Phylogenetics of a Fungal InvasionOrigins and Widespread Dispersal ofWhite-Nose Syndrome

Kevin P Dreesab Jeffrey M Lorchc Sebastien J Puechmailled Katy L Pariseab

Gudrun Wibbelte Joseph R Hoytf Keping Sung Ariunbold Jargalsaikhanhi

Munkhnast Dalannasti Jonathan M Palmerj Daniel L Lindnerj

A Marm Kilpatrickf Talima Pearsona Paul S Keima David S Blehertc

Jeffrey T Fosterab

Pathogen and Microbiome Institute Northern Arizona University Flagstaff Arizona USAa Department ofMolecular Cellular and Biomedical Sciences University of New Hampshire Durham New Hampshire USAbUS Geological Survey National Wildlife Health Center Madison Wisconsin USAc School of Biological andEnvironmental Sciences University College Dublin Belfield Dublin Irelandd Leibniz Institute for Zoo andWildlife Research Berlin Germanye Department of Ecology and Evolutionary Biology University of CaliforniaSanta Cruz California USAf Urban and Environmental Science College Changchun Normal UniversityChangchun Peoplersquos Republic of Chinag Department of Biology School of Mathematics and Natural SciencesThe Mongolian National University of Education Ulaanbaatar Mongoliah Bats Research Center of MongoliaUlaanbaatar Mongoliai US Forest Service Northern Research Station Center for Forest Mycology ResearchMadison Wisconsin USAj

ABSTRACT Globalization has facilitated the worldwide movement and introductionof pathogens but epizoological reconstructions of these invasions are often hin-dered by limited sampling and insufficient genetic resolution among isolates Pseud-ogymnoascus destructans a fungal pathogen causing the epizootic of white-nosesyndrome in North American bats has exhibited few genetic polymorphisms in pre-vious studies presenting challenges for both epizoological tracking of the spread ofthis fungus and for determining its evolutionary history We used single nucleotidepolymorphisms (SNPs) from whole-genome sequencing and microsatellites to con-struct high-resolution phylogenies of P destructans Shallow genetic diversity andthe lack of geographic structuring among North American isolates support a recentintroduction followed by expansion via clonal reproduction across the epizooticzone Moreover the genetic relationships of isolates within North America suggestwidespread mixing and long-distance movement of the fungus Genetic diversityamong isolates of P destructans from Europe was substantially higher than in thosefrom North America However genetic distance between the North American iso-lates and any given European isolate was similar to the distance between the indi-vidual European isolates In contrast the isolates we examined from Asia werehighly divergent from both European and North American isolates Although the de-finitive source for introduction of the North American population has not been con-clusively identified our data support the origin of the North American invasion byP destructans from Europe rather than Asia

IMPORTANCE This phylogenetic study of the bat white-nose syndrome agentP destructans uses genomics to elucidate evolutionary relationships among popula-tions of the fungal pathogen to understand the epizoology of this biological inva-sion We analyze hypervariable and abundant genetic characters (microsatellites andgenomic SNPs respectively) to reveal previously uncharacterized diversity amongpopulations of the pathogen from North America and Eurasia We present new evi-dence supporting recent introduction of the fungus to North America from a diverseEurasian population with limited increase in genetic variation in North Americasince that introduction

Received 31 October 2017 Accepted 2November 2017 Published 12 December2017

Citation Drees KP Lorch JM Puechmaille SJParise KL Wibbelt G Hoyt JR Sun KJargalsaikhan A Dalannast M Palmer JMLindner DL Marm Kilpatrick A Pearson T KeimPS Blehert DS Foster JT 2017 Phylogenetics ofa fungal invasion origins and widespreaddispersal of white-nose syndrome mBio8e01941-17 httpsdoiorg101128mBio01941-17

Editor Joseph Heitman Duke University

Copyright copy 2017 Drees et al This is an open-access article distributed under the terms ofthe Creative Commons Attribution 40International license

Address correspondence to Jeffrey T Fosterjefffosternauedu

Present address Sebastien J PuechmailleZoological Institute amp Museum GreifswaldUniversity Greifswald Germany

This article is a direct contribution from aFellow of the American Academy ofMicrobiology Solicited external reviewersMatthew Fisher Imperial College LondonJason Stajich University of California Riverside

RESEARCH ARTICLE

crossm

NovemberDecember 2017 Volume 8 Issue 6 e01941-17 reg mbioasmorg 1

KEYWORDS Chiroptera Pseudogymnoascus destructans emerging infectious diseaseepizootic microsatellite whole-genome sequencing wildlife

Fungal diseases are emerging as major threats to ecosystem integrity at localregional and global levels (1) For example introduced fungal phytopathogens

such as Cryphonectria parasitica (agent of chestnut blight) and Cronartium ribicola(agent of white pine blister rust) have caused dramatic declines in foundation treespecies of American forests with severe implications for regional forest structure andfood webs (2 3) Several fungal pathogens of vertebrates have also recently emergedand are of major conservation concern for wildlife including Batrachochytrium dendro-batidis and Batrachochytrium salamandrivorans (agents of amphibian chytridiomycosis)as well as Ophidiomyces ophiodiicola (agent of snake fungal disease) (4ndash6) In particularthe ongoing chytridiomycosis panzootic ranks among the most devastating wildlifediseases due to its broad host range high virulence and rapid worldwide invasion (7)In 2006 another severe fungal epizootic white-nose syndrome (WNS) emerged amonghibernating bats in upstate New York in the United States This disease was named forthe white growth of the psychrophilic fungus Pseudogymnoascus destructans on theskin especially muzzles of infected bats (8) The pathogen has colonized nearly allsurveyed bat hibernacula in the eastern United States and Canada over the last 10 years(9 10) Millions of bats have died as a result of this disease and several species arethreatened with extinction including Myotis septentrionalis (northern long-eared bat)the once plentiful Myotis lucifugus (little brown bat) and the endangered Myotis sodalis(Indiana bat) (11 12) Bats provide widespread insect suppression services to naturaland agricultural ecosystems and are primary suppliers of nutrients to unique caveecosystems through deposition of guano (13) Thus this catastrophic reduction in thebat populations of North America will likely have pervasive ecologic repercussions

Rachowicz et al (14) proposed the novel and endemic pathogen hypothesis for theorigin of B dendrobatidis and emphasized differences in management and researchpriorities for pathogens of different origins A novel pathogen population would beexpected to have limited diversity compared to its source population reflecting arecent introduction and demographic bottleneck of the introduced organism (14 15)Conversely a pathogen with greater diversity implies a longer natural history in an areaand supports the endemic pathogen hypothesis Many bacterial viral parasitic andfungal diseases of epizoological importance have expanded clonally (16) and geneticanalyses of P destructans have been consistent with this pattern

Pseudogymnoascus destructans primarily reproduces asexually producing abundanthaploid conidia (17 18) Although it is theoretically possible for P destructans toreproduce sexually via a heterothallic mating system this has not yet been observedand only one of two required mating types has to date been detected in the genomesof North American fungal isolates (19) Previous studies of the origin and spread of WNSrevealed little or no genetic diversity in North American isolates of P destructans (2021) However these previous studies were limited by low discriminatory power Onlyeight loci were sequenced and they did not exhibit sufficient polymorphisms toidentify the exact source population or to distinguish among isolates of a recentlyemerged pathogen during an infectious disease outbreak (22)

In contrast other genetic markers such as microsatellites (23) and whole-genomesingle nucleotide polymorphism (SNP) genotyping (24) use many loci from throughoutthe genome to increase the resolution of genotypes The high variability of microsat-ellite loci leads to more heterogeneity than point mutations in genes often allowing fordiscrimination between closely related or recently diverged isolates Furthermore thehallmark of whole-genome SNP genotyping is the large number of characters (rangingfrom hundreds to hundreds of thousands of SNP loci in a typical analysis) that can beused to construct a robust phylogeny of clonal organisms (22)

We report the results of a high-resolution phylogenetic analysis of P destructansusing both microsatellite and whole-genome SNP loci to complement the benefits and

Drees et al reg

NovemberDecember 2017 Volume 8 Issue 6 e01941-17 mbioasmorg 2

minimize the drawbacks of each approach Our analysis includes isolates from through-out the WNS epizootic zone in North America that were collected from 2008 to 2014We also included isolates from Europe Mongolia and China (where P destructans wasrecently found to be widely distributed [25]) to more thoroughly investigate thegenetic diversity of putative origin populations of the North American population

RESULTSSNP phylogeny Illumina paired-end whole-genome shotgun sequences were ob-

tained for 26 isolates of P destructans from the North American WNS epizootic zonefrom the years 2008 to 2014 5 isolates from Europe and 3 isolates from Asia Thesereads as well as publicly available P destructans and near-relative Pseudogymnoascusand Geomyces species assemblies were aligned to a reference genome from P destruc-tans 20631-21 (NCBI accession no GCA_0016412651) (26) Oidiodendron maius Zn(Ascomycota Leotiomycetes) was used as an outgroup based on our phylogeneticanalyses of fungi closely related to Pseudogymnoascus (data not shown) Descriptivestatistics of the alignments as well as NCBI accession numbers of all samples analyzedare displayed in Table 1 Duplicatedrepeat regions accounted for 4325 of thegenome and were excluded from our SNP-based analyses Only ~01 of the referencegenome was orthologous in all samples resulting in 4757 SNP loci of which 2479 weresynapomorphic (ie shared between one or more samples and useful to define clades)Maximum parsimony analysis resulted in four trees with a low rescaled consistency (RC)index (RC of 04704) indicating a high degree of homoplasy that one would expectfrom a data set representing a long evolutionary history (data not shown) Maximumlikelihood analysis (Fig 1) indicated strong bootstrap support for a single P destructansclade that is sister to other all currently recognized members of the genus Pseudogym-noascus including ancient samples from Russian permafrost Moreover in this analysisisolate JH15CN0111a from China is basal to P destructans isolates from MongoliaEurope and North America allowing us to root the P destructans phylogeny (Fig 2)

As previously determined by multilocus sequence typing (MLST) (27) members ofthe genus Pseudogymnoascus are not all closely related to each other We thus limitedthe sample set to just P destructans and aligned only reads from this species to identifySNP loci shared specifically among P destructans isolates This resulted in the detectionof 37752 core SNP loci within the 20-Mb core genome alignment Maximum parsimonyproduced one tree with an RC of 07729 (data not shown) Maximum likelihood analysis(Fig 2) distinguishes three well-supported clades among the P destructans isolatesChina Mongolia and Europe The North American isolates are nearly identical in thistree and are therefore represented as a group by the type isolate 20631-21 obtainedin New York in 2008 This phylogeny clearly shows the North American clade ofP destructans as a member of a greater European clade and distinct from the easternand central Asian isolates The tree also shows that North American P destructansisolate is most closely related to a Ukrainian isolate (Gd44) in our sample set and leastrelated to an isolate from France (Gd41) This is in contrast to a previous MLST study(28) which showed the opposite trend using many of the same isolates However lowsample sizes long branch lengths and poor bootstrap support for the branchesbetween members of the European clade preclude stronger conclusions about theregion of Europe that gave rise to North American P destructans With our current dataset the possibility that North American P destructans was introduced from Asiahowever seems unlikely

We reduced the sample set once again to include only isolates of P destructans fromNorth America and reanalyzed the alignments for SNPs Only 51 core SNP loci wereshared among the 26 North American isolates Maximum parsimony produced one treewith an RC of 100 (data not shown) Thus all homoplasy observed among P destruc-tans phylogenies was due to the Eurasian genomes Only three SNPs were synapomor-phic Two SNPs show differences between the type isolate 20631-21 and all othersamples A single SNP shared by samples 23414-1W and 23434-1W both obtained inIndiana in 2011 suggest the formation of a geographically isolated lineage Less

Genomics of White-Nose Syndrome Invasion reg

NovemberDecember 2017 Volume 8 Issue 6 e01941-17 mbioasmorg 3

TABLE 1 Whole-genome sequences aligned to the reference genome of Pseudogymnoascus destructans (NCBI accession noGCA_0016412651)

Isolate ID no Speciesa Sourceb Locationc Date

Readlength(bp)

Avg readdepth

of coregenomecoveredd Accession no

20631-21 P destructans M lucifugus NY Feb 2008 NAe NA 10000 GCA_0016412651GU999986 P destructans M myotis DEU Mar 2009 2 101 15583 9873 SRR6011467GU350433 P destructans M myotis CHE Apr 2009 2 101 18797 9893 SRR6011468GU350434 P destructans M myotis HUN Mar 2009 2 101 16929 9900 SRR601146520693-1 P destructans M lucifugus MA Mar 2008 2 101 20639 9979 SRR601146622429-8 P destructans M septentrionalis WV Jan 2009 2 101 20862 9981 SRR601147122971-3 P destructans M lucifugus ON Mar 2010 2 101 12324 9973 SRR601147222504-1 P destructans M lucifugus PA Mar 2009 2 101 19273 9967 SRR601146922442-2 P destructans M lucifugus NJ Feb 2009 2 101 20079 9971 SRR601147022948-1 P destructans M septentrionalis TN Mar 2010 2 101 18047 9949 SRR601147320674-9 P destructans M septentrionalis VT Mar 2008 2 98 10025 9946 SRR601147420682-10 P destructans M septentrionalis MA Mar 2008 2 101 15600 9911 SRR601147722004-1 P destructans M lucifugus CT Apr 2008 2 101 21378 9920 SRR601147822426-2 P destructans M lucifugus CT Jan 2009 2 101 17049 9942 SRR601147522469-1 P destructans P subflavus VA Mar 2009 2 101 16709 9942 SRR601147622480-1 P destructans E fuscus NY Mar 2009 2 98 8585 9943 SRR601148122884-4W P destructans M lucifugus VT Jan 2010 2 101 14188 9943 SRR601148222930-2 P destructans P subflavus TN Feb 2010 2 101 8298 9903 SRR601147922949-4 P destructans M septentrionalis MD Mar 2010 2 103 21159 9954 SRR601148022972-2W P destructans M lucifugus ON Mar 2010 2 101 8248 9903 SRR601148322997-1 P destructans M septentrionalis TN Apr 2010 2 101 27120 9997 SRR601148423414-1W P destructans M lucifugus IN Jan 2011 2 98 12000 9959 SRR601149323434-1W P destructans M lucifugus IN Jan 2011 2 101 25507 9996 SRR601149223444-1 P destructans M lucifugus TN Feb 2011 2 98 9667 9949 SRR601149123455-1 P destructans M lucifugus VA Feb 2011 2 101 16317 9970 SRR6011490Gd41 P destructans M myotis FRA Mar 2009 2 251 8904 9931 SRR6011497Gd44 P destructans M myotis UKR Feb 2011 2 301 9268 9871 SRR601149623874-1 P destructans M lucifugus ME Dec 2011 2 101 20076 9997 SRR601149523877-1 P destructans M septentrionalis DE Mar 2012 2 101 17732 9997 SRR601149423897-2 P destructans P subflavus MO Mar 2012 2 251 11980 9993 SRR6011489W41203 P destructans Sub NB Apr 2012 2 251 7431 9954 SRR6011488JH15CN0111a P destructans M petax CHN Mar 2015 2 150 7431 9678 SRR6011485JH16MG088 P destructans P ognevi MNG 2016 2 150 7884 9731 SRR6011486JH16MG093 P destructans P ognevi MNG 2016 2 150 1528 5310 SRR601148703VT05 Pseudogymnoascus sp Sed VT 2008 NA NA 7663 GCA_0016626451UAMH10579 P verrucosus Peat AB 2002 NA NA 7692 GCA_01662655105NY08 Pseudogymnoascus sp Sed NY 2008 NA NA 7668 GCA_001662605123342-1-I1 Pseudogymnoascus sp P subflavus WI 2008 NA NA 6120 GCA_001662575124MN13 Pseudogymnoascus sp Sed MN 2008 NA NA 6928 GCA_0016625951WSF3629 Pseudogymnoascus sp Peat WI 1960 NA NA 7702 GCA_0016625851F-103 Pseudogymnoascus sp Soil NY Contemporary NA NA 7676 GCA_0007508951F-3557 Pseudogymnoascus sp Pine post SWE Contemporary NA NA 5204 GCA_0007506651F-3775 Pseudogymnoascus sp Soil DEU Contemporary NA NA 5060 GCA_0007507151F-3808 Pseudogymnoascus sp M glareolus RUS Contemporary NA NA 5251 GCA_0007506751F-4246 Pseudogymnoascus sp Sed MNG Contemporary NA NA 5377 GCA_0007507351F-4281 Pseudogymnoascus sp Cryopeg RUS 012ndash02 myaf NA NA 5709 GCA_0007507451F-4513 Pseudogymnoascus sp Permafrost RUS 18ndash30 mya NA NA 5409 GCA_0007507551F-4514 Pseudogymnoascus sp Permafrost RUS 18ndash30 mya NA NA 5180 GCA_0007507951F-4515 Pseudogymnoascus sp Permafrost RUS 18ndash30 mya NA NA 6124 GCA_0007508051F-4516 Pseudogymnoascus sp Permafrost RUS 18ndash30 mya NA NA 5386 GCA_0007508151F-4517 Pseudogymnoascus sp Permafrost RUS 18ndash30 mya NA NA 6086 GCA_0007508751F-4518 Pseudogymnoascus sp Soil RUS Contemporary NA NA 6747 GCA_0007509251F-4519 Pseudogymnoascus sp Soil RUS Contemporary NA NA 7704 GCA_0007509351F-4520 Pseudogymnoascus sp Soil RUS Contemporary NA NA 6701 GCA_0007509351ATCC 16222 P pannorum Soil DEU NA NA NA 5808 GCA_0016306051M1372 P pannorum Soil CA Jun 1961 NA NA 5855 GCA_0004973051Zn O maius NA NA NA NA NA 029 GCA_0008273251aThe species shown are Pseudogymnoascus destructans Pseudogymnoascus verrucosus Pseudogymnoascus sp Pseudogymnoascus pannorum and Oidiodendron maiusbThe species shown are Myotis myotis Myotis lucifugus Myotis septentrionalis Perimyotis subflavus Eptesicus fuscus Myotis petax Plecotus ognevi and Myodes glareolus(bank vole) Sub hibernaculum substrate (ie roosting surface) Sed hibernaculum sediment

cNY New York DEU Germany CHE Chechnya HUN Hungary MA Massachusetts WV West Virginia ON Ontario Canada PA Pennsylvania NJ New Jersey TNTennessee VT Vermont CT Connecticut VA Virginia MD Maryland IN Indiana FRA France UKR Ukraine ME Maine DE Delaware MO Missouri NB NewBrunswick CHN China MNG Montenegro AB Alberta WI Wisconsin MN Minnesota SWE Sweden RUS Russia CA California

dCoverage reported as percentage of core genome (ie portion of genome shared by all samples that is not in a duplicated region) that passed 10 minimum depthof coverage and 90 of read agreement filters

eNA not availablefmya million years ago

Drees et al reg

NovemberDecember 2017 Volume 8 Issue 6 e01941-17 mbioasmorg 4

conservative filtering steps for SNP quality and read depth and removing the require-ment for a locus to be present in all genomes failed to substantially increase thenumber of synapomorphies Of the 51 SNP loci none of the mutations were in codingsequences (Table 2) but rather were from intergenic sequences introns or untrans-lated regions (UTRs) of exons

Isolate mating type was determined from whole-genome sequence alignments tothe reference genome sequence of type isolate 20631-21 which was previously deter-mined to mating type MAT1-1 (19) All isolates from North America in this studypossessed the MAT1-1 mating type Most Eurasian isolates did as well although anisolate from Switzerland (GU350433) and an isolate from Mongolia (JH16MG088) weremating type MAT1-2

Microsatellite phylogeny Insufficient synapomorphic SNPs were discoveredamong the North American P destructans whole-genome sequences to produce aninformative phylogeny such that analyses produced only a star-shaped phylogeny(inset in Fig 3) Therefore 96 North American isolates of P destructans including thoseanalyzed by whole-genome sequencing were genotyped with a 23-locus microsatellitepanel (see Table S1 in the supplemental material) Thirty-one unique genotypes wereidentified with one dominant genotype for 13 of the isolates A phylogeny wasconstructed by the neighbor-joining (NJ) method from the microsatellite data (Fig 3)These data although based upon fewer genetic loci contain many more samples andreveal far more genetic diversity than the SNP-based phylogeny of North American

0008

Oidiodendron maius

Pd Gd44 UKR 2011

Pd GU350433 CHE 2009Pd GU350434 HUN 2009

Pd JH16MG088 MNG 2016

Pd 20631 21 NY 2008

Pd Gd41 FRA 2009

Pd JH16MG093 MNG 2016Pd JH15CN0111a CHN 2015

Pd GU999986 DEU 2009

Psp 03VT05 VT 2008

Psp F3557 SWE contemporary

Psp 23342 1 I1 WI 2008Psp F4518 RUS contemporary

Psp 24MN13 MN 2008

Psp BL549 TN 2009

Psp 04NY16 NY 2009

Psp F4513 RUS 3mya

Psp F 103 NY contemporary

Psp WSF3629 WI 1960

Psp F4281 RUS 200kyaPp M1372 CA 1961

Psp F4246 MNG contemporary

Psp F4514 RUS 3mya

Pp ATCC 16222 DEU contemporaryPsp F3775 DEU contemporary

Psp 05NY08 NY 2008

Psp F4517 RUS 3myaPsp BL308 TN 2009

Psp F4519 RUS contemporary

Psp F4516 RUS 3mya

Psp F3808 RUS contemporary

Pv UAMH10579 AB 2002

Psp F4515 RUS 3mya

Psp F4520 RUS contemporary

100

51

98

73

100

100

100

10045

100

96

43

100

100

100

100

100

100

100

100

100

97

100

100

74

Pseudogymnoascus destructans

FIG 1 Maximum likelihood phylogenetic tree of the genus Pseudogymnoascus (previously Geomyces) based upon 4757 SNPs(of which 2479 are synapomorphic) Branches are labeled with bootstrap support values Oidiodendron maius serves asoutgroup Pseudogymnoascus destructans is a sister clade to other members of the genus Psp Pseudogymnoascus sp PdP destructans Pp P pannorum Pv P verrucosus Additional data for each sample include isolate name countrystate of originand date of collection (estimated as preservation date for samples taken from permafrost) Samples without a precisely knowncollection date are indicated as ldquocontemporaryrdquo

Genomics of White-Nose Syndrome Invasion reg

NovemberDecember 2017 Volume 8 Issue 6 e01941-17 mbioasmorg 5

P destructans This tree supports the single clade containing isolates 23431-1W and23434-1W from Indiana identified in the whole-genome phylogeny but includes anumber of other isolates that did not share synapomorphic SNPs with the Indianaisolates Other clades are evident as well but they do not seem to correspond togeographic regions or sampling dates A Mantel test comparing microsatellite-basedgenetic distance to geographic distance between isolates showed no correlation (r

0016 n 99 replicates P 037) We note however that despite the appearance ofdefined groupings throughout the tree there was limited bootstrap support for all ofthe branches within this phylogeny (all bootstrap values were 70)

Population genetics Indices of genetic diversity and linkage were calculated forSNP genotypes Asian samples were left out of this analysis due to the low sample sizeThe Simpsonrsquos index of genetic diversity was reduced in North America ( 0690standard error [SE] 0 130) compared to Europe ( 0800) the putative sourcepopulation for P destructans Average genomic linkage represented by standardizedindex of association (29) was close to zero for both populations Among NorthAmerican isolatesrD 00585 whereas for EuroperD 000812 Thus recombinationdoes not appear to be a significant factor in either populationrsquos genetic structure andP destructans isolates in both regions are reproducing clonally The evenness of oursampling was not sufficient nor did our data conform to various model assumptions toproduce reliable population structure estimates (using models such as STRUCTURE andSNMF) from our SNP or microsatellite data (30)

DISCUSSIONPhylogeography The two genetic markers used in this study SNPs and microsat-

ellites provide complementary information regarding the phylogeography of P de-

GU350433 MMYO CHE 2009

GU350434 MMYO HUN 2009

Gd44 MMYO UKR 2011

GU999986 MMYO DEU 2009

Gd41 MMYO FRA 2009

JH15CN0111a MYPX CHN 2015

JH16MG088 PLOG MNG 2016

20631-21 MYLU NY 2008

JH16MG093 PLOG MNG 20161517 [756 - 2439]

103 [53 - 166]

1972 [989 -3165]

3408 [1689 - 5452]

2011 [1006 - 3227]

66 [33 - 107]

FIG 2 Maximum clade credibility tree for isolates of Pseudogymnoascus destructans from North America (n 1)Europe (n 5) and Asia (n 3) based upon 37752 core SNP sites (of which 22594 are synapomorphic) All nodeshad 100 posterior probabilities Nodes are labeled with time (years before present) followed by the 95high-probability density interval in brackets dates on all shorter branches are not shown North American isolatesare nearly indistinguishable at these SNP loci and so were represented in this tree by a single isolate (20631-21)The tree is rooted on the Chinese isolate based upon the Fig 1 phylogeny that shows other Pseudogymnoascusspp as a sister clade to P destructans Data for each sample include isolate name bat host species codecountrystate of origin and date of collection

Drees et al reg

NovemberDecember 2017 Volume 8 Issue 6 e01941-17 mbioasmorg 6

structans Whole-genome sequencing revealed three distinct populations of P destruc-tans among the isolates we tested one consisting of European isolates that includedWNS epizootic isolates from North America another of Mongolian isolates and a thirdrepresented by an isolate from China The phylogeny of European isolates determinedin this study with 58707 SNP loci was consistent with that described previously using14 SNPs in an eight-member multilocus sequence typing analysis (MLST) (28) specifi-cally the genetic diversity of P destructans was 14 higher in Europe than in NorthAmerica In this study we identified only 51 core SNPs among North American isolateswhich clearly demonstrates the homogeneity of P destructans population structureacross the epizootic zone and is consistent with previous reports of clonal spread (2021 28 31 32) North American isolates formed a monophyletic group but appear to berepresentative of diversity from Europe (ie the branch lengths between the Europeansamples were comparable between the European and North American isolates) This aswell as the 99995 reduction in core SNP loci when non-American isolates areremoved represents a loss of genetic diversity consistent with previous conclusions ofa recent introduction of a novel pathogen (33 34) Moreover star-shaped phylogeniescontaining few synapomorphies are typical in recent pathogen introductions (35ndash37)

TABLE 2 Annotation of core SNP loci from isolates of P destructans from North America

Accession no Position Type Variant isolate(s) Protein ID no Product

KV4413861 1202839 UTRa 20693-1_MA_2008 OAF634131 NAD synthaseKV4413861 2276958 Intergenic 22948-1_TN_2010 NAb NAKV4413951 783753 UTR 22948-1_TN_2010 OAF589581 Hypothetical proteinKV4413961 546602 Intergenic 23444-1_TN_2011 NA NAKV4413981 551631 UTR 23877-1_DE_2012 OAF579761 Hypothetical proteinKV4413991 235966 Intergenic 23414-1W_IN_2011

23434-1W_IN_2011NA NA

KV4414001 640730 UTR 22442-2_NJ_2009 OAF572731 Hypothetical proteinKV4414011 139816 Intergenic 22948-1_TN_2010 NA NAKV4414021 72139 Intergenic 23897-2_MO_2012 NA NAKV4414021 226767 UTR 22948-1_TN_2010 OAF568521 Hypothetical proteinKV4414021 510412 Intergenic 20693-1_MA_2008 NA NAKV4414041 262446 UTR 22442-2_NJ_2009 OAF564191 Hypothetical proteinKV4413871 430984 Intergenic 23874-1_ME_2012 NA NAKV4413871 820735 UTR 22426-2_CT_2009 OAF628081 Hypothetical proteinKV4413871 1825781 Intergenic 22948-1_TN_2010 NA NAKV4413871 2082665 UTR 22442-2_NJ_2009 OAF625741 ESCRT-I complex subunit VPS28KV4413871 685734 Intergenic 22442-2_NJ_2009 NA NAKV4413871 785423 Intergenic 23897-2_MO_2012 NA NAKV4413871 54855 Intergenic 22971-3_ON_2010 NA NAKV4413871 193542 Intergenic 22948-1_TN_2010 NA NAKV4413871 253155 Intergenic 22442-2_NJ_2009 NA NAKV4413871 77282 Intergenic 23455-1_VA_2011 NA NAKV4413871 120236 Intergenic 23897-2_MO_2012 NA NAKV4413881 775461 Intragenic 22972-2W_ON_2010 OAF617441 Hypothetical proteinKV4413881 950597 Intron 23897-2_MO_2012 OAF619151 Hypothetical proteinKV4413881 120202 UTR 22469-1_VA_2009 OAF620441 Hypothetical proteinKV4413881 57283 UTR 20631-21_NY_2008 OAF618131 Hypothetical proteinKV4413891 343287 Intron 23455-1_VA_2011 OAF616881 AGCAKT protein kinaseKV4413891 419134 Intergenic 23414-1W_IN_2011 NA NAKV4413891 867065 UTR 22004-1_CT_2008 OAF613681 Actin cytoskeleton-regulatory complex protein end3KV4413891 1173430 Intergenic 22971-3_ON_2010 NA NAKV4413891 1173432 Intergenic 22971-3_ON_2010 NA NAKV4413891 50002 UTR 22948-1_TN_2010 OAF613941 Hypothetical proteinKV4413901 643943 Intergenic 23897-2_MO_2012 NA NAKV4413901 659862 UTR 22948-1_TN_2010 OAF612161 Hypothetical proteinKV4413901 861423 UTR 22948-1_TN_2010 OAF609331 Hypothetical proteinKV4413901 1243398 UTR 23434-1W_IN_2011 OAF607711 Hypothetical proteinKV4413901 1524359 UTR 22948-1_TN_2010 OAF608721 Hypothetical proteinKV4413911 151986 UTR 20693-1_MA_2008 OAF603661 Hypothetical proteinKV4413911 1184294 Intergenic 22442-2_NJ_2009 NA NAKV4413921 712894 Intergenic 23414-1W_IN_2011 NA NAaUTR untranslated region of exonbNA not available

Genomics of White-Nose Syndrome Invasion reg

NovemberDecember 2017 Volume 8 Issue 6 e01941-17 mbioasmorg 7

FIG 3 Phylogeny of 96 North American Pseudogymnoascus destructans isolates constructed from alleles of 23 microsatellite loci Isolates that were includedin whole-genome sequence phylogenies are colored red The scale bar indicates genetic distance Shown is a neighbor-joining tree of 96 North Americanisolates rooted with GU350434 (host M myotis [MMYO] location Hungary [HUN] date 2009) Samples from the epicenter of the WNS epizootic are coloredblue (Inset) Star-shaped maximum parsimony tree from comparisons of whole genomes constructed from 51 SNPs only 3 of which are synapomorphic

Drees et al reg

NovemberDecember 2017 Volume 8 Issue 6 e01941-17 mbioasmorg 8

with lack of rapid diversification into distinct lineages likely representing the normrather than the rule A recent report on the spread of WNS also identified few mutationsof P destructans upon introduction to North America (32) A strict interpretation of thedates in Fig 2 would suggest that all P destructans strains in North America and Europediverged from each other within the past ~100 years and from the ancestor to Asianisolates roughly 3400 years ago While these estimates are plausible we believe a moreaccurate estimation will require increased sampling of isolates from Eurasian popula-tions Perhaps analyses using P destructans genomes from samples preserved long agoas we did with Pseudogymnoascus sp genomes from ancient Russian permafrostsamples in Fig 1 or detection of WNS-induced bat die-offs in Eurasia from the fossilrecord will enable more accurate dating Given that P destructans has likely been a batpathogen for millions of years (73) the lack of mortality in European bats considerablegenetic diversity in microsatellite loci and the broad distribution of the fungus acrossthe palearcticmdashall changes that we envision taking millennia to evolvemdashit is likely thatP destructans is much older than a few thousand years old Clearly more detailedmolecular dating analyses are needed to resolve the question of the emergence anddiversification of this species

The microsatellite-based phylogeny containing more isolates illustrates some addi-tional epizoological features with relevance to pathogen invasion and establishmentWhat is most striking about these data is the large amount of genetic diversity revealedamong the North American isolates Earlier studies indicated that 73 isolates of P de-structans from New York Vermont Pennsylvania Ohio West Virginia North CarolinaOntario New Brunswick Nova Scotia and Prince Edward Island were geneticallyidentical via MLST (20 21 31) Genotyping of these samples plus 78 others from easternCanada and the United States resulted in a single genotype in 109 samples plus twosingleton genotypes from New Brunswick and one from Ontario In contrast microsat-ellite loci from our study defined 31 different genotypes among the isolates from NorthAmerica Another hallmark of these data is the lack of geographic or temporal corre-spondence between clades evident in the tree For example the Fig 3 phylogenyshows that isolates collected from New York in 2008 at the beginning of the WNSoutbreak are distributed throughout the tree Some isolates from the same samplinglocation and date (eg the Williams Hotel Mine) occur in different clades indicatingrelatively high genetic diversity from this one region compared to other regions inNorth America Unfortunately these data do not indicate whether this diversity waspresent in the founding population or arose soon after introduction a commonproblem with population genetic analyses of pathogens (38) New Brunswick and NovaScotia isolates also occur in many clades associated with isolates from throughout theepizootic zone A possible exception to the lack of geographic structuring we observedare several basal clades containing only northeastern Canadian isolates or isolates fromNew England and New York early in the outbreak This region is relatively isolated andmay represent an area that received P destructans isolates geographically spreadingfrom New England early in the outbreak and has had time since then to diversifygenetically Intense sampling in this region may also be contributing sampling bias tothis observation however

Clonality Questions regarding clonal expansion of P destructans in North Americacan be answered more thoroughly by this study due to the large number of charactersused in this data set The strongest evidence for clonal reproduction of an ascomycetefungus is the lack of both mating types in a population whereas an even distributionof mating types in a population would suggest the prevalence of sexual recombinationIn a previous study (19) 5 out of 23 (22) of isolates from Eastern Europe possessed theMAT1-2 locus With the addition of our samples from Eurasia the proportion of MAT1-2to MAT1-1 mating types is 7 out of 30 isolates (23) In contrast all 26 of the isolatesfrom North America in this study possessed the MAT1-1 mating type Additionalanalysis of P destructans mating types in an eastern European population showed amore equal distribution of mating types (586 MAT1-1 and 413 MAT1-2 n 41)

Genomics of White-Nose Syndrome Invasion reg

NovemberDecember 2017 Volume 8 Issue 6 e01941-17 mbioasmorg 9

(39) The absence of the MAT1-2 mating type in North American P destructans isevidence of both asexual reproduction and likely a recent genetic bottleneck

The overrepresentation of widespread and persistent genotypes is also evidence ofclonal reproduction as opposed to sexual reproduction (40) as was indicated by bothSNP and microsatellite analyses in this study Based upon SNP genotyping 8 of the 26isolates sequenced (31) were found to be clonal ranging from New England toTennessee and sampled between 2008 and 2010 Similarly two microsatellite geno-types account for 15 and 6 samples respectively out of a total of 55 North Americanisolates for which we obtained complete microsatellite genotypes (38) Quantitativelythe Simpsonrsquos index of genetic diversity was slightly reduced in North Americaalthough indexes of genetic diversity are highly dependent upon sample size despiterarefaction and the number of isolates from Europe compared to the number ofisolates in North America analyzed in this study is very small Average genomic linkageas measured by the standardized index of association indicates that both the Europeanand North American populations of P destructans are at linkage equilibrium suggestingboth populations reproduce primarily as clones

Introduction of P destructans to North America Warnecke et al (41) hypothe-sized that P destructans was a novel pathogen introduced to North America fromEurope based upon evidence that North American little brown bats developed WNSwhen inoculated with a European isolate of P destructans Their findings were furtherstrengthened by Leopardi et al (28) who demonstrated genetic similarity based onMLST between the North American and some European fungal populations indicatingthe likely source population for this introduction to be from Europe SubsequentlyP destructans has been isolated from bats throughout Europe (39 42ndash46) and can alsocause histopathological lesions consistent with WNS in European species of bats (4748) However widespread mortality of bats from WNS has not been documented on theEuropean continent (49) This suggests European bat species may have developedresistance or tolerance to infection by P destructans presumably due to coevolutionbetween hosts and the fungal pathogen (41 50) In contrast the high mortality of NorthAmerican bats infected with P destructans is consistent with exposure of naive hostspecies to a pathogen preadapted to similar hosts and environmental conditions

Our work supports the hypothesis that P destructans is a novel pathogen recentlyintroduced to North America by demonstrating the relationship of North Americanisolates of the fungus to a population dominated by European isolates but geneticallydistant from isolates from Asia Using high-resolution genetic markers we document aloss of genetic diversity in epizootic P destructans consistent with a recent introductionof this pathogen to North America

Similarities to other fungal invasions Pseudogymnoascus destructans follows apattern of emergence and spread similar to other invasive fungal pathogens In thecase of Cryphonectria parasitica the causative agent of chestnut blight (38) highgenetic diversity in putative source populations coupled with a significant decrease inallelic richness in microsatellite and other multilocus genotypes among North Americanisolates suggested a founder effect associated with the introduction of the organismfrom Asia North American isolates of C parasitica were as genetically distant fromAsian populations as the Asian populations were from each other Analysis of popula-tion structure could narrow the source population down to a broad region (ie HonshuJapan) but required the admixture of an unsampled population significantly differentfrom the other Asian samples A more detailed genomic analysis of an invading funguswas conducted with isolates of B dendrobatidis originating from multiple continentsand revealed a considerably more complex phylogeny of that fungus than was previ-ously determined A virulent panzootic strain apparently emerged and rapidly spreadacross the globe yet the original source population of the lineage remains ambiguousdespite a comprehensive sampling effort and thorough sequencing analyses (51)Consistent with a pattern also seen in our study haplotypes from B dendrobatidis didnot correlate well with host or geography suggesting other drivers of differentiation

Drees et al reg

NovemberDecember 2017 Volume 8 Issue 6 e01941-17 mbioasmorg 10

Additionally phylogenies constructed from different types of genetic markers yieldeddifferent sometimes contradictory results for clones of the potato blight agent Phy-tophthora infestans (52) A common thread in all of these case studies is that moregenetic information lends complexity but not necessarily clarity to the natural historyof an invading fungal pathogen In terms of the natural history of P destructans it willrequire extensive sampling in Europe to find the population that was introduced toNorth America

Using two high-resolution genetic charactersmdashSNPs within whole-genome se-quences and microsatellitesmdashwe have demonstrated that isolates of P destructans inNorth America form a single clade much more closely related to isolates from Europethan to genetically distant populations sampled in Asia The North American isolatesrepresent a loss of genetic diversity in comparison to isolates from Europe addingfurther support to the hypothesis that P destructans was recently introduced to NorthAmerica from Europe (28) Future sampling in Europe will be needed to more preciselydefine the origin of P destructans although the substantial diversity of P destructans inEurope coupled with results from other fungal invasion studies suggests that identi-fication of the exact source population may be challenging Microsatellite loci wereparticularly suitable for distinguishing isolates from throughout the North AmericanWNS epizootic zone but the few synapomorphic SNPs found among these isolatescould not resolve their phylogenetic relationships However sufficient SNPs werepresent to differentiate isolates of P destructans from Europe China Mongolia andNorth America This work elevates our understanding of the origin and spread ofP destructans to a similar level to that achieved for B dendrobatidis and C parasiticaproviding a framework for a global pathogen population structure that can be used forfuture epizoological investigations

MATERIALS AND METHODSSampling and culturing P destructans Skin samples from bats or swab samples of bats and

substrates were cultured on Sabouraud dextrose agar or potato dextrose agar and incubated at 7 to 10degCas previously described (42 53)

Whole-genome sequencing Genomic DNA was extracted from P destructans by a variety ofmethods including the OmniPrep for Fungi kit (G-Biosciences St Louis MO) the DNeasy blood andtissue kit (Qiagen Hilden Germany) using the supplementary protocol from the manufacturer ldquoPurifi-cation of total DNA from yeast using the DNeasy Blood and Tissue kit (DIY13 Aug-06)rdquo as describedpreviously (54) or a phenol-chloroform extraction Preparation of libraries for Illumina whole-genomesequencing was based upon a previously published method (55) but was modified as sequencingtechnology evolved during the course of the project In summary approximately 1 to 10 g of DNAsample in 200 l Tris-EDTA buffer was sheared with a SonicMan microplate sonicator (Brooks Automa-tion Chelmsford MA) to produce fragments 200 to 1000 bp in length with the average fragment lengthbeing 600 bp The shearing protocol was as follows 75 s at 0degC prechill followed by 20 cycles ofsonication for 100 s at full power 75 s at 0degC lid chill 10 s at 0degC plate chill and 75 s at 0degC postchillEnd repair dA-tailing adapter ligation and indexing followed the standard Illumina protocol ldquoPreparingsamples for multiplexed paired-end sequencingrdquo (part no 1005361) using reagents from the NEBNextDNA library prep master mix set for Illumina (New England Biolabs Ipswich MA) or the KAPA high-throughput library preparation kit ldquowith beadrdquo (Kapa Biosystems Wilmington MA) Libraries wereindexed with the Illumina multiplexing sample preparation oligonucleotide kit 6-bp indices (Illumina SanDiego CA) or custom 8-bp indices for higher index read discrimination (56) Size selection of 600-bpfragments was accomplished by excision from 2 agarose gels followed by purification with a QIAquickgel purification kit (Qiagen Hilden Germany) E-Gel SizeSelect gel (Life Technologies Inc Grand IslandNY) or Agencourt AMPure XP SPRI beads (Beckman Coulter Inc Indianapolis IN) using the KapaBiosystems ldquoHigh-throughput NGS library preparation technical guide Illumina platformsrdquo (57) Productsfrom each step of the library preparation were purified with either Agencourt AMPure XP SPRI beadsusing the same protocol or the QIAquick 96 PCR purification kit (Qiagen Hilden Germany) Libraries werequantified via quantitative PCR with a KAPA library quantification kit (Kapa Biosystems Wilmington MA)for the ABI Prism 9600 real-time PCR system (Life Technologies Inc Grand Island NY) Prior to loadingon the sequencer the library fragment size distribution was qualitatively confirmed with an Agilent DNAhigh-sensitivity kit for the 2100 Bioanalyzer (Agilent Santa Clara CA) Individual libraries were run on anIllumina GAIIx sequencer (V2 chemistry) or an Illumina MiSeq (V3 chemistry) to produce 100-bppaired-end reads or 250-bp paired-end reads respectively Libraries sequenced on a HiSeq 2000 weremultiplexed with a maximum of five libraries per lane to produce 150-bp paired-end reads with V3chemistry

Whole-genome sequence analysis The sequence from resequencing and assembly of the genomeof P destructans 20631-21 collected in New York in 2008 (26) (NCBI GenBank assembly accession noGCA_0016412651) was used as a reference for SNP discovery using the Northern Arizona SNP Pipeline

Genomics of White-Nose Syndrome Invasion reg

NovemberDecember 2017 Volume 8 Issue 6 e01941-17 mbioasmorg 11

(NASP) version 102 (58) With NASP Illumina paired-end reads were aligned to the reference genomewith the Burrows-Wheeler Aligner (59) version 075a mem algorithm SNPs were detected in thealignments with the Unified Genotyper of the Genome Analysis Toolkit (60) build 25-2-gf57256bfollowing recommended best practices (61 62) Duplicated regions of the reference genome includingrepeat regions and multiple gene copies were determined by aligning the reference sequence to itselfusing the nucmer algorithm of MUMmer version 323 (63) SNPs that fell within these duplicate regionswere excluded from further analysis to avoid false SNP calls due to ambiguous read alignment SNP lociwere also excluded if at least one sample lacked a base call at an SNP locus) had read coverage of lessthan 10 in at least one sample and less than 90 of the reads agreed with the SNP call Default settingsfor each software package were used unless otherwise noted Mating type was determined from theprevalence of reads mapping to the MAT1-1 mating type locus in read alignments to the referencesequence (NCBI GenBank accession no KV4413901 bases 187500 to 188500) SNPs were annotated withSnpEff version 43q (64)

Microsatellite genotyping DNA samples from P destructans were tested with the 23-locus multi-locus variable number tandem-repeat analysis (MLVA) panel described by Drees et al (65) Briefly weidentified 2- to 6-bp repeats in the genome sequence of P destructans type strain 20631-21 (GenBankaccession no GL573169 to GL575015) We then tested 127 primer pairs for microsatellites with lengthpolymorphisms using a diverse set of DNA extracts from 39 isolates of P destructans collected from NorthAmerica and Europe We selected 23 loci containing repeat number polymorphisms Using these primersets we conducted fragment analysis using the Terminator v31 cycle sequencing kit on a 3130xl Geneticanalyzer (Life Technologies Inc Grand Island NY) Allele sizes were determined using the LIZ 1200 sizestandard in GeneMapper version 40

Phylogenetics PAUP version 40 a150 (66) was used to conduct maximum parsimony analysis onSNP data and neighbor-joining analysis (based on mean character difference) on microsatellite dataExtended majority rule maximum likelihood phylogenetic trees were created from concatenated SNPsequences with RAxML version 827 (67) using the ASC_GTRGAMMA substitution model with ascertain-ment bias correction for each base character to account for invariant loci and the autoMRE bootstoppingcriterion with a maximum of 1000 bootstraps Trees were plotted with FigTree version 140 (A Rambaut2012 httptreebioedacuksoftwarefigtree)

BEAST2 version 245 (68) was used to create maximum clade credibility trees from SNP locus dataBriefly the SNP data were first filtered to remove SNPs within 50 bp of each other to minimize the effectsof linkage on the analyses Optimum base substitution models for SNP data were determined with theR package phangorn version 211 (69) resulting in the generalized time-reversible (GTR) model for thefull analysis and the JC69 model for the North American isolates alone Both analyses used a strictmolecular clock and a coalescent constant population tree model Skyline demographic models whichmay be more appropriate for the expanding populations in North America as well as a relaxed log normalmolecular clock were attempted for comparison but failed to coalesce Because only core SNPs werebeing analyzed the analyses were corrected for ascertainment bias with a count of invariant bases in thenonduplicated alignments to the reference genome The Markov chain Monte Carlo (MCMC) chain lengthwas set to 10 million with 10 burn-in and logging every 1000 steps Five chains were run producinga total of 50000 trees which were combined and subsampled to 10000 trees with LogCombiner version243 and used to create an annotated maximum clade credibility tree with TreeAnnotator version 243

Population genetics Simpsonrsquos index of genetic diversity and index of association were calculatedwith the R package poppr version 230 (70) as follows SNP allele information was preprocessed bydetermining genetic distances between individuals with bitwisedist calculating the threshold distancefor clustering isolates with cutoff_predictor and assigning isolates to genotypes with mlgfilter Simp-sonrsquos index of genetic diversity was determined for both SNP and microsatellite genotypes withdiversity_ci with 1000000 bootstraps North American isolates were rarefied to equal the number ofEuropean isolates in the SNP analysis which enabled the comparison of estimated Simpsonrsquos indicesbetween the populations but prevented the calculation of standard error for the European populationestimate Bootstrapping with 1000 samples to determine the mean standard index of association fromSNP allele data was conducted with sampia whereas the standard index of association and one-sidedpermutation test were calculated directly for microsatellite genotypes with ia

Bruvorsquos genetic distance (which incorporates the number of repeats at a locus rather than justdifferences) for North American microsatellite genotypes was determined with popprrsquos bruvodistfunction A pairwise geographic distance matrix between sampling locations was created with the Rpackage geosphere v15-5 (71) The Mantel test was conducted with the R package ade4 v17-5 (72)Significance for this test was determined with 99 replicates of a Monte Carlo permutation test

Accession number(s) Accession numbers for the sequences of the isolates examined in this studyare listed in Tables 1 and 2

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at httpsdoiorg101128mBio

01941-17TABLE S1 XLS file 01 MB

ACKNOWLEDGMENTSFunding was provided by the US Geological Survey the US Fish amp Wildlife Service

(grant no 50120-B-G004) Bat Conservation International and the National Science

Drees et al reg

NovemberDecember 2017 Volume 8 Issue 6 e01941-17 mbioasmorg 12

FoundationmdashEcology of Infectious Diseases (DEB-1115895 and DEB-1336290 to AMKand JTF)

We thank Hugh Broders Karen Vanderwolf Kate Langwig and Chris Dobony forgenerously sharing isolates of P destructans used in this analysis Stephanie RivasLindsey Lovell John Gillece Remy Hilsabeck James Schupp Lela Andrews StephenBeckstrom-Sternberg Jamie Beckstrom-Sternberg Nicolette Janke Adina Doyle Sa-brina German Colin Sobek and Adam Triplett provided invaluable technical support

The use of trade product or firm names is for descriptive purposes only and doesnot imply endorsement by the US Government

REFERENCES1 Fisher MC Henk DA Briggs CJ Brownstein JS Madoff LC McCraw SL

Gurr SJ 2012 Emerging fungal threats to animal plant and ecosystemhealth Nature 484186 ndash194 httpsdoiorg101038nature10947

2 Ellison AM Bank MS Clinton BD Colburn EA Elliott K Ford CR Foster DRKloeppel BD Knoepp JD Lovett GM Mohan J Orwig DA RodenhouseNL Sobczak WV Stinson KA Stone JK Swan CM Thompson J Von HolleB Webster JR 2005 Loss of foundation species consequences for thestructure and dynamics of forested ecosystems Front Ecol Environ3479ndash486 httpsdoiorg1018901540-9295(2005)003[0479LOFSCF]20CO2

3 Loo JA 2009 Ecological impacts of non-indigenous invasive fungi asforest pathogens Biol Invasions 1181ndash96 httpsdoiorg101007s10530-008-9321-3

4 Berger L Speare R Daszak P Green DE Cunningham AA Goggin CLSlocombe R Ragan MA Hyatt AD McDonald KR Hines HB Lips KRMarantelli G Parkes H 1998 Chytridiomycosis causes amphibian mor-tality associated with population declines in the rain forests of Australiaand Central America Proc Natl Acad Sci U S A 959031ndash9036 httpsdoiorg101073pnas95159031

5 Bohuski E Lorch JM Griffin KM Blehert DS 2015 TaqMan real-timepolymerase chain reaction for detection of Ophidiomyces ophiodiicolathe fungus associated with snake fungal disease BMC Vet Res 1195httpsdoiorg101186s12917-015-0407-8

6 Stegen G Pasmans F Schmidt BR Rouffaer LO Van Praet S Schaub MCanessa S Laudelout A Kinet T Adriaensen C Haesebrouck F Bert WBossuyt F Martel A 2017 Drivers of salamander extirpation mediatedby Batrachochytrium salamandrivorans Nature 544353ndash356 httpsdoiorg101038nature22059

7 Skerratt LF Berger L Speare R Cashins S McDonald KR Phillott ADHines HB Kenyon N 2007 Spread of chytridiomycosis has caused therapid global decline and extinction of frogs Ecohealth 4125ndash134 httpsdoiorg101007s10393-007-0093-5

8 Blehert DS Hicks AC Behr M Meteyer CU Berlowski-Zier BM Buckles ELColeman JT Darling SR Gargas A Niver R Okoniewski JC Rudd RJ StoneWB 2009 Bat white-nose syndrome an emerging fungal pathogenScience 323227 httpsdoiorg101126science1163874

9 Frick WF Puechmaille SJ Hoyt JR Nickel BA Langwig KE Foster JTBarlow KE Bartonicka T Feller D Haarsma A-J Herzog C Horaacutecek I vander Kooij J Mulkens B Petrov B Reynolds R Rodrigues L Stihler CWTurner GG Kilpatrick AM 2015 Disease alters macroecological patternsof North American bats Glob Ecol Biogeogr 24741ndash749 httpsdoiorg101111geb12290

10 Langwig KE Frick WF Reynolds R Parise KL Drees KP Hoyt JR Cheng TLKunz TH Foster JT Kilpatrick AM 2015 Host and pathogen ecologydrive the seasonal dynamics of a fungal disease white-nose syndromeProc Biol Sci 28220142335 httpsdoiorg101098rspb20142335

11 Frick WF Pollock JF Hicks AC Langwig KE Reynolds DS Turner GGButchkoski CM Kunz TH 2010 An emerging disease causes regionalpopulation collapse of a common North American bat species Science329679 ndash 682 httpsdoiorg101126science1188594

12 Langwig KE Frick WF Bried JT Hicks AC Kunz TH Kilpatrick AM 2012Sociality density-dependence and microclimates determine the persis-tence of populations suffering from a novel fungal disease white-nosesyndrome Ecol Lett 151050 ndash1057 httpsdoiorg101111j1461-0248201201829x

13 Kunz TH Braun de Torrez E Bauer D Lobova T Fleming TH 2011Ecosystem services provided by bats Ann N Y Acad Sci 12231ndash38httpsdoiorg101111j1749-6632201106004x

14 Rachowicz LJ Hero J-M Alford RA Taylor JW Morgan JAT VredenburgVT Collins JP Briggs CJ 2005 The novel and endemic pathogenhypotheses competing explanations for the origin of emerging infec-tious diseases of wildlife Conserv Biol 191441ndash1448 httpsdoiorg101111j1523-1739200500255x

15 Kilpatrick AM Briggs CJ Daszak P 2010 The ecology and impact ofchytridiomycosis an emerging disease of amphibians Trends Ecol Evol25109 ndash118 httpsdoiorg101016jtree200907011

16 Tibayrenc M Ayala FJ 2012 Reproductive clonality of pathogens aperspective on pathogenic viruses bacteria fungi and parasitic proto-zoa Proc Natl Acad Sci U S A 109E3305ndashE3313 httpsdoiorg101073pnas1212452109

17 Chaturvedi V Springer DJ Behr MJ Ramani R Li X Peck MK Ren P BoppDJ Wood B Samsonoff WA Butchkoski CM Hicks AC Stone WB RuddRJ Chaturvedi S 2010 Morphological and molecular characterizations ofpsychrophilic fungus Geomyces destructans from New York bats withwhite nose syndrome (WNS) PLoS One 5e10783 httpsdoiorg101371journalpone0010783

18 Meteyer CU Buckles EL Blehert DS Hicks AC Green DE Shearn-BochslerV Thomas NJ Gargas A Behr MJ 2009 Histopathologic criteria toconfirm white-nose syndrome in bats J Vet Diagn Invest 21411ndash 414httpsdoiorg101177104063870902100401

19 Palmer JM Kubatova A Novakova A Minnis AM Kolarik M Lindner DL2014 Molecular characterization of a heterothallic mating system inPseudogymnoascus destructans the fungus causing white-nose syn-drome of bats G3 41755ndash1763 httpsdoiorg101534g3114012641

20 Khankhet J Vanderwolf KJ McAlpine DF McBurney S Overy DP SlavicD Xu J 2014 Clonal expansion of the Pseudogymnoascus destructansgenotype in North America is accompanied by significant variation inphenotypic expression PLoS One 9e104684 httpsdoiorg101371journalpone0104684

21 Ren P Haman KH Last LA Rajkumar SS Keel MK Chaturvedi V 2012Clonal spread of Geomyces destructans among bats midwestern andsouthern United States Emerg Infect Dis 18883ndash 885 httpsdoiorg103201eid1805111711

22 Pearson T Okinaka RT Foster JT Keim P 2009 Phylogenetic understand-ing of clonal populations in an era of whole genome sequencing InfectGenet Evol 91010 ndash1019 httpsdoiorg101016jmeegid200905014

23 Van Belkum A 2007 Tracing isolates of bacterial species by multilocusvariable number of tandem repeat analysis (MLVA) FEMS Immunol MedMicrobiol 4922ndash27 httpsdoiorg101111j1574-695X200600173x

24 Etienne KA Gillece J Hilsabeck R Schupp JM Colman R Lockhart SRGade L Thompson EH Sutton DA Neblett-Fanfair R Park BJ TurabelidzeG Keim P Brandt ME Deak E Engelthaler DM 2012 Whole genomesequence typing to investigate the Apophysomyces outbreak following atornado in Joplin Missouri 2011 PLoS One 7e49989 httpsdoiorg101371journalpone0049989

25 Hoyt JR Sun K Parise KL Lu G Langwig KE Jiang TT Yang S Frick WFKilpatrick AM Foster JT Feng J 2016 Widespread bat white-nose syn-drome fungus northeastern China Emerg Infect Dis 22140 ndash142 httpsdoiorg103201eid2201151314

26 Drees KP Palmer JM Sebra R Lorch JM Chen C Wu CC Bok JW KellerNP Blehert DS Cuomo CA Lindner DL Foster JT 2016 Use of multiplesequencing technologies to produce a high-quality genome of thefungus Pseudogymnoascus destructans the causative agent of bat white-nose syndrome Genome Announc 4e00445-16 httpsdoiorg101128genomeA00445-16

27 Minnis AM Lindner DL 2013 Phylogenetic evaluation of Geomyces and

Genomics of White-Nose Syndrome Invasion reg

NovemberDecember 2017 Volume 8 Issue 6 e01941-17 mbioasmorg 13

allies reveals no close relatives of Pseudogymnoascus destructans combnov in bat hibernacula of eastern North America Fungal Biol 117638 ndash 649 httpsdoiorg101016jfunbio201307001

28 Leopardi S Blake D Puechmaille SJ 2015 White-nose syndrome fungusintroduced from Europe to North America Curr Biol 25R217ndashR219httpsdoiorg101016jcub201501047

29 Agapow P-M Burt A 2001 Indices of multilocus linkage disequilibriumMol Ecol Notes 1101ndash102 httpsdoiorg101046j1471-8278200000014x

30 Puechmaille SJ 2016 The program Structure does not reliably recoverthe correct population structure when sampling is uneven subsamplingand new estimators alleviate the problem Mol Ecol Resour 16608 ndash 627httpsdoiorg1011111755-099812512

31 Rajkumar SS Li X Rudd RJ Okoniewski JC Xu J Chaturvedi S ChaturvediV 2011 Clonal genotype of Geomyces destructans among bats withwhite nose syndrome New York USA Emerg Infect Dis 171273ndash1276httpsdoiorg103201eid1707102056

32 Trivedi J Lachapelle J Vanderwolf KJ Misra V Willis CKR Ratcliffe JMNess RW Anderson JB Kohn LM 2017 Fungus causing white-nosesyndrome in bats accumulates genetic variability in North America withno sign of recombination mSphere 2e00271-17 httpsdoiorg101128mSphereDirect00271-17

33 Fontaine MC Austerlitz F Giraud T Labbeacute F Papura D Richard-CerveraS Delmotte F 2013 Genetic signature of a range expansion and leap-frog event after the recent invasion of Europe by the grapevine downymildew pathogen Plasmopara viticola Mol Ecol 222771ndash2786 httpsdoiorg101111mec12293

34 Gladieux P Feurtey A Hood ME Snirc A Clavel J Dutech C Roy MGiraud T 2015 The population biology of fungal invasions Mol Ecol241969 ndash1986 httpsdoiorg101111mec13028

35 Eppinger M Pearson T Koenig SS Pearson O Hicks N Agrawal S SanjarF Galens K Daugherty S Crabtree J Hendriksen RS Price LB UpadhyayBP Shakya G Fraser CM Ravel J Keim PS 2014 Genomic epidemiologyof the Haitian cholera outbreak a single introduction followed by rapidextensive and continued spread characterized the onset of the epi-demic mBio 5e01721 httpsdoiorg101128mBio01721-14

36 Frost SD Volz EM 2013 Modelling tree shape and structure in viralphylodynamics Philos Trans R Soc Lond B Biol Sci 36820120208 httpsdoiorg101098rstb20120208

37 Poon AFY Walker LW Murray H McCloskey RM Harrigan PR Liang RH2013 Mapping the shapes of phylogenetic trees from human andzoonotic RNA viruses PLoS One 8e78122 httpsdoiorg101371journalpone0078122

38 Dutech C Barregraves B Bridier J Robin C Milgroom MG Ravigneacute V 2012 Thechestnut blight fungus world tour successive introduction events fromdiverse origins in an invasive plant fungal pathogen Mol Ecol 213931ndash3946 httpsdoiorg101111j1365-294X201205575x

39 Zukal J Bandouchova H Brichta J Cmokova A Jaron KS Kolarik MKovacova V Kubaacutetovaacute A Novaacutekovaacute A Orlov O Pikula J Presetnik P ŠubaJ Zahradniacutekovaacute A Jr Martiacutenkovaacute N 2016 White-nose syndrome withoutborders Pseudogymnoascus destructans infection tolerated in Europeand Palearctic Asia but not in North America Sci Rep 619829 httpsdoiorg101038srep19829

40 Tibayrenc M Kjellberg F Arnaud J Oury B Breniegravere SF Dardeacute ML AyalaFJ 1991 Are eukaryotic microorganisms clonal or sexual A populationgenetics vantage Proc Natl Acad Sci U S A 885129 ndash5133 httpsdoiorg101073pnas88125129

41 Warnecke L Turner JM Bollinger TK Lorch JM Misra V Cryan PMWibbelt G Blehert DS Willis CKR 2012 Inoculation of bats with Euro-pean Geomyces destructans supports the novel pathogen hypothesis forthe origin of white-nose syndrome Proc Natl Acad Sci U S A 1096999 ndash7003 httpsdoiorg101073pnas1200374109

42 Puechmaille SJ Verdeyroux P Fuller H Gouilh MA Bekaert M Teeling EC2010 White-nose syndrome fungus (Geomyces destructans) in batFrance Emerg Infect Dis 16290 ndash293 httpsdoiorg103201eid1602091391

43 Wibbelt G Kurth A Hellmann D Weishaar M Barlow A Veith M PruumlgerJ Goumlrfoumll T Grosche L Bontadina F Zoumlphel U Seidl HP Seidl HP BlehertDS 2010 White-nose syndrome fungus (Geomyces destructans) in batsEurope Emerg Infect Dis 161237ndash1243 httpsdoiorg103201eid1608100002

44 Barlow AM Worledge L Miller H Drees KP Wright P Foster JT Sobek CBorman AM Fraser M 2015 First confirmation of Pseudogymnoascus

destructans in British bats and hibernacula Vet Rec 17773 httpsdoiorg101136vr102923

45 Paiva-Cardoso MdN Morinha F Barros P Vale-Gonccedilalves H Coelho ACFernandes L Travassos P Faria AS Bastos E Santos M Cabral JA 2014First isolation of Pseudogymnoascus destructans in bats from PortugalEur J Wildl Res 60645ndash 649 httpsdoiorg101007s10344-014-0831-2

46 Pavlinic I ETHakovic M Lojkic I 2015 Pseudogymnoascus destructans inCroatia confirmed Eur J Wildl Res 61325ndash328 httpsdoiorg101007s10344-014-0885-1

47 Bandouchova H Bartonicka T Berkova H Brichta J Cerny J Kovacova VKolarik M Koumlllner B Kulich P Martiacutenkovaacute N Rehak Z Turner GG ZukalJ Pikula J 2015 Pseudogymnoascus destructans evidence of virulent skininvasion for bats under natural conditions Europe Transbound EmergDis 621ndash5 httpsdoiorg101111tbed12282

48 Pikula J Bandouchova H Novotny L Meteyer CU Zukal J Irwin NR ZimaJ Martiacutenkovaacute N 2012 Histopathology confirms white-nose syndrome inbats in Europe J Wildl Dis 48207ndash211 httpsdoiorg1075890090-3558-481207

49 Puechmaille SJ Wibbelt G Korn V Fuller H Forget F Muumlhldorfer K KurthA Bogdanowicz W Borel C Bosch T Cherezy T Drebet M Goumlrfoumll T HaarsmaAJ Herhaus F Hallart G Hammer M Jungmann C Le Bris Y Lutsar L MasingM Mulkens B Passior K Starrach M Wojtaszewski A Zoumlphel U Teeling EC2011 Pan-European distribution of white-nose syndrome fungus (Geomycesdestructans) not associated with mass mortality PLoS One 6e19167 httpsdoiorg101371journalpone0019167

50 Puechmaille SJ Frick WF Kunz TH Racey PA Voigt CC Wibbelt GTeeling EC 2011 White-nose syndrome is this emerging disease athreat to European bats Trends Ecol Evol 26570 ndash576 httpsdoiorg101016jtree201106013

51 Rosenblum EB James TY Zamudio KR Poorten TJ Ilut D Rodriguez DEastman JM Richards-Hrdlicka K Joneson S Jenkinson TS Longcore JEParra Olea G Toledo LF Arellano ML Medina EM Restrepo S FlechasSV Berger L Briggs CJ Stajich JE 2013 Complex history of theamphibian-killing chytrid fungus revealed with genome resequenc-ing data Proc Natl Acad Sci U S A 1109385ndash9390 httpsdoiorg101073pnas1300130110

52 Knaus BJ Tabima JF Davis CE Judelson HS Gruumlnwald NJ 2016 Genomicanalyses of dominant US clonal lineages of Phytophthora infestansreveals a shared common ancestry for clonal lineages US11 and US18and a lack of recently shared ancestry among all other US lineagesPhytopathology 1061393ndash1403 httpsdoiorg101094PHYTO-10-15-0279-R

53 Gargas A Trest MT Christensen M Volk TJ Blehert DS 2009 Geomycesdestructans sp nov associated with bat white-nose syndrome Myco-taxon 108147ndash154 httpsdoiorg105248108147

54 Shuey MM Drees KP Lindner DL Keim P Foster JT 2014 Highlysensitive quantitative PCR for the detection and differentiation of Pseu-dogymnoascus destructans and other Pseudogymnoascus species ApplEnviron Microbiol 801726 ndash1731 httpsdoiorg101128AEM02897-13

55 Gillece JD Schupp JM Balajee SA Harris J Pearson T Yan Y Keim PDeBess E Marsden-Haug N Wohrle R Engelthaler DM Lockhart SR2011 Whole genome sequence analysis of Cryptococcus gattii from thePacific Northwest reveals unexpected diversity PLoS One 6e28550httpsdoiorg101371journalpone0028550

56 Kozarewa I Turner DJ 2011 96-plex molecular barcoding for the Illu-mina genome analyzer p 279 ndash298 Humana Press New York NYhttpsdoiorg101007978-1-61779-089-8_20

57 Kapa Biosystems 2012 High-throughput NGS library preparation tech-nical guide Illumina platforms KR0427 112 Kapa Biosystems Wilming-ton MA

58 Sahl JW Lemmer D Travis J Schupp JM Gillece JD Aziz M Driebe EMDrees KP Hicks ND Williamson CHD Hepp CM Smith DE Roe CEngelthaler DM Wagner DM Keim P 2016 NASP an accurate rapidmethod for the identification of SNPs in WGS datasets that supportsflexible input and output formats Microbial Genomics 2e000074httpsdoiorg101099mgen0000074

59 Li H Durbin R 2010 Fast and accurate long-read alignment withBurrows-Wheeler transform Bioinformatics 26589 ndash595 httpsdoiorg101093bioinformaticsbtp698

60 McKenna A Hanna M Banks E Sivachenko A Cibulskis K Kernytsky AGarimella K Altshuler D Gabriel S Daly M DePristo MA 2010 TheGenome Analysis Toolkit a MapReduce framework for analyzing next-generation DNA sequencing data Genome Res 201297ndash1303 httpsdoiorg101101gr107524110

Drees et al reg

NovemberDecember 2017 Volume 8 Issue 6 e01941-17 mbioasmorg 14

61 Van der Auwera GA Carneiro MO Hartl C Poplin R del Angel GLevy-Moonshine A Jordan T Shakir K Roazen D Thibault J Banks EGarimella KV Altshuler D Gabriel S DePristo MA 2013 From FastQ datato high-confidence variant calls the Genome Analysis Toolkit best prac-tices pipeline Curr Protoc Bioinformatics 4311101ndash111033 httpsdoiorg1010020471250953bi1110s43

62 DePristo MA Banks E Poplin R Garimella KV Maguire JR Hartl CPhilippakis AA del Angel G Rivas MA Hanna M McKenna A Fennell TJKernytsky AM Sivachenko AY Cibulskis K Gabriel SB Altshuler D DalyMJ 2011 A framework for variation discovery and genotyping usingnext-generation DNA sequencing data Nat Genet 43491ndash 498 httpsdoiorg101038ng806

63 Kurtz S Phillippy A Delcher AL Smoot M Shumway M Antonescu CSalzberg SL 2004 Versatile and open software for comparing largegenomes Genome Biol 5R12 httpsdoiorg101186gb-2004-5-2-r12

64 Cingolani P Platts A Wang LL Coon M Nguyen T Wang L Land SJ LuX Ruden DM 2012 A program for annotating and predicting the effectsof single nucleotide polymorphisms SnpEff SNPs in the genome ofDrosophila melanogaster strain w1118 iso-2 iso-3 Fly 680 ndash92 httpsdoiorg104161fly19695

65 Drees KP Parise KL Rivas SM Felton LL Puechmaille SJ Keim P FosterJT 2017 Characterization of microsatellites in Pseudogymnoascus

destructans for white-nose syndrome genetic analysis J Wildl Dis53869 ndash 874 httpsdoiorg1075892016-09-217

66 Swofford DL 2003 PAUP phylogenetic analysis using parsimony ( andother methods) version 40 b10 Sinauer Associates Sunderland MA

67 Stamatakis A Blagojevic F Nikolopoulos DS Antonopoulos CD 2007Exploring new search algorithms and hardware for phylogeneticsRAxML meets the IBM cell J VLSI Signal Process Syst Signal Image VideoTechnol 48271ndash286 httpsdoiorg101007s11265-007-0067-4

68 Drummond AJ Rambaut A 2007 BEAST Bayesian evolutionary analysisby sampling trees BMC Evol Biol 7214 httpsdoiorg1011861471-2148-7-214

69 Schliep KP 2011 phangorn phylogenetic analysis in R Bioinformatics27592ndash593 httpsdoiorg101093bioinformaticsbtq706

70 Kamvar ZN Brooks JC Gruumlnwald NJ 2015 Novel R tools for analysis ofgenome-wide population genetic data with emphasis on clonality FrontGenet 6208 httpsdoiorg103389fgene201500208

71 Karney CFF 2013 Algorithms for geodesics J Geodesy 8743ndash55 httpsdoiorg101007s00190-012-0578-z

72 Chessel D Dufour AB Thioulouse J 2004 The ade4 packagemdashI one-table methods R News 45ndash10

73 Palmer JM Drees KP Foster JT Lindner DL Extreme sensitivity toultra-violet light in the fungal pathogen causing white-nose syndromeof bats Nature Communications in press

Genomics of White-Nose Syndrome Invasion reg

NovemberDecember 2017 Volume 8 Issue 6 e01941-17 mbioasmorg 15