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Title:
Phylogenetics of a fungal invasion: origins and widespread dispersal of white-nose syndrome
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mBio
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Drees, Kevin P.
Lorch, Jeffrey M.
Puechmaille, Sebastien J.
Parise, Katy L.
Wibbelt, Gudrun
Hoyt, Joseph R.
Sun, Keping
Jargalsaikhan, Ariunbold
Dalannast, Munkhnast
Palmer, Jonathan M.
Lindner, Daniel L.
Kilpatrick, A. Marm
Pearson, Talima
Keim, Paul S.
Blehert, David S.
Foster, Jeffrey T.
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American Society for Microbiology
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Phylogeny ( lcsh )
White-nose syndrome ( lcsh )
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Globalization has facilitated the worldwide movement and introduction of pathogens, but epizoological reconstructions of these invasions are often hindered by limited sampling and insufficient genetic resolution among isolates. Pseudogymnoascus destructans, a fungal pathogen causing the epizootic of white-nose syndrome in North American bats, has exhibited few genetic polymorphisms in previous studies, presenting challenges for both epizoological tracking of the spread of this fungus and for determining its evolutionary history. We used single nucleotide polymorphisms (SNPs) from whole-genome sequencing and microsatellites to construct high-resolution phylogenies of P. destructans. Shallow genetic diversity and the lack of geographic structuring among North American isolates support a recent introduction followed by expansion via clonal reproduction across the epizootic zone. Moreover, the genetic relationships of isolates within North America suggest widespread mixing and long-distance movement of the fungus. Genetic diversity among isolates of P. destructans from Europe was substantially higher than in those from North America. However, genetic distance between the North American isolates and any given European isolate was similar to the distance between the individual European isolates. In contrast, the isolates we examined from Asia were highly divergent from both European and North American isolates. Although the definitive source for introduction of the North American population has not been conclusively identified, our data support the origin of the North American invasion by P. destructans from Europe rather than Asia.
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Volume 8, Issue 6

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PhylogeneticsofaFungalInvasion: OriginsandWidespreadDispersalof White-NoseSyndromeKevinP.Drees,a,bJeffreyM.Lorch,cSebastienJ.Puechmaille,d* KatyL.Parise,a,bGudrunWibbelt,eJosephR.Hoyt,fKepingSun,gAriunboldJargalsaikhan,h,iMunkhnastDalannast,i JonathanM.Palmer,jDanielL.Lindner,jA.MarmKilpatrick,fTalimaPearson,a PaulS.Keim,aDavidS.Blehert,c JeffreyT.Fostera,bPathogenandMicrobiomeInstitute,NorthernArizonaUniversity,Flagstaff,Arizona,USAa;Departmentof Molecular,CellularandBiomedicalSciences,UniversityofNewHampshire,Durham,NewHampshire,USAb; U.S.GeologicalSurvey,NationalWildlifeHealthCenter,Madison,Wisconsin,USAc;SchoolofBiologicaland EnvironmentalSciences,UniversityCollegeDublin,Beleld,Dublin,Irelandd;LeibnizInstituteforZooand WildlifeResearch,Berlin,Germanye;DepartmentofEcologyandEvolutionaryBiology,UniversityofCalifornia, SantaCruz,California,USAf;UrbanandEnvironmentalScienceCollege,ChangchunNormalUniversity, Changchun,PeoplesRepublicofChinag;DepartmentofBiology,SchoolofMathematicsandNaturalSciences, TheMongolianNationalUniversityofEducation,Ulaanbaatar,Mongoliah;BatsResearchCenterofMongolia, Ulaanbaatar,Mongoliai;U.S.ForestService,NorthernResearchStation,CenterforForestMycologyResearch, Madison,Wisconsin,USAjABSTRACTGlobalizationhasfacilitatedtheworldwidemovementandintroduction ofpathogens,butepizoologicalreconstructionsoftheseinvasionsareoftenhinderedbylimitedsamplingandinsufcientgeneticresolutionamongisolates. Pseudogymnoascusdestructans,afungalpathogencausingtheepizooticofwhite-nose syndromeinNorthAmericanbats,hasexhibitedfewgeneticpolymorphismsinpreviousstudies,presentingchallengesforbothepizoologicaltrackingofthespreadof thisfungusandfordeterminingitsevolutionaryhistory.Weusedsinglenucleotide polymorphisms(SNPs)fromwhole-genomesequencingandmicrosatellitestoconstructhigh-resolutionphylogeniesof P.destructans.Shallowgeneticdiversityand thelackofgeographicstructuringamongNorthAmericanisolatessupportarecent introductionfollowedbyexpansionviaclonalreproductionacrosstheepizootic zone.Moreover,thegeneticrelationshipsofisolateswithinNorthAmericasuggest widespreadmixingandlong-distancemovementofthefungus.Geneticdiversity amongisolatesof P.destructans fromEuropewassubstantiallyhigherthaninthose fromNorthAmerica.However,geneticdistancebetweentheNorthAmericanisolatesandanygivenEuropeanisolatewassimilartothedistancebetweentheindividualEuropeanisolates.Incontrast,theisolatesweexaminedfromAsiawere highlydivergentfrombothEuropeanandNorthAmericanisolates.Althoughthede- nitivesourceforintroductionoftheNorthAmericanpopulationhasnotbeenconclusivelyidentied,ourdatasupporttheoriginoftheNorthAmericaninvasionby P.destructans fromEuroperatherthanAsia.IMPORTANCEThisphylogeneticstudyofthebatwhite-nosesyndromeagent, P.destructans,usesgenomicstoelucidateevolutionaryrelationshipsamongpopulationsofthefungalpathogentounderstandtheepizoologyofthisbiologicalinvasion.Weanalyzehypervariableandabundantgeneticcharacters(microsatellitesand genomicSNPs,respectively)torevealpreviouslyuncharacterizeddiversityamong populationsofthepathogenfromNorthAmericaandEurasia.WepresentnewevidencesupportingrecentintroductionofthefungustoNorthAmericafromadiverse Eurasianpopulation,withlimitedincreaseingeneticvariationinNorthAmerica sincethatintroduction.Received 31October2017 Accepted 2 November2017 Published 12December 2017 Citation DreesKP,LorchJM,PuechmailleSJ, PariseKL,WibbeltG,HoytJR,SunK, JargalsaikhanA,DalannastM,PalmerJM, LindnerDL,MarmKilpatrickA,PearsonT,Keim PS,BlehertDS,FosterJT.2017.Phylogeneticsof afungalinvasion:originsandwidespread dispersalofwhite-nosesyndrome.mBio 8:e01941-17. https://doi.org/10.1128/mBio .01941-17. Editor Joseph Heitman,DukeUniversity Copyright 2017Dreesetal.Thisisanopenaccessarticledistributedunderthetermsof the CreativeCommonsAttribution4.0 International license. Address correspondencetoJeffreyT.Foster, jeff.foster@nau.edu. Presentaddress:SebastienJ.Puechmaille, ZoologicalInstitute&Museum,Greifswald University,Greifswald,Germany. Thisarticleisadirectcontributionfroma FellowoftheAmericanAcademyof Microbiology.Solicitedexternalreviewers: MatthewFisher,ImperialCollegeLondon; JasonStajich,UniversityofCalifornia,Riverside.RESEARCHARTICLEcrossm November/December2017Volume8Issue6e01941-17 mbio.asm.org 1 mbio.asm.org on May 23, 2018 Published by mbio.asm.org Downloaded from

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KEYWORDSChiroptera, Pseudogymnoascusdestructans ,emerginginfectiousdisease, epizootic,microsatellite,whole-genomesequencing,wildlifeFungaldiseasesareemergingasmajorthreatstoecosystemintegrityatlocal, regional,andgloballevels(1).Forexample,introducedfungalphytopathogens such as Cryphonectriaparasitica (agentofchestnutblight)and Cronartiumribicola (agentofwhitepineblisterrust)havecauseddramaticdeclinesinfoundationtree speciesofAmericanforests,withsevereimplicationsforregionalforeststructureand foodwebs( 2, 3).Severalfungalpathogensofvertebrateshavealsorecentlyemerged and areofmajorconservationconcernforwildlife,including Batrachochytriumdendrobatidis and Batrachochytriumsalamandrivorans (agentsofamphibianchytridiomycosis), aswellas Ophidiomycesophiodiicola (agentofsnakefungaldisease)( 4).Inparticular, the ongoingchytridiomycosispanzooticranksamongthemostdevastatingwildlife diseasesduetoitsbroadhostrange,highvirulence,andrapidworldwideinvasion( 7). In 2006,anotherseverefungalepizootic,white-nosesyndrome(WNS),emergedamong hibernatingbatsinupstateNewYorkintheUnitedStates.Thisdiseasewasnamedfor thewhitegrowthofthepsychrophilicfungus Pseudogymnoascusdestructans onthe skin,especiallymuzzles,ofinfectedbats( 8).Thepathogenhascolonizednearlyall surveyed bathibernaculaintheeasternUnitedStatesandCanadaoverthelast10years ( 9, 10).Millionsofbatshavediedasaresultofthisdisease,andseveralspeciesare threatened withextinction,including Myotisseptentrionalis (northernlong-earedbat), theonceplentiful Myotislucifugus (littlebrownbat),andtheendangered Myotissodalis (Indianabat)( 11, 12).Batsprovidewidespreadinsectsuppressionservicestonatural and agriculturalecosystemsandareprimarysuppliersofnutrientstouniquecave ecosystemsthroughdepositionofguano( 13).Thus,thiscatastrophicreductioninthe bat populationsofNorthAmericawilllikelyhavepervasiveecologicrepercussions. Rachowiczetal.( 14)proposedthenovelandendemicpathogenhypothesisforthe origin of B.dendrobatidis andemphasizeddifferencesinmanagementandresearch prioritiesforpathogensofdifferentorigins.Anovelpathogenpopulationwouldbe expectedtohavelimiteddiversitycomparedtoitssourcepopulation,reectinga recentintroductionanddemographicbottleneckoftheintroducedorganism( 14, 15). Conversely, apathogenwithgreaterdiversityimpliesalongernaturalhistoryinanarea andsupportstheendemicpathogenhypothesis.Manybacterial,viral,parasitic,and fungaldiseasesofepizoologicalimportancehaveexpandedclonally( 16),andgenetic analyses of P.destructans havebeenconsistentwiththispattern. Pseudogymnoascusdestructans primarilyreproducesasexually,producingabundant haploidconidia( 17, 18).Althoughitistheoreticallypossiblefor P. destructans to reproducesexuallyviaaheterothallicmatingsystem,thishasnotyetbeenobserved, andonlyoneoftworequiredmatingtypeshastodatebeendetectedinthegenomes ofNorthAmericanfungalisolates( 19).PreviousstudiesoftheoriginandspreadofWNS revealed littleornogeneticdiversityinNorthAmericanisolatesof P.destructans (20, 21).However,thesepreviousstudieswerelimitedbylowdiscriminatorypower.Only eight lociweresequenced,andtheydidnotexhibitsufcientpolymorphismsto identifytheexactsourcepopulationortodistinguishamongisolatesofarecently emergedpathogenduringaninfectiousdiseaseoutbreak( 22). In contrast,othergeneticmarkerssuchasmicrosatellites( 23)andwhole-genome single nucleotidepolymorphism(SNP)genotyping( 24)usemanylocifromthroughout the genometoincreasetheresolutionofgenotypes.Thehighvariabilityofmicrosatellitelocileadstomoreheterogeneitythanpointmutationsingenes,oftenallowingfor discriminationbetweencloselyrelatedorrecentlydivergedisolates.Furthermore,the hallmarkofwhole-genomeSNPgenotypingisthelargenumberofcharacters(ranging fromhundredstohundredsofthousandsofSNPlociinatypicalanalysis)thatcanbe usedtoconstructarobustphylogenyofclonalorganisms( 22). We reporttheresultsofahigh-resolutionphylogeneticanalysisof P.destructans usingbothmicrosatelliteandwhole-genomeSNPlocitocomplementthebenetsandDreesetal. November/December2017Volume8Issue6e01941-17 mbio.asm.org 2 mbio.asm.org on May 23, 2018 Published by mbio.asm.org Downloaded from

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minimizethedrawbacksofeachapproach.OuranalysisincludesisolatesfromthroughouttheWNSepizooticzoneinNorthAmericathatwerecollectedfrom2008to2014. WealsoincludedisolatesfromEurope,Mongolia,andChina(where P.destructans was recentlyfoundtobewidelydistributed[ 25])tomorethoroughlyinvestigatethe genetic diversityofputativeoriginpopulationsoftheNorthAmericanpopulation. RESULTS SNPphylogeny. Illumina paired-endwhole-genomeshotgunsequenceswereobtainedfor26isolatesof P.destructans fromtheNorthAmericanWNSepizooticzone fromtheyears2008to2014,5isolatesfromEurope,and3isolatesfromAsia.These reads,aswellaspubliclyavailable P.destructans andnear-relative Pseudogymnoascus and Geomyces speciesassemblies,werealignedtoareferencegenomefrom P.destructans 20631-21(NCBIaccessionno.GCA_001641265.1)( 26). Oidiodendron maius Zn (Ascomycota, Leotiomycetes)wasusedasanoutgroupbasedonourphylogenetic analysesoffungicloselyrelatedto Pseudogymnoascus (datanotshown).Descriptive statisticsofthealignments,aswellasNCBIaccessionnumbersofallsamplesanalyzed, aredisplayedin Table1.Duplicated/repeatregionsaccountedfor43.25%ofthe genome andwereexcludedfromourSNP-basedanalyses.Only~0.1%ofthereference genomewasorthologousinallsamples,resultingin4,757SNPloci,ofwhich2,479were synapomorphic(i.e.,sharedbetweenoneormoresamplesandusefultodeneclades). Maximumparsimonyanalysisresultedinfourtreeswithalowrescaledconsistency(RC) index(RCof0.4704),indicatingahighdegreeofhomoplasythatonewouldexpect fromadatasetrepresentingalongevolutionaryhistory(datanotshown).Maximum likelihoodanalysis( Fig.1)indicatedstrongbootstrapsupportforasingle P. destructans cladethatissistertootherallcurrentlyrecognizedmembersofthegenus Pseudogymnoascus,includingancientsamplesfromRussianpermafrost.Moreover,inthisanalysis, isolateJH15CN0111afromChinaisbasalto P.destructans isolatesfromMongolia, Europe,andNorthAmerica,allowingustorootthe P.destructans phylogeny( Fig.2). As previouslydeterminedbymultilocussequencetyping(MLST)( 27),membersof the genus Pseudogymnoascus arenotallcloselyrelatedtoeachother.Wethuslimited thesamplesettojust P.destructans andalignedonlyreadsfromthisspeciestoidentify SNPlocisharedspecicallyamong P.destructans isolates.Thisresultedinthedetection of37,752coreSNPlociwithinthe20-Mbcoregenomealignment.Maximumparsimony producedonetreewithanRCof0.7729(datanotshown).Maximumlikelihoodanalysis ( Fig.2)distinguishesthreewell-supportedcladesamongthe P. destructans isolates: China,Mongolia,andEurope.TheNorthAmericanisolatesarenearlyidenticalinthis treeandarethereforerepresentedasagroupbythetypeisolate20631-21,obtained inNewYorkin2008.ThisphylogenyclearlyshowstheNorthAmericancladeof P.destructans asamemberofagreaterEuropeancladeanddistinctfromtheeastern andcentralAsianisolates.ThetreealsoshowsthatNorthAmerican P.destructans isolateismostcloselyrelatedtoaUkrainianisolate(Gd44)inoursamplesetandleast relatedtoanisolatefromFrance(Gd41).ThisisincontrasttoapreviousMLSTstudy ( 28),whichshowedtheoppositetrendusingmanyofthesameisolates.However,low sample sizes,longbranchlengths,andpoorbootstrapsupportforthebranches betweenmembersoftheEuropeancladeprecludestrongerconclusionsaboutthe regionofEuropethatgaverisetoNorthAmerican P.destructans.Withourcurrentdata set,thepossibilitythatNorthAmerican P.destructans wasintroducedfromAsia, however,seemsunlikely. Wereducedthesamplesetonceagaintoincludeonlyisolatesof P.destructans from NorthAmericaandreanalyzedthealignmentsforSNPs.Only51coreSNPlociwere sharedamongthe26NorthAmericanisolates.Maximumparsimonyproducedonetree withanRCof1.00(datanotshown).Thus,allhomoplasyobservedamong P.destructans phylogenieswasduetotheEurasiangenomes.OnlythreeSNPsweresynapomorphic.TwoSNPsshowdifferencesbetweenthetypeisolate20631-21andallother samples.AsingleSNPsharedbysamples23414-1Wand23434-1W,bothobtainedin Indianain2011,suggesttheformationofageographicallyisolatedlineage.LessGenomicsofWhite-NoseSyndromeInvasion November/December2017Volume8Issue6e01941-17 mbio.asm.org 3 mbio.asm.org on May 23, 2018 Published by mbio.asm.org Downloaded from

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TABLE1 Whole-genomesequencesalignedtothereferencegenomeof Pseudogymnoascusdestructans (NCBIaccessionno. GCA_001641265.1) IsolateIDno.SpeciesaSourcebLocationcDate Read length (bp) Avgread depth %ofcore genome covereddAccessionno. 20631-21 P.destructansM.lucifugus NYFeb2008NAeNA100.00GCA_001641265.1 GU999986 P.destructansM.myotis DEUMar20092 101155.8398.73SRR6011467 GU350433 P.destructansM.myotis CHEApr20092 101187.9798.93SRR6011468 GU350434 P.destructansM.myotis HUNMar20092 101169.2999.00SRR6011465 20693-1 P.destructansM.lucifugus MAMar20082 101206.3999.79SRR6011466 22429-8 P.destructansM.septentrionalis WVJan20092 101208.6299.81SRR6011471 22971-3 P.destructansM.lucifugus ONMar20102 101123.2499.73SRR6011472 22504-1 P.destructansM.lucifugus PAMar20092 101192.7399.67SRR6011469 22442-2 P.destructansM.lucifugus NJFeb20092 101200.7999.71SRR6011470 22948-1 P.destructansM.septentrionalis TNMar20102 101180.4799.49SRR6011473 20674-9 P.destructansM.septentrionalis VTMar20082 98100.2599.46SRR6011474 20682-10 P.destructansM.septentrionalis MAMar20082 101156.0099.11SRR6011477 22004-1 P.destructansM.lucifugus CTApr20082 101213.7899.20SRR6011478 22426-2 P.destructansM.lucifugus CTJan20092 101170.4999.42SRR6011475 22469-1 P.destructansP.subavus VAMar20092 101167.0999.42SRR6011476 22480-1 P.destructansE.fuscus NYMar20092 9885.8599.43SRR6011481 22884-4W P.destructansM.lucifugus VTJan20102 101141.8899.43SRR6011482 22930-2 P.destructansP.subavus TNFeb20102 10182.9899.03SRR6011479 22949-4 P.destructansM.septentrionalis MDMar20102 103211.5999.54SRR6011480 22972-2W P.destructansM.lucifugus ONMar20102 10182.4899.03SRR6011483 22997-1 P.destructansM.septentrionalis TNApr20102 101271.2099.97SRR6011484 23414-1W P.destructansM.lucifugus INJan20112 98120.0099.59SRR6011493 23434-1W P.destructansM.lucifugus INJan20112 101255.0799.96SRR6011492 23444-1 P.destructansM.lucifugus TNFeb20112 9896.6799.49SRR6011491 23455-1 P.destructansM.lucifugus VAFeb20112 101163.1799.70SRR6011490 Gd41 P.destructansM.myotis FRAMar20092 25189.0499.31SRR6011497 Gd44 P.destructansM.myotis UKRFeb20112 30192.6898.71SRR6011496 23874-1 P.destructansM.lucifugus MEDec20112 101200.7699.97SRR6011495 23877-1 P.destructansM.septentrionalis DEMar20122 101177.3299.97SRR6011494 23897-2 P.destructansP.subavus MOMar20122 251119.8099.93SRR6011489 W41203 P.destructans SubNBApr20122 25174.3199.54SRR6011488 JH15CN0111a P.destructansM.petax CHNMar20152 15074.3196.78SRR6011485 JH16MG088 P.destructansP.ognevi MNG20162 15078.8497.31SRR6011486 JH16MG093 P.destructansP.ognevi MNG20162 15015.2853.10SRR6011487 03VT05 Pseudogymnoascus sp.SedVT2008NANA76.63GCA_001662645.1 UAMH10579 P.verrucosus PeatAB2002NANA76.92GCA_01662655.1 05NY08 Pseudogymnoascus sp.SedNY2008NANA76.68GCA_001662605.1 23342-1-I1 Pseudogymnoascus sp. P.subavus WI2008NANA61.20GCA_001662575.1 24MN13 Pseudogymnoascus sp.SedMN2008NANA69.28GCA_001662595.1 WSF3629 Pseudogymnoascus sp.PeatWI1960NANA77.02GCA_001662585.1 F-103 Pseudogymnoascus sp.SoilNYContemporaryNANA76.76GCA_000750895.1 F-3557 Pseudogymnoascus sp.PinepostSWEContemporaryNANA52.04GCA_000750665.1 F-3775 Pseudogymnoascus sp.SoilDEUContemporaryNANA50.60GCA_000750715.1 F-3808 Pseudogymnoascus sp. M.glareolus RUSContemporaryNANA52.51GCA_000750675.1 F-4246 Pseudogymnoascus sp.SedMNGContemporaryNANA53.77GCA_000750735.1 F-4281 Pseudogymnoascus sp.CryopegRUS0.12.2myafNANA57.09GCA_000750745.1 F-4513 Pseudogymnoascus sp.PermafrostRUS1.8.0myaNANA54.09GCA_000750755.1 F-4514 Pseudogymnoascus sp.PermafrostRUS1.8.0myaNANA51.80GCA_000750795.1 F-4515 Pseudogymnoascus sp.PermafrostRUS1.8.0myaNANA61.24GCA_000750805.1 F-4516 Pseudogymnoascus sp.PermafrostRUS1.8.0myaNANA53.86GCA_000750815.1 F-4517 Pseudogymnoascus sp.PermafrostRUS1.8.0myaNANA60.86GCA_000750875.1 F-4518 Pseudogymnoascus sp.SoilRUSContemporaryNANA67.47GCA_000750925.1 F-4519 Pseudogymnoascus sp.SoilRUSContemporaryNANA77.04GCA_000750935.1 F-4520 Pseudogymnoascus sp.SoilRUSContemporaryNANA67.01GCA_000750935.1 ATCC16222 P.pannorum SoilDEUNANANA58.08GCA_001630605.1 M1372 P.pannorum SoilCAJun1961NANA58.55GCA_000497305.1 Zn O.maius NANANANANA0.29GCA_000827325.1 aThespeciesshownare Pseudogymnoascusdestructans, Pseudogymnoascusverrucosus, Pseudogymnoascus sp., Pseudogymnoascuspannorum,and Oidiodendronmaius.bThespeciesshownare Myotismyotis, Myotislucifugus, Myotisseptentrionalis, Perimyotissubavus, Eptesicusfuscus, Myotispetax, Plecotusognevi,and Myodesglareolus (bankvole).Sub,hibernaculumsubstrate(i.e.,roostingsurface);Sed,hibernaculumsediment.cNY,NewYork;DEU,Germany;CHE,Chechnya;HUN,Hungary;MA,Massachusetts;WV,WestVirginia;ON,Ontario,Canada;PA,Pennsylvania;NJ,NewJersey;TN, Tennessee;VT,Vermont;CT,Connecticut;VA,Virginia;MD,Maryland;IN,Indiana;FRA,France;UKR,Ukraine;ME,Maine;DE,Delaware;MO,Missouri;NB,New Brunswick;CHN,China;MNG,Montenegro;AB,Alberta;WI,Wisconsin;MN,Minnesota;SWE,Sweden;RUS,Russia;CA,California.dCoveragereportedaspercentageofcoregenome(i.e.,portionofgenomesharedbyallsamplesthatisnotinaduplicatedregion)thatpassed10 minimumdepth ofcoverageand90%ofreadagreementlters.eNA,notavailable.fmya,millionyearsago. Dreesetal. November/December2017Volume8Issue6e01941-17 mbio.asm.org 4 mbio.asm.org on May 23, 2018 Published by mbio.asm.org Downloaded from

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conservativelteringstepsforSNPqualityandreaddepthandremovingtherequirementforalocustobepresentinallgenomesfailedtosubstantiallyincreasethe numberofsynapomorphies.Ofthe51SNPloci,noneofthemutationswereincoding sequences( Table2),butratherwerefromintergenicsequences,introns,oruntranslated regions(UTRs)ofexons. Isolatematingtypewasdeterminedfromwhole-genomesequencealignmentsto thereferencegenomesequenceoftypeisolate20631-21,whichwaspreviouslydeterminedtomatingtypeMAT1-1( 19).AllisolatesfromNorthAmericainthisstudy possessed theMAT1-1matingtype.MostEurasianisolatesdidaswell,althoughan isolatefromSwitzerland(GU350433)andanisolatefromMongolia(JH16MG088)were matingtypeMAT1-2. Microsatellitephylogeny. InsufcientsynapomorphicSNPswerediscovered amongtheNorthAmerican P.destructans whole-genomesequencestoproducean informativephylogeny,suchthatanalysesproducedonlyastar-shapedphylogeny (insetin Fig.3).Therefore,96NorthAmericanisolatesof P. destructans,includingthose analyzedbywhole-genomesequencing,weregenotypedwitha23-locusmicrosatellite panel(seeTableS1inthesupplementalmaterial).Thirty-oneuniquegenotypeswere identied,withonedominantgenotypefor13oftheisolates.Aphylogenywas constructedbytheneighbor-joining(NJ)methodfromthemicrosatellitedata( Fig.3). These data,althoughbaseduponfewergeneticloci,containmanymoresamplesand revealfarmoregeneticdiversitythantheSNP-basedphylogenyofNorthAmerican 0.008 Oidiodendron maiu s Pd Gd44 UKR 2011 Pd GU350433 CHE 2009 Pd GU350434 HUN 2009 Pd JH16MG088 MNG 2016 Pd 20631 21 NY 2008 Pd Gd41 FRA 2009 Pd JH16MG093 MNG 2016 Pd JH15CN0111a CHN 2015 Pd GU999986 DEU 2009 Psp 03VT05 VT 2008 Psp F3557 SWE contemporary Psp 23342 1 I1 WI 2008 Psp 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 200kya Pp M1372 CA 1961 Psp F4246 MNG contemporary Psp F4514 RUS 3mya Pp ATCC 16222 DEU contemporary Psp F3775 DEU contemporary Psp 05NY08 NY 2008 Psp F4517 RUS 3mya Psp 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 contemporary100 51 98 73 100 100 100 100 45 100 96 43 100 100 100 100 100 100 100 100 100 97 100 100 74Pseudogymnoascus destructans FIG1 Maximumlikelihoodphylogenetictreeofthegenus Pseudogymnoascus (previously Geomyces),basedupon4,757SNPs (ofwhich2,479aresynapomorphic).Branchesarelabeledwithbootstrapsupportvalues. Oidiodendronmaius servesas outgroup. Pseudogymnoascusdestructans isasistercladetoothermembersofthegenus.Psp, Pseudogymnoascus sp.;Pd, P.destructans;Pp, P.pannorum;Pv, P.verrucosus.Additionaldataforeachsampleincludeisolatename,country/stateoforigin, anddateofcollection(estimatedaspreservationdateforsamplestakenfrompermafrost).Sampleswithoutapreciselyknown collectiondateareindicatedascontemporary.GenomicsofWhite-NoseSyndromeInvasion November/December2017Volume8Issue6e01941-17 mbio.asm.org 5 mbio.asm.org on May 23, 2018 Published by mbio.asm.org Downloaded from

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P.destructans.Thistreesupportsthesinglecladecontainingisolates23431-1Wand 23434-1WfromIndianaidentiedinthewhole-genomephylogeny,butincludesa numberofotherisolatesthatdidnotsharesynapomorphicSNPswiththeIndiana isolates.Othercladesareevidentaswell,buttheydonotseemtocorrespondto geographicregionsorsamplingdates.AManteltestcomparingmicrosatellite-based geneticdistancetogeographicdistancebetweenisolatesshowednocorrelation( r 0.016, n 99replicates, P 0.37).Wenote,however,thatdespitetheappearanceof denedgroupingsthroughoutthetree,therewaslimitedbootstrapsupportforallof thebrancheswithinthisphylogeny(allbootstrapvalueswere 70). Populationgenetics. Indicesofgeneticdiversityandlinkagewerecalculatedfor SNPgenotypes.Asiansampleswereleftoutofthisanalysisduetothelowsamplesize. TheSimpsonsindexofgeneticdiversitywasreducedinNorthAmerica( 0.690, standarderror[SE] 0.130)comparedtoEurope( 0.800),theputativesource populationfor P.destructans.Averagegenomiclinkage,representedbystandardized indexofassociation( 29),wasclosetozeroforbothpopulations.AmongNorth American isolates, r D 0.0585,whereasforEurope, r D 0.00812.Thus,recombination doesnotappeartobeasignicantfactorineitherpopulationsgeneticstructure,and P.destructans isolatesinbothregionsarereproducingclonally.Theevennessofour samplingwasnotsufcient,nordidourdataconformtovariousmodelassumptionsto producereliablepopulationstructureestimates(usingmodelssuchasSTRUCTUREand SNMF)fromourSNPormicrosatellitedata( 30). DISCUSSION Phylogeography. The twogeneticmarkersusedinthisstudy,SNPsandmicrosatellites,providecomplementaryinformationregardingthephylogeographyof P.deGU350433 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 2016 1,517 [756 2,439] 103 [53 166] 1,972 [989 -3,165] 3,408 [1,689 5,452] 2,011 [1,006 3,227] 66 [33 107] FIG2 Maximumcladecredibilitytreeforisolatesof Pseudogymnoascusdestructans fromNorthAmerica(n 1), Europe(n 5),andAsia(n 3)basedupon37,752coreSNPsites(ofwhich22,594aresynapomorphic).Allnodes had100%posteriorprobabilities.Nodesarelabeledwithtime(yearsbeforepresent),followedbythe95% high-probabilitydensityintervalinbrackets;datesonallshorterbranchesarenotshown.NorthAmericanisolates arenearlyindistinguishableattheseSNPloci,andsowererepresentedinthistreebyasingleisolate(20631-21). ThetreeisrootedontheChineseisolatebaseduponthe Fig.1 phylogenythatshowsother Pseudogymnoascus spp. asasistercladeto P.destructans.Dataforeachsampleincludeisolatename,bathostspeciescode, country/stateoforigin,anddateofcollection.Dreesetal. November/December2017Volume8Issue6e01941-17 mbio.asm.org 6 mbio.asm.org on May 23, 2018 Published by mbio.asm.org Downloaded from

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structans.Whole-genomesequencingrevealedthreedistinctpopulationsof P.destructans amongtheisolateswetested:oneconsistingofEuropeanisolatesthatincluded WNSepizooticisolatesfromNorthAmerica,anotherofMongolianisolates,andathird representedbyanisolatefromChina.ThephylogenyofEuropeanisolatesdetermined inthisstudywith58,707SNPlociwasconsistentwiththatdescribedpreviouslyusing 14SNPsinaneight-membermultilocussequencetypinganalysis(MLST)( 28):specically, thegeneticdiversityof P.destructans was14%higherinEuropethaninNorth America.Inthisstudy,weidentiedonly51coreSNPsamongNorthAmericanisolates, whichclearlydemonstratesthehomogeneityof P.destructans populationstructure acrosstheepizooticzoneandisconsistentwithpreviousreportsofclonalspread( 20, 21, 28, 31, 32).NorthAmericanisolatesformedamonophyleticgroupbutappeartobe representative ofdiversityfromEurope(i.e.,thebranchlengthsbetweentheEuropean sampleswerecomparablebetweentheEuropeanandNorthAmericanisolates).This,as wellasthe99.995%reductionincoreSNPlociwhennon-Americanisolatesare removed,representsalossofgeneticdiversityconsistentwithpreviousconclusionsof arecentintroductionofanovelpathogen( 33, 34).Moreover,star-shapedphylogenies containing fewsynapomorphiesaretypicalinrecentpathogenintroductions( 35),TABLE2 AnnotationofcoreSNPlocifromisolatesof P.destructans fromNorthAmerica Accessionno.PositionTypeVariantisolate(s)ProteinIDno.Product KV441386.1 1202839UTRa20693-1_MA_2008OAF63413.1NADsynthase KV441386.1 2276958Intergenic22948-1_TN_2010NAbNA KV441395.1 783753UTR22948-1_TN_2010OAF58958.1Hypotheticalprotein KV441396.1 546602Intergenic23444-1_TN_2011NA NA KV441398.1 551631UTR23877-1_DE_2012OAF57976.1Hypotheticalprotein KV441399.1 235966Intergenic23414-1W_IN_2011, 23434-1W_IN_2011 NA NA KV441400.1 640730UTR22442-2_NJ_2009OAF57273.1Hypotheticalprotein KV441401.1 139816Intergenic22948-1_TN_2010NA NA KV441402.1 72139Intergenic23897-2_MO_2012NA NA KV441402.1 226767UTR22948-1_TN_2010OAF56852.1Hypotheticalprotein KV441402.1 510412Intergenic20693-1_MA_2008NA NA KV441404.1 262446UTR22442-2_NJ_2009OAF56419.1Hypotheticalprotein KV441387.1 430984Intergenic23874-1_ME_2012NA NA KV441387.1 820735UTR22426-2_CT_2009OAF62808.1Hypotheticalprotein KV441387.1 1825781Intergenic22948-1_TN_2010NA NA KV441387.1 2082665UTR22442-2_NJ_2009OAF62574.1ESCRT-IcomplexsubunitVPS28 KV441387.1 685734Intergenic22442-2_NJ_2009NA NA KV441387.1 785423Intergenic23897-2_MO_2012NA NA KV441387.1 54855Intergenic22971-3_ON_2010NA NA KV441387.1 193542Intergenic22948-1_TN_2010NA NA KV441387.1 253155Intergenic22442-2_NJ_2009NA NA KV441387.1 77282Intergenic23455-1_VA_2011NA NA KV441387.1 120236Intergenic23897-2_MO_2012NA NA KV441388.1 775461Intragenic22972-2W_ON_2010OAF61744.1Hypotheticalprotein KV441388.1 950597Intron23897-2_MO_2012OAF61915.1Hypotheticalprotein KV441388.1 120202UTR22469-1_VA_2009OAF62044.1Hypotheticalprotein KV441388.1 57283UTR20631-21_NY_2008OAF61813.1Hypotheticalprotein KV441389.1 343287Intron23455-1_VA_2011OAF61688.1AGC/AKTproteinkinase KV441389.1 419134Intergenic23414-1W_IN_2011NA NA KV441389.1 867065UTR22004-1_CT_2008OAF61368.1Actincytoskeleton-regulatorycomplexproteinend3 KV441389.1 1173430Intergenic22971-3_ON_2010NA NA KV441389.1 1173432Intergenic22971-3_ON_2010NA NA KV441389.1 50002UTR22948-1_TN_2010OAF61394.1Hypotheticalprotein KV441390.1 643943Intergenic23897-2_MO_2012NA NA KV441390.1 659862UTR22948-1_TN_2010OAF61216.1Hypotheticalprotein KV441390.1 861423UTR22948-1_TN_2010OAF60933.1Hypotheticalprotein KV441390.1 1243398UTR23434-1W_IN_2011OAF60771.1Hypotheticalprotein KV441390.1 1524359UTR22948-1_TN_2010OAF60872.1Hypotheticalprotein KV441391.1 151986UTR20693-1_MA_2008OAF60366.1Hypotheticalprotein KV441391.1 1184294Intergenic22442-2_NJ_2009NA NA KV441392.1 712894Intergenic23414-1W_IN_2011NA NA aUTR,untranslatedregionofexon.bNA,notavailable. GenomicsofWhite-NoseSyndromeInvasion November/December2017Volume8Issue6e01941-17 mbio.asm.org 7 mbio.asm.org on May 23, 2018 Published by mbio.asm.org Downloaded from

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FIG3 Phylogenyof96NorthAmerican Pseudogymnoascusdestructans isolatesconstructedfromallelesof23microsatelliteloci.Isolatesthatwereincluded inwhole-genomesequencephylogeniesarecoloredred.Thescalebarindicatesgeneticdistance.Shownisaneighbor-joiningtreeof96NorthAmerican isolatesrootedwithGU350434(host, M.myotis [MMYO];location,Hungary[HUN];date,2009).SamplesfromtheepicenteroftheWNSepizooticarecolored blue.(Inset)Star-shapedmaximumparsimonytreefromcomparisonsofwholegenomes,constructedfrom51SNPs,only3ofwhicharesynapomorphic.Dreesetal. November/December2017Volume8Issue6e01941-17 mbio.asm.org 8 mbio.asm.org on May 23, 2018 Published by mbio.asm.org Downloaded from

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withlackofrapiddiversicationintodistinctlineageslikelyrepresentingthenorm ratherthantherule.ArecentreportonthespreadofWNSalsoidentiedfewmutations of P.destructans uponintroductiontoNorthAmerica( 32).Astrictinterpretationofthe dates in Fig.2 wouldsuggestthatall P. destructans strainsinNorthAmericaandEurope divergedfromeachotherwithinthepast~100yearsandfromtheancestortoAsian isolatesroughly3,400yearsago.Whiletheseestimatesareplausible,webelieveamore accurateestimationwillrequireincreasedsamplingofisolatesfromEurasianpopulations.Perhapsanalysesusing P.destructans genomesfromsamplespreservedlongago, aswedidwith Pseudogymnoascus sp.genomesfromancientRussianpermafrost samplesin Fig.1,ordetectionofWNS-inducedbatdie-offsinEurasiafromthefossil record willenablemoreaccuratedating.Giventhat P.destructans haslikelybeenabat pathogenformillionsofyears( 73),thelackofmortalityinEuropeanbats,considerable genetic diversityinmicrosatelliteloci,andthebroaddistributionofthefungusacross thepalearcticallchangesthatweenvisiontakingmillenniatoevolveitislikelythat P.destructans ismucholderthanafewthousandyearsold.Clearlymoredetailed moleculardatinganalysesareneededtoresolvethequestionoftheemergenceand diversicationofthisspecies. Themicrosatellite-basedphylogenycontainingmoreisolatesillustratessomeadditionalepizoologicalfeatureswithrelevancetopathogeninvasionandestablishment. Whatismoststrikingaboutthesedataisthelargeamountofgeneticdiversityrevealed amongtheNorthAmericanisolates.Earlierstudiesindicatedthat73isolatesof P.destructans fromNewYork,Vermont,Pennsylvania,Ohio,WestVirginia,NorthCarolina, Ontario,NewBrunswick,NovaScotia,andPrinceEdwardIslandweregenetically identicalviaMLST( 20, 21, 31).Genotypingofthesesamplesplus78othersfromeastern Canada andtheUnitedStatesresultedinasinglegenotypein109samples,plustwo singletongenotypesfromNewBrunswickandonefromOntario.Incontrast,microsatellitelocifromourstudydened31differentgenotypesamongtheisolatesfromNorth America.Anotherhallmarkofthesedataisthelackofgeographicortemporalcorrespondencebetweencladesevidentinthetree.Forexample,the Fig.3 phylogeny shows thatisolatescollectedfromNewYorkin2008,atthebeginningoftheWNS outbreak,aredistributedthroughoutthetree.Someisolatesfromthesamesampling locationanddate(e.g.,theWilliamsHotelMine)occurindifferentclades,indicating relativelyhighgeneticdiversityfromthisoneregioncomparedtootherregionsin NorthAmerica.Unfortunately,thesedatadonotindicatewhetherthisdiversitywas presentinthefoundingpopulationorarosesoonafterintroduction,acommon problemwithpopulationgeneticanalysesofpathogens( 38).NewBrunswickandNova Scotia isolatesalsooccurinmanycladesassociatedwithisolatesfromthroughoutthe epizooticzone.Apossibleexceptiontothelackofgeographicstructuringweobserved areseveralbasalcladescontainingonlynortheasternCanadianisolatesorisolatesfrom NewEnglandandNewYorkearlyintheoutbreak.Thisregionisrelativelyisolatedand mayrepresentanareathatreceived P.destructans isolatesgeographicallyspreading fromNewEnglandearlyintheoutbreakandhashadtimesincethentodiversify genetically.Intensesamplinginthisregionmayalsobecontributingsamplingbiasto thisobservation,however. Clonality. Questionsregardingclonalexpansionof P.destructans inNorthAmerica canbeansweredmorethoroughlybythisstudyduetothelargenumberofcharacters usedinthisdataset.Thestrongestevidenceforclonalreproductionofanascomycete fungusisthelackofbothmatingtypesinapopulation,whereasanevendistribution ofmatingtypesinapopulationwouldsuggesttheprevalenceofsexualrecombination. Inapreviousstudy( 19),5outof23(22%)ofisolatesfromEasternEuropepossessedthe MAT1-2 locus. WiththeadditionofoursamplesfromEurasia,theproportionofMAT1-2 toMAT1-1matingtypesis7outof30isolates(23%).Incontrast,all26oftheisolates fromNorthAmericainthisstudypossessedtheMAT1-1matingtype.Additional analysisof P.destructans matingtypesinaneasternEuropeanpopulationshoweda moreequaldistributionofmatingtypes(58.6%MAT1-1and41.3%MAT1-2; n 41)GenomicsofWhite-NoseSyndromeInvasion November/December2017Volume8Issue6e01941-17 mbio.asm.org 9 mbio.asm.org on May 23, 2018 Published by mbio.asm.org Downloaded from

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(39).TheabsenceoftheMAT1-2matingtypeinNorthAmerican P. destructans is evidenceofbothasexualreproductionandlikelyarecentgeneticbottleneck. Theoverrepresentationofwidespreadandpersistentgenotypesisalsoevidenceof clonalreproductionasopposedtosexualreproduction( 40),aswasindicatedbyboth SNP andmicrosatelliteanalysesinthisstudy.BaseduponSNPgenotyping,8ofthe26 isolatessequenced(31%)werefoundtobeclonal,rangingfromNewEnglandto Tennesseeandsampledbetween2008and2010.Similarly,twomicrosatellitegenotypesaccountfor15and6samples,respectively,outofatotalof55NorthAmerican isolatesforwhichweobtainedcompletemicrosatellitegenotypes(38%).Quantitatively, theSimpsonsindexofgeneticdiversitywasslightlyreducedinNorthAmerica, althoughindexesofgeneticdiversityarehighlydependentuponsamplesize,despite rarefaction,andthenumberofisolatesfromEuropecomparedtothenumberof isolatesinNorthAmericaanalyzedinthisstudyisverysmall.Averagegenomiclinkage asmeasuredbythestandardizedindexofassociationindicatesthatboththeEuropean andNorthAmericanpopulationsof P.destructans areatlinkageequilibrium,suggesting bothpopulationsreproduceprimarilyasclones. Introductionof P.destructans toNorthAmerica. Warneckeetal.( 41)hypothesized that P.destructans wasanovelpathogenintroducedtoNorthAmericafrom EuropebaseduponevidencethatNorthAmericanlittlebrownbatsdevelopedWNS wheninoculatedwithaEuropeanisolateof P.destructans.Theirndingswerefurther strengthenedbyLeopardietal.( 28),whodemonstratedgeneticsimilarity,basedon MLST, betweentheNorthAmericanandsomeEuropeanfungalpopulations,indicating thelikelysourcepopulationforthisintroductiontobefromEurope.Subsequently, P.destructans hasbeenisolatedfrombatsthroughoutEurope( 39, 42)andcanalso cause histopathologicallesionsconsistentwithWNSinEuropeanspeciesofbats( 47, 48).However,widespreadmortalityofbatsfromWNShasnotbeendocumentedonthe European continent(49).ThissuggestsEuropeanbatspeciesmayhavedeveloped resistance ortolerancetoinfectionby P.destructans,presumablyduetocoevolution betweenhostsandthefungalpathogen( 41, 50).Incontrast,thehighmortalityofNorth American batsinfectedwith P.destructans isconsistentwithexposureofnaivehost speciestoapathogenpreadaptedtosimilarhostsandenvironmentalconditions. Ourworksupportsthehypothesisthat P.destructans isanovelpathogenrecently introducedtoNorthAmericabydemonstratingtherelationshipofNorthAmerican isolatesofthefungustoapopulationdominatedbyEuropeanisolatesbutgenetically distantfromisolatesfromAsia.Usinghigh-resolutiongeneticmarkers,wedocumenta lossofgeneticdiversityinepizootic P.destructans consistentwitharecentintroduction ofthispathogentoNorthAmerica. Similaritiestootherfungalinvasions. Pseudogymnoascusdestructans followsa patternofemergenceandspreadsimilartootherinvasivefungalpathogens.Inthe caseof Cryphonectriaparasitica,thecausativeagentofchestnutblight( 38),high genetic diversityinputativesourcepopulationscoupledwithasignicantdecreasein allelicrichnessinmicrosatelliteandothermultilocusgenotypesamongNorthAmerican isolatessuggestedafoundereffectassociatedwiththeintroductionoftheorganism fromAsia.NorthAmericanisolatesof C.parasitica wereasgeneticallydistantfrom AsianpopulationsastheAsianpopulationswerefromeachother.Analysisofpopulationstructurecouldnarrowthesourcepopulationdowntoabroadregion(i.e.,Honshu, Japan),butrequiredtheadmixtureofanunsampledpopulationsignicantlydifferent fromtheotherAsiansamples.Amoredetailedgenomicanalysisofaninvadingfungus wasconductedwithisolatesof B.dendrobatidis originatingfrommultiplecontinents andrevealedaconsiderablymorecomplexphylogenyofthatfungusthanwaspreviouslydetermined.Avirulentpanzooticstrainapparentlyemergedandrapidlyspread acrosstheglobe,yettheoriginalsourcepopulationofthelineageremainsambiguous despiteacomprehensivesamplingeffortandthoroughsequencinganalyses( 51). Consistent withapatternalsoseeninourstudy,haplotypesfrom B.dendrobatidis did notcorrelatewellwithhostorgeography,suggestingotherdriversofdifferentiation.Dreesetal. November/December2017Volume8Issue6e01941-17 mbio.asm.org 10 mbio.asm.org on May 23, 2018 Published by mbio.asm.org Downloaded from

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Additionally,phylogeniesconstructedfromdifferenttypesofgeneticmarkersyielded different,sometimescontradictoryresultsforclonesofthepotatoblightagent, Phytophthorainfestans ( 52).Acommonthreadinallofthesecasestudiesisthatmore genetic informationlendscomplexity,butnotnecessarilyclarity,tothenaturalhistory ofaninvadingfungalpathogen.Intermsofthenaturalhistoryof P.destructans,itwill requireextensivesamplinginEuropetondthepopulationthatwasintroducedto NorthAmerica. Usingtwohigh-resolutiongeneticcharactersSNPswithinwhole-genomesequencesandmicrosatelliteswehavedemonstratedthatisolatesof P.destructans in NorthAmericaformasingleclademuchmorecloselyrelatedtoisolatesfromEurope thantogeneticallydistantpopulationssampledinAsia.TheNorthAmericanisolates representalossofgeneticdiversityincomparisontoisolatesfromEurope,adding furthersupporttothehypothesisthat P.destructans wasrecentlyintroducedtoNorth AmericafromEurope( 28).FuturesamplinginEuropewillbeneededtomoreprecisely dene theoriginof P.destructans,althoughthesubstantialdiversityof P.destructans in Europe,coupledwithresultsfromotherfungalinvasionstudies,suggeststhatidenti- cationoftheexactsourcepopulationmaybechallenging.Microsatellitelociwere particularlysuitablefordistinguishingisolatesfromthroughouttheNorthAmerican WNSepizooticzone,butthefewsynapomorphicSNPsfoundamongtheseisolates couldnotresolvetheirphylogeneticrelationships.However,sufcientSNPswere presenttodifferentiateisolatesof P.destructans fromEurope,China,Mongolia,and NorthAmerica.Thisworkelevatesourunderstandingoftheoriginandspreadof P.destructans toasimilarleveltothatachievedfor B.dendrobatidis and C.parasitica, providingaframeworkforaglobalpathogenpopulationstructurethatcanbeusedfor futureepizoologicalinvestigations. MATERIALSANDMETHODSSamplingandculturing P.destructans. Skinsamplesfrombatsorswabsamplesofbatsand substrateswereculturedonSabourauddextroseagarorpotatodextroseagarandincubatedat7to10C, aspreviouslydescribed( 42, 53). Whole-genome sequencing. GenomicDNAwasextractedfrom P.destructans byavarietyof methods,includingtheOmniPrepforFungikit(G-Biosciences,St.Louis,MO),theDNeasybloodand tissuekit(Qiagen,Hilden,Germany)usingthesupplementaryprotocolfromthemanufacturerPuricationoftotalDNAfromyeastusingtheDNeasyBloodandTissuekit(DIY13Aug-06)asdescribed previously( 54),oraphenol-chloroformextraction.PreparationoflibrariesforIlluminawhole-genome sequencing wasbaseduponapreviouslypublishedmethod(55)butwasmodiedassequencing technology evolvedduringthecourseoftheproject.Insummary,approximately1to10gofDNA samplein200lTris-EDTAbufferwasshearedwithaSonicManmicroplatesonicator(BrooksAutomation,Chelmsford,MA)toproducefragments200to1,000bpinlength,withtheaveragefragmentlength being600bp.Theshearingprotocolwasasfollows:75sat0Cprechill,followedby20cyclesof sonicationfor10.0satfullpower,75sat0Clidchill,10sat0Cplatechill,and75sat0Cpostchill. Endrepair,dA-tailing,adapterligation,andindexingfollowedthestandardIlluminaprotocolPreparing samplesformultiplexedpaired-endsequencing(partno.1005361)usingreagentsfromtheNEBNext DNAlibraryprepmastermixsetforIllumina(NewEnglandBiolabs,Ipswich,MA)ortheKAPAhighthroughputlibrarypreparationkitwithbead(KapaBiosystems,Wilmington,MA).Librarieswere indexedwiththeIlluminamultiplexingsamplepreparationoligonucleotidekit6-bpindices(Illumina,San Diego,CA)orcustom8-bpindicesforhigherindexreaddiscrimination( 56).Sizeselectionof600-bp fragments wasaccomplishedbyexcisionfrom2%agarosegelsfollowedbypuricationwithaQIAquick gelpuricationkit(Qiagen,Hilden,Germany),E-GelSizeSelectgel(LifeTechnologies,Inc.,GrandIsland, NY),orAgencourtAMPureXPSPRIbeads(BeckmanCoulter,Inc.,Indianapolis,IN)usingtheKapa BiosystemsHigh-throughputNGSlibrarypreparationtechnicalguide,Illuminaplatforms( 57).Products from eachstepofthelibrarypreparationwerepuriedwitheitherAgencourtAMPureXPSPRIbeads usingthesameprotocolortheQIAquick96PCRpuricationkit(Qiagen,Hilden,Germany).Librarieswere quantiedviaquantitativePCRwithaKAPAlibraryquanticationkit(KapaBiosystems,Wilmington,MA) fortheABIPrism9600real-timePCRsystem(LifeTechnologies,Inc.,GrandIsland,NY).Priortoloading onthesequencer,thelibraryfragmentsizedistributionwasqualitativelyconrmedwithanAgilentDNA high-sensitivitykitforthe2100Bioanalyzer(Agilent,SantaClara,CA).Individuallibrarieswererunonan IlluminaGAIIxsequencer(V2chemistry)oranIlluminaMiSeq(V3chemistry)toproduce100-bp paired-endreadsor250-bppaired-endreads,respectively.LibrariessequencedonaHiSeq2000were multiplexedwithamaximumofvelibrariesperlanetoproduce150-bppaired-endreadswithV3 chemistry. Whole-genomesequenceanalysis. Thesequencefromresequencingandassemblyofthegenome of P.destructans 20631-21,collectedinNewYorkin2008( 26)(NCBIGenBankassemblyaccessionno. GCA_001641265.1), wasusedasareferenceforSNPdiscoveryusingtheNorthernArizonaSNPPipelineGenomicsofWhite-NoseSyndromeInvasion November/December2017Volume8Issue6e01941-17 mbio.asm.org 11 mbio.asm.org on May 23, 2018 Published by mbio.asm.org Downloaded from

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(NASP)version1.0.2(58).WithNASP,Illuminapaired-endreadswerealignedtothereferencegenome with theBurrows-WheelerAligner(59)version0.7.5amemalgorithm.SNPsweredetectedinthe alignments withtheUniedGenotyperoftheGenomeAnalysisToolkit(60)build2.5-2-gf57256b following recommendedbestpractices( 61, 62).Duplicatedregionsofthereferencegenome,including repeat regionsandmultiplegenecopies,weredeterminedbyaligningthereferencesequencetoitself usingthenucmeralgorithmofMUMmerversion3.23( 63).SNPsthatfellwithintheseduplicateregions were excludedfromfurtheranalysistoavoidfalseSNPcallsduetoambiguousreadalignment.SNPloci werealsoexcludedifatleastonesamplelackedabasecallatanSNPlocus),hadreadcoverageofless than10 inatleastonesample,andlessthan90%ofthereadsagreedwiththeSNPcall.Defaultsettings foreachsoftwarepackagewereusedunlessotherwisenoted.Matingtypewasdeterminedfromthe prevalenceofreadsmappingtothe MAT1-1 matingtypelocusinreadalignmentstothereference sequence(NCBIGenBankaccessionno. KV441390.1,bases187500to188500).SNPswereannotatedwith SnpEff version4.3q(64). Microsatellite genotyping. DNAsamplesfrom P.destructans weretestedwiththe23-locusmultilocusvariablenumbertandem-repeatanalysis(MLVA)paneldescribedbyDreesetal.( 65).Briey,we identied 2-to6-bprepeatsinthegenomesequenceof P.destructans typestrain20631-21(GenBank accessionno. GL573169 to GL575015).Wethentested127primerpairsformicrosatelliteswithlength polymorphisms usingadiversesetofDNAextractsfrom39isolatesof P.destructans collectedfromNorth AmericaandEurope.Weselected23locicontainingrepeatnumberpolymorphisms.Usingtheseprimer sets,weconductedfragmentanalysisusingtheTerminatorv3.1cyclesequencingkitona3130xlGenetic analyzer(LifeTechnologies,Inc.,GrandIsland,NY).AllelesizesweredeterminedusingtheLIZ1200size standardinGeneMapperversion4.0. Phylogenetics. PAUP* version4.0a150( 66)wasusedtoconductmaximumparsimonyanalysison SNP data,andneighbor-joininganalysis(basedonmeancharacterdifference)onmicrosatellitedata. ExtendedmajorityrulemaximumlikelihoodphylogenetictreeswerecreatedfromconcatenatedSNP sequenceswithRAxMLversion8.2.7( 67)usingtheASC_GTRGAMMAsubstitutionmodelwithascertainment biascorrectionforeachbasecharactertoaccountforinvariantlociandtheautoMREbootstopping criterionwithamaximumof1,000bootstraps.TreeswereplottedwithFigTreeversion1.4.0(A.Rambaut, 2012; http://tree.bio.ed.ac.uk/software/gtree/). BEAST2 version2.4.5(68)wasusedtocreatemaximumcladecredibilitytreesfromSNPlocusdata. Briey, theSNPdatawererstlteredtoremoveSNPswithin50bpofeachothertominimizetheeffects oflinkageontheanalyses.OptimumbasesubstitutionmodelsforSNPdataweredeterminedwiththe Rpackagephangornversion2.1.1( 69),resultinginthegeneralizedtime-reversible(GTR)modelforthe full analysisandtheJC69modelfortheNorthAmericanisolatesalone.Bothanalysesusedastrict molecularclockandacoalescentconstantpopulationtreemodel.Skylinedemographicmodels,which maybemoreappropriatefortheexpandingpopulationsinNorthAmericaaswellasarelaxedlognormal molecularclock,wereattemptedforcomparisonbutfailedtocoalesce.BecauseonlycoreSNPswere beinganalyzed,theanalyseswerecorrectedforascertainmentbiaswithacountofinvariantbasesinthe nonduplicatedalignmentstothereferencegenome.TheMarkovchainMonteCarlo(MCMC)chainlength wassetto10million,with10%burn-inandloggingevery1,000steps.Fivechainswererun,producing atotalof50,000trees,whichwerecombinedandsubsampledto10,000treeswithLogCombinerversion 2.4.3andusedtocreateanannotatedmaximumcladecredibilitytreewithTreeAnnotatorversion2.4.3. Populationgenetics. Simpsonsindexofgeneticdiversityandindexofassociationwerecalculated withtheRpackagepopprversion2.3.0( 70)asfollows.SNPalleleinformationwaspreprocessedby determining geneticdistancesbetweenindividualswithbitwise.dist,calculatingthethresholddistance forclusteringisolateswithcutoff_predictor,andassigningisolatestogenotypeswithmlg.lter.SimpsonsindexofgeneticdiversitywasdeterminedforbothSNPandmicrosatellitegenotypeswith diversity_ciwith1,000,000bootstraps.NorthAmericanisolateswererareedtoequalthenumberof EuropeanisolatesintheSNPanalysis,whichenabledthecomparisonofestimatedSimpsonsindices betweenthepopulationsbutpreventedthecalculationofstandarderrorfortheEuropeanpopulation estimate.Bootstrappingwith1,000samplestodeterminethemeanstandardindexofassociationfrom SNPalleledatawasconductedwithsamp.ia,whereasthestandardindexofassociationandone-sided permutationtestwerecalculateddirectlyformicrosatellitegenotypeswithia. Bruvosgeneticdistance(whichincorporatesthenumberofrepeatsatalocusratherthanjust differences)forNorthAmericanmicrosatellitegenotypeswasdeterminedwithpopprsbruvo.dist function.ApairwisegeographicdistancematrixbetweensamplinglocationswascreatedwiththeR packagegeospherev.1.5-5( 71).TheManteltestwasconductedwiththeRpackageade4v.1.7-5(72). Signicance forthistestwasdeterminedwith99replicatesofaMonteCarlopermutationtest. Accessionnumber(s). Accessionnumbersforthesequencesoftheisolatesexaminedinthisstudy arelistedin Tables1 and 2.SUPPLEMENTALMATERIAL Supplementalmaterialforthisarticlemaybefoundat https://doi.org/10.1128/mBio .01941-17. TABLE S1,XLSle,0.1MB. ACKNOWLEDGMENTS FundingwasprovidedbytheU.S.GeologicalSurvey,theU.S.Fish&WildlifeService (grant no.50120-B-G004),BatConservationInternational,andtheNationalScienceDreesetal. November/December2017Volume8Issue6e01941-17 mbio.asm.org 12 mbio.asm.org on May 23, 2018 Published by mbio.asm.org Downloaded from

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FoundationEcologyofInfectiousDiseases(DEB-1115895andDEB-1336290,toA.M.K. andJ.T.F.). WethankHughBroders,KarenVanderwolf,KateLangwig,andChrisDobonyfor generouslysharingisolatesof P.destructans usedinthisanalysis.StephanieRivas, LindseyLovell,JohnGillece,RemyHilsabeck,JamesSchupp,LelaAndrews,Stephen Beckstrom-Sternberg,JamieBeckstrom-Sternberg,NicoletteJanke,AdinaDoyle,SabrinaGerman,ColinSobek,andAdamTriplettprovidedinvaluabletechnicalsupport. Theuseoftrade,product,orrmnamesisfordescriptivepurposesonlyanddoes notimplyendorsementbytheU.S.Government. REFERENCES1.FisherMC,HenkDA,BriggsCJ,BrownsteinJS,MadoffLC,McCrawSL, GurrSJ.2012.Emergingfungalthreatstoanimal,plantandecosystem health.Nature484:186. https://doi.org/10.1038/nature10947. 2. 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