ anthocyanins present

A- anthocyanins absent

R regulator

ABP anthocyanin biosynthetic pathway

X mutation

expression

Anthocyanin loss in A. pubescens

► Completion of the genetic map (13 microsattelite loci in an F2 population of 250 individuals)

► New floral whorls in Aquilegia – Gene expression in Staminodia as compared to other whorls? (high density oligonucleotide arrays, designed by J. Borevitz, Chicago and W. Rensink, TIGR, printed by NimbleGen)

► Identify and sequence BACs with loci of interest (e.g. ABP loci)

Down the road...

► Expression of regulatory loci in A. pubescens & A. formosa (EST database + phylogenetic analysis for identification of regulatory loci)

► Localize ABP & regulatory loci on a genetic map (A. pubescens x A. formosa) (SNPs in EST database) Does any of them map to a QTL for spur chroma?

► Sequence alleles of ABP & regulatory loci from A. pubescens and A. formosa (EST database)

Questions?