Range-wide genetic analysis of little brown bat (myotis lucifugus) populations: estimating the risk of spread of white-nose syndrome

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Range-wide genetic analysis of little brown bat (myotis lucifugus) populations: estimating the risk of spread of white-nose syndrome

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Range-wide genetic analysis of little brown bat (myotis lucifugus) populations: estimating the risk of spread of white-nose syndrome
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PLOS One
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Vonhof, Maarten J.
Russell, Amy L.
Miller-Butterworth, Cassandra M.
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Bats ( lcsh )
White-nose syndrome ( lcsh )
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serial ( sobekcm )

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The little brown bat (Myotis lucifugus) is one of the most widespread bat species in North America and is experiencing severe population declines because of an emerging fungal disease, white-nose syndrome (WNS). To manage and conserve this species effectively it is important to understand patterns of gene flow and population connectivity to identify possible barriers to disease transmission. However, little is known about the population genetic structure of little brown bats, and to date, no studies have investigated population structure across their entire range. We examined mitochondrial DNA and nuclear microsatellites in 637 little brown bats (including all currently recognized subspecific lineages) from 29 locations across North America, to assess levels of genetic variation and population differentiation across the range of the species, including areas affected by WNS and those currently unaffected. We identified considerable spatial variation in patterns of female dispersal and significant genetic variation between populations in eastern versus western portions of the range. Overall levels of nuclear genetic differentiation were low, and there is no evidence for any major barriers to gene flow across their range. However, patterns of mtDNA differentiation are highly variable, with high ΦST values between most sample pairs (including between all western samples, between western and eastern samples, and between some eastern samples), while low mitochondrial differentiation was observed within two groups of samples found in central and eastern regions of North America. Furthermore, the Alaskan population was highly differentiated from all others, and western populations were characterized by isolation by distance while eastern populations were not. These data raise the possibility that the current patterns of spread of WNS observed in eastern North America may not apply to the entire range and that there may be broad-scale spatial variation in the risk of WNS transmission and occurrence if the disease continues to spread west.

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RESEARCHARTICLERange-WideGeneticAnalysisofLittleBrown Bat( Myotislucifugus )Populations: EstimatingtheRiskofSpreadofWhite-Nose SyndromeMaartenJ.Vonhof1,2* ,AmyL.Russell3,CassandraM.Miller-Butterworth41 DepartmentofBiologicalSciences,WesternMichiganUniversity,Kalamazoo,Michigan,UnitedStatesof America, 2 EnvironmentalandSustainabilityStudiesProgram,WesternMichiganUniversity,Kalamazoo, Michigan,UnitedStatesofAmerica, 3 DepartmentofBiology,GrandValleyStateUniversity,Allendale, Michigan,UnitedStatesofAmerica, 4 PennStateBeaver,Monaca,Pennsylvania,UnitedStatesofAmerica maarten.vonhof@wmich.eduAbstractThelittlebrownbat( Myotislucifugus )isoneofthemostwidespreadbatspeciesinNorth Americaandisexperiencingseverepopulationdeclinesbecauseofanemergingfungaldisease,white-nosesyndrome(WNS).Tomanageandconservethisspecieseffectivelyitis importanttounderstandpatternsofgeneflowandpopulationconnectivitytoidentifypossiblebarrierstodiseasetransmission.However,littleisknownaboutthepopulationgenetic structureoflittlebrownbats,andtodate,nostudieshaveinvestigatedpopulationstructure acrosstheirentirerange.WeexaminedmitochondrialDNAandnuclearmicrosatellitesin 637littlebrownbats(includingallcurrentlyrecognizedsubspecificlineages)from29locationsacrossNorthAmerica,toassesslevelsofgeneticvariationandpopulationdifferentiationacrosstherangeofthespecies,includingareasaffectedbyWNSandthosecurrently unaffected.Weidentifiedconsiderablespatialvariationinpatternsoffemaledispersaland significantgeneticvariationbetweenpopulationsineasternversuswesternportionsofthe range.Overalllevelsofnucleargeneticdifferentiationwerelow,andthereisnoevidencefor anymajorbarrierstogeneflowacrosstheirrange.However,patternsofmtDNAdifferentiationarehighlyvariable,withhigh STvaluesbetweenmostsamplepairs(including betweenallwesternsamples,betweenwesternandeasternsamples,andbetweensome easternsamples),whilelowmitochondrialdifferentiationwasobservedwithintwogroupsof samplesfoundincentralandeasternregionsofNorthAmerica.Furthermore,theAlaskan populationwashighlydifferentiatedfromallothers,andwesternpopulationswerecharacterizedbyisolationbydistancewhileeasternpopulationswerenot.Thesedataraisethe possibilitythatthecurrentpatternsofspreadofWNSobservedineasternNorthAmerica maynotapplytotheentirerangeandthattheremaybebroad-scalespatialvariationinthe riskofWNStransmissionandoccurrenceifthediseasecontinuestospreadwest. PLOSONE|DOI:10.1371/journal.pone.0128713July8,2015 1/23 a11111 OPENACCESS Citation: VonhofMJ,RussellAL,Miller-Butterworth CM(2015)Range-WideGeneticAnalysisofLittle BrownBat( Myotislucifugus )Populations:Estimating theRiskofSpreadofWhite-NoseSyndrome.PLoS ONE10(7):e0128713.doi:10.1371/journal. pone.0128713 AcademicEditor: FilipposA.Aravanopoulos, AristotleUniversityofThessaloniki,GREECE Received: November19,2014 Accepted: April29,2015 Published: July8,2015 Copyright: 2015Vonhofetal.Thisisanopen accessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense ,whichpermits unrestricteduse,distribution,andreproductioninany medium,providedtheoriginalauthorandsourceare credited. DataAvailabilityStatement: Allsequencedatahas beensubmittedtoGenbank(accessionnumbers KM363877-KM363977andKM382071-382147).All otherrelevantdataareinthepaperandits SupportingInformationfiles. Funding: Fundingforthisstudywasprovidedby WesternMichiganUniversity,thePennsylvaniaGame CommissionStateWildlifeGrantProgram (CooperativeAgreementNo.231100),andthe PennsylvaniaStateUniversityResearch DevelopmentGrants.

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IntroductionUnderstandinghowhostmovementpatternsinfluencethetransmissionofpathogensiscritical tothedevelopmentofeffectivepreventionandcontrolstrategies,andtotheconservationand managementofhostpopulationsduringandafterdiseaseoutbreaks.However,formanyhost species,dataonindividualmovementsandcontactratesaredifficultorimpossibletocollect becauseofcrypticbehavior,thegeographicscaleofmovements,ormethodologicalconsiderationsthatlimitourabilitytofollowindividualsthroughtimeandspace.Evidencefromempiricalstudiesemployingpopulationandlandscapegeneticapproacheshasdemonstratedthat landscapefeatures,suchasmountainsandriversthatlimithostgeneflow,oftenrepresentbarrierstodiseasetransmission[ 1 – 6 ],althoughalternativemechanismsofpathogendispersal, includinghumansandotherhighlymobileintermediatehosts,mayoverridetheinfluenceof primaryhostpopulationgeneticstructure[ 1 ].Nevertheless,wheretheyexist,suchbarriersto hostgeneflowcanhaveadramaticimpactoninitialdiseaseestablishment,therateanddirectionofdiseasespread,spatialpatternsofhostresistance,anddynamicsandgeneticstructureof pathogenpopulations[ 1 – 6 ].Assumingthatratesofcontactamongindividualsleadingtogene flowareindicativeofcontactsthatcouldresultindiseasetransmission,geneticmethodsprovideausefulalternativetotraditionaldemographicapproachesasameansofexamininghost movementsandtheirimpactondiseasetransmission[ 1 ]. White-nosesyndrome(WNS)isanemergingfungaldiseasecausinghighlevelsofmortality inhibernatingNorthAmericanbats[ 7 – 9 ].Thecausativeagent, Pseudogymnoascusdestructans (hereafter Pd ),isacold-lovingfungusthataffectsbatsduringhibernationandsubsequent arousal. Pd causescharacteristiccup-likeerosionsoftheepidermisofthewingsandmuzzle, andmayinvadesebaceousglandsandhairfollicles[ 10 ].Mortalityofbatslikelyoccursthrough thelossofphysiologicalhomeostasis[ 11 ],possiblyassociatedwithdehydrationandelectrolyte depletion[ 12 13 ],leadingtomorefrequentarousalbehaviorandprematurelossoffatreserves [ 14 15 ].SinceitwasfirstdiscoveredinNewYorkStateduringthewinterof2006 – 07,WNShas sincespreadto27additionalstatesandfiveCanadianprovinces,andisknowntoaffectatleast sevenspeciesofhibernatingbats[ 16 ].Mortalityratesvaryconsiderablyamongspeciesbutcan beveryhigh( > 90%forlittlebrownbats, Myotislucifugus ,andnorthernlong-earedbats, M septentrionalis [ 9 ]),andcumulativemortalityofallaffectedbatspecieshasbeenestimatedat 5.7to6.7millionindividualsasofJanuary2012[ 17 ].Therapidemergence,andthegeographic andtaxonomicspreadofthediseasehaveraisedseriousconcernsaboutthelong-termsurvival ofhibernatingbatspeciesineasternNorthAmerica,andhavehighlightedourlackofknowledgeofthefactorsthatmayinfluenceWNStransmissionandspreadtocurrentlyunaffected regions. LittlebrownbatswereamongthefirstspeciestobediagnosedwithWNS[ 7 ],andpopulationmodelsindicatethatifmortalityratesstayconstant,thisspeciescouldbeextirpatedfrom thenortheasternUnitedStateswithin16years[ 18 ].Hibernatingpopulationsofallsizeshave beenaffectedbyWNS,buttheprobabilityofinfectionincreaseswithincreasingcolonysize [ 19 20 ],althoughmortalitywithinpopulationsisdensity-independentandcharacterizedby frequency-dependenttransmission[ 21 ].Thus,thereisahighprobabilitythatlittlebrownbat populationsincurrentlyaffectedregionswillbehighlyreducedandpossiblybeextirpatedin comingdecades.Additionally,WNSmayposeathreattotheentirespeciesifthediseasecontinuestospreadacrossthespecies ’ range.Further,becauselittlebrownbatsareoneoftwo affectedbatspecieswhosegeographicrangesspantemperateNorthAmerica,theymaydrive transmissionof Pd toanovelsuiteofwesternNorthAmericanhibernatingbatspeciesthat mightotherwiseremaingeographicallyisolatedfromthedisease.Therefore,itiscrucialthat Range-WidePopulationGeneticAnalysisofLittleBrownBats PLOSONE|DOI:10.1371/journal.pone.0128713July8,2015 2/23 CompetingInterests: Theauthorshavedeclared thatnocompetinginterestsexist.

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weunderstandtheprobabilityof Pd transmissionacrosstherangeoflittlebrownbats,and whethertherearebarrierstogeneflowthatcouldrestrictthegeographicspreadofWNS. Hereweapplygeneticapproachestounderstandlevelsofgeneflowandpopulationconnectivityinthelittlebrownbat.Thissmall(6 – 10g)insectivorousbatspeciesisamongthemost widespread( Fig1 )andwell-studiedinNorthAmerica[ 22 23 ].Duringthesummer,reproductivefemalesformmaternitycoloniesinbuildings,trees,orcreviceswhereparturitionandpostnatalcaretakeplace,whilemalesandnon-reproductivefemalestypicallyroostsolitarily[ 22 ]. Inwinter,bothsexescongregateinhibernacula,andmatingtakesplaceduringthepre-hibernationswarmingperiod,orduringhibernationitself[ 24 25 ].Thesizeofhibernatingpopulationsmayvaryconsiderably,ontheorderof10 ’ sto100,000 ’ s,butmostofthelargerknown hibernaculaoccurinkarstregionsofeasternNorthAmerica,andverylittleisknownaboutthe distributionorsizeofhibernaculainwesternNorthAmerica.Becauseindividualsfrommany breedinggroupscometogetheratswarmingorhibernationsiteswithmalesthatmayormay nothaveoriginatedfromthesamebreedinggroup[ 26 27 ],thesesiteshavebeensuggestedto represent ‘ hotspots ’ ofgeneflowfortemperatebats[ 28 – 30 ].Thus,patternsofgeneflowwill representtheinterplayofmovementsofindividualsbetweensummerand/orwinterpopulations,andlevelsandspatialpatternsofconnectivityamongsummerandwinterpopulations thatdeterminethecompositionofmatingaggregations. Therearecurrentlyfiverecognizedsubspeciesoflittlebrownbats( M l alascensis M l carissima M l lucifugus M l pernox ,and M l relictus [ 22 31 ];see Fig1 )basedonmorphology, buttheextenttowhichthesesubspeciesdivergegeneticallyisunclear.Coalescentanalysesof nuclearDNA(nucDNA)andmitochondrialDNA(mtDNA)suggestthatsomesubspeciesmay representindependentevolutionarylineages,butthat M lucifugus maybeparaphyleticwith respecttothewesternlong-earedbat, M evotis [ 32 ].Inaddition,twodistinctmtDNAlineages (correspondingto M. l carissima and M l lucifugus )co-occurinsouthernAlbertaandnorthcentralMontana,butthesetwogroupsofbatsarenotdifferentiatedbasedonnuclearmicrosatelliteDNAormorphology,suggestingthatthesubspeciesinquestionmayinterbreedand exchangegenes[ 33 ].Asinglemitochondriallineagecorrespondingto M l lucifugus was observedintheMinnesotapopulations,andtherewasastrongsignalofpopulationexpansion datingto18kya[ 34 ].EnvironmentalnichemodelingbasedonconditionsduringtheLastGlacialMaximum(LGM)indicatedthepresenceofasinglelargerefugiumextendingacrossthe southeasternandsouth-centralUnitedStates,andmorefragmentedrefugiainthesouthern portionofthemountainouswesternUnitedStates[ 34 ],suggestingapossiblemechanismfor lineagedifferentiationwithinthisspecieswhereseparationintodisjunctglacialrefugiawasfollowedbysubsequentpost-glacialrangeexpansionandsecondarycontact. Fewstudieshaveexaminedgeneticvariationinlittlebrownbats,andtherehasbeenno comprehensiverange-widepopulationgeneticanalysisofthisspecies.Fine-scalegeneticstudiesinMinnesotadescribedhighlevelsofmtDNAstructureandweakbutsignificantnucDNA structureamongmaternitycolonies[ 35 ],apatternconsistentwithmanyothertemperatebat speciescharacterizedbyfemalephilopatrytosummerbreedinghabitatandextensivegeneflow viamatingatswarmingsitesduringtheautumn[ 36 – 41 ].InsouthesternCanada,highlevelsof geneticconnectivitywereidentifiedamongswarmingsites,howeverminimalstructuringat bothmtDNAandnucDNAmarkerssuggesteddispersalmayoccurinbothsexes,althoughit maybemale-biased[ 42 ].InPennsylvania,nosignificantnuclearstructurewasidentified amonghibernatingpopulations,butthesepopulationswerestructuredmatrilineally.This mtDNAstructurewascorrelatedwithlocaltopography,whichmayhavedelayedthespreadof WNStowesternpartsofthestate[ 6 ]. TherapidspreadofWNSthrougheasternNorthAmericanpopulationsoflittlebrownbats (andotheraffectedspecies)suggeststhatfewbarrierstotransmissionexistwithinthecurrent Range-WidePopulationGeneticAnalysisofLittleBrownBats PLOSONE|DOI:10.1371/journal.pone.0128713July8,2015 3/23

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Fig1.MapofCanadaandtheUnitedStatesshowingthedistributionofdescribed Myotislucifugus subspecies(modifiedfrom[ 17 ])andsamplinglocations. a)showssamplinglocationswithpiecharts indicatingfrequenciesofmtDNAsubspecificclades(subspecificdesignationsareindicatedinthelegendand colorsfollowthoseusedin Fig2 )ineachpopulation,whileb)showsgroupingsofpopulations(orangeand bluedots)withinwhichpairwise STvaluesbasedonmtDNAhaplotypefrequencieswerelowversus populationsthatweresignificantlydifferentiatedfromallotherpopulations(purpledots;highpairwise STvalueswithallothersampledpopulations).OnesampledpopulationinMichigan(shownwithablackdotina) Range-WidePopulationGeneticAnalysisofLittleBrownBats PLOSONE|DOI:10.1371/journal.pone.0128713July8,2015 4/23

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rangeofthedisease.HereweutilizemtDNAsequenceandnucDNAmicrosatellitevariation fromalargesampleoflittlebrownbatscollectedacrosstherangeofthespeciestoaddressthe followingobjectives:1)assesslevelsofgeneticvariationinlittlebrownbatpopulations,includingareasaffectedbyWNSandthosecurrentlyunaffected;2)quantifygeneticdifferentiation amongpopulationssampledacrosstherangeofthespecies,includingpopulationsineastern NorthAmericawithinthecurrentrangeofWNS,aswellasadditionalpopulationssituated botheastandwestofthetransitionbetweentheGreatPlainsandRockyMountains;and3) assessthecurrentgeographicdistributionofandlevelsofgeneticdifferentiationamongcurrently-recognizedsubspecificlineages. Therearefewphysiographicbarriersthatwouldlimitmovementofhighlyvagileorganisms eastoftheRockyMountains.PhylogeographicstudiesofwidespreadbatsandbirdsinNorth wasnotincludedinmtDNAanalyses.Populationabbreviationsaredetailedin Table1 ,andcolorsinpie chartsina)correspondtocladesshownin Fig2 .Datasourcesforthemapinclude:nationalatlas.gov, iucnredlist.org,andESRIData&Maps2006throughArcGIS( S1File ). doi:10.1371/journal.pone.0128713.g001 Fig2.PhylogenetictreeshowingrelationshipsbetweenNearctic Myotis (sensu[ 58 ]),andthepresenceofthreedistinctcladeswithin M lucifugus basedonmaximumlikelihoodanalysisofpartialCOIsequencesinPhyML3.0. AmemberoftheNeotropical Myotis clade( M austroriparius )was includedastheoutgroup.Leavesarecollapsedtohighlightwell-supportedclades,andtheverticaldimensionofthetrianglesisproportionaltoth enumberof samplesincluded.SH-likebranchsupportvaluesareprovidedforallmajorclades.Cladescontaining M lucifugus aredesignatedbythespecific abbreviationfollowedbythesubspeciesname(e.g., M l lucifugus referstothenominalsubspecies).Notethatoneclade(including M l carissima )also containsmembersofotherspecies(including M evotis M keenii ,and M thysanodes )aspreviouslydescribed[ 32 ]. doi:10.1371/journal.pone.0128713.g002 Range-WidePopulationGeneticAnalysisofLittleBrownBats PLOSONE|DOI:10.1371/journal.pone.0128713July8,2015 5/23

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Americatypicallyreportlittledifferentiationamongpopulationswithineasternandcentral portionsofNorthAmerica,significantdifferentiationamongeasternandwesternpopulations, andhigherlevelsofdifferentiationamongpopulationswithinthemountainouswest[ 41 43 – 46 ].WepredictthattheRockyMountainswillrepresentabarriertogeneflow,andthatwe willthereforeobservegeneticdifferentiationbetweensamplesiteseastversuswestoftheGreat Plains-RockyMountainstransition.Further,becauseofhighertopographicvariability,wepredicthigherlevelsofgeneticdifferentiationamongsamplesitesinthemountainouswestthan ineasternNorthAmerica.Ifsubspecificlineagesrepresentreproductively-isolatedunitsthat aroseduringtheLGM,thenwepredictthatpatternsofdifferentiationatbothnucDNAand mtDNAmarkerswillmatchthedescribedgeographicdistributionofsubspecies.Ourstudy providesvaluabledataonpopulationconnectivityandhenceopportunitiesforWNStransmissionacrosstherangeoflittlebrownbatsthatmaybeusedtoinformthemanagementandconservationofaffectedspecies.Methods SamplecollectionTissuesampleswereobtainedduringthesummer(betweenMayandAugust)from637individualsat29locationsacrosstherangeoflittlebrownbats( Table1 S1Table ,and Fig1 ).Two 3mmbiopsypunches,onefromeachwing,weretakenfromeachbatandstoredin5MNaCl with20%DMSO[ 47 ].Thebatswerereleasedaftersampling.Themajorityofpopulationsampleswerecollectedatmaternitycolonies(N=16)orsingleorseveralclosely-spaced( < 10km) nettingsites(N=12).However,theIdahosampleconstitutedbatscollectedin8different countiesinthesoutheasternportionofthestate.Whensamplescamefrommorethanonecapturelocation,centroidswerecalculatedandusedasapproximatesamplelocations.EthicsstatementOneauthor(MJV)collectedsamplesforthisstudyfromonepopulationinMichigan.ThesampleswerecollectedunderpermissiongrantedbytheStateofMichiganDepartmentofNatural Resources(PermitSC1257),andthemethodswereapprovedbytheWesternMichiganUniversityInstitutionalAnimalCareandUseCommitteeProtocolNumber08-05-03.Allother sampleswerecollectedbyuniversityandgovernmentresearchersperformingotherresearch whowererequiredtohaveappropriatepermitsandothernecessarypermissiontoundertake theirwork.MitochondrialDNAsequencingandanalysisTotalgenomicDNAwasisolatedusingDNeasyTissueKits(Qiagen,ValenciaCA).Weamplifiedandsequenceda636bpfragmentofthemitochondrialcytochromecoxidasesubunitI (COI)geneusingprimersHCO2198andLCO1490[ 48 ]orprimersVF1andVR1[ 49 ].Bats fromallsamplesitesexceptMichiganweresequenced,foratotalof617individuals.PCRs wereconductedin25 lvolumescontaining0.4 Mofeachprimerand20 – 50ngofDNAtemplate,usingIllustraPuReTaqReady-To-GoPCRbeads(GEHealthcareLifeSciences,PittsburghPA).Whenreconstitutedto25 lwithwater,thesebeadscontained2.5unitsPuReTaq DNApolymerase,200 MeachdNTPin10mMTris-HCl(pH9),50mMKCl,1.5mM MgCl2,andanunspecifiedconcentrationofbovineserumalbumin(BSA).Nootheradditives wereaddedtothesolution.Cyclingconditionsconsistedofonecycleof5minat94C,30 cyclesof30secat94C,45secat68Cand1minat72C,andafinalcycleof2minat72C. PCRproductswerepurifiedbydigestionwithexonucleaseIandshrimpalkalinephosphatase Range-WidePopulationGeneticAnalysisofLittleBrownBats PLOSONE|DOI:10.1371/journal.pone.0128713July8,2015 6/23

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Table1.SampledlittlebrownbatpopulationsanddiversitystatisticsformitochondrialCOIsequences( NSEQ,numberofindividualssequenced; NHAP,numberofhaplotypes; NHAP-UN,numberofhaplotypesuniquetoasite; h ,haplotypediversity; ,nucleotidediversity),andmicrosatellite genotypes( NGEN,numberofindividualsgenotyped; HO,observedheterozygosity; HE,expectedheterozygosity; NA,meannumberofallelesper locus; AR ,allelicrichness; AR-P ,privateallelicrichness; FIS,inbreedingcoefficient). Provinceor State CountyAbbrev.WNS-Status NSEQNHAPNHAP-UNh NGENHOHENAARAR-PFISAlaskaKenai Peninsula AKNeg18550.650.0017180.8640.8148.07.670.01-0.061 CaliforniaMariposaCA-MaNeg10210.200.00160 CaliforniaMonoCA-MoNeg15210.480.00080 CaliforniaSiskiyouCA-SiNeg20760.840.0026200.9220.92013.212.200.12-0.003 CaliforniaShastaCA-ShNeg11210.180.00140 WashingtonSkagitWANeg17860.640.0175170.8690.91512.211.800.170.050 British ColumbiaSouth BC-SNeg30760.610.0157300.8890.90314.611.800.120.015 IdahoMultipleIDNeg26640.520.0038230.8600.90413.611.890.260.049 WyomingCarbonWYNeg30970.790.0148300.8700.90415.012.090.020.037 AlbertaSouth AB-SNeg28960.660.0103290.9200.91816.112.920.12-0.002 AlbertaNorth AB-NNeg28950.790.0045290.9000.91615.912.760.260.017 British ColumbiaNorth BC-NNeg15850.700.0040150.9190.91513.313.330.14-0.003 ManitobaMBNeg11410.750.00440 OntarioON-1Neg15770.890.00660 MinnesotaStLouisMNNeg20730.730.0046200.9170.89413.812.290.29-0.025 WisconsinMarquetteWI-MaNeg20940.870.0057200.8890.89513.612.040.390.007 WisconsinSaukWI-SaNeg20720.800.0045160.8680.90512.011.700.430.041 MichiganCassMINeg0200.8280.88113.011.640.650.061 KentuckyRowanKYPos2913110.840.0043330.8860.90015.712.190.330.016 OhioFair eldOHPos311150.880.0059300.8630.88114.311.270.260.021 TennesseeBlountTNPos32410.460.0008300.8520.90514.811.910.230.059 West Virginia RaleighWVPos20730.740.00330 OntarioON-2Pos301240.800.00310 Pennsylvania BlairPAPos211240.880.0035210.8280.89413.611.900.160.018 MarylandWashingtonMDPos3412100.910.0041300.8860.89616.012.150.220.004 NewYorkJeffersonNYPos201380.930.0036180.8630.90013.012.080.180.026 NewJerseyMorrisNJ-MoPos301240.900.0041310.8520.89415.011.930.290.045 NewJerseySalemNJ-SaPos20720.780.00240 QuebecQBPos16740.780.0019300.8480.89014.311.430.10.047 Overall6177.84.50.720.00515100.8760.89713.911.900.230.020 Populationsareorderedwesttoeast.WNS-StatusdenotessamplinglocalitieswithinstatesorprovincesthatwerepositiveornegativeforWNSasof 2012. doi:10.1371/journal.pone.0128713.t001 Range-WidePopulationGeneticAnalysisofLittleBrownBats PLOSONE|DOI:10.1371/journal.pone.0128713July8,2015 7/23

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(EXOSAP),andweresequencedinbothdirections,usingtheamplificationprimers,attheUniversityofArizonaGeneticsCoreFacility.SequenceswereeditedusingCodonCodeAligner3.0 (GeneCodesCorp.)andalignedusingthedefaultsettingsinMAFFT[ 50 ].MicrosatellitegenotypingandanalysisWegenotypedindividualsatelevenhighlyvariablemicrosatellitelociusingprimerspreviously developedforothervespertilionidbats(IBatCA5,CA11,CA43,CA47,andM23[ 51 ];MS3D02 andMS3F05[ 52 ];E24andG9[ 53 ];Cora_F11_C04[ 54 ];Coto_G02F_H10R[ 55 ]).Wedidnot genotypepopulationsampleswith 15individuals(CA-Ma,CA-Mo,CA-Sh,MB-1,MB-2; Table1 ),withtheexceptionofBC-Nthathadbeengenotypedforanotherstudy.BasedonpreliminaryresultsoflowlevelsofdifferentiationineasternNorthAmericaandtoreducecosts, wedidnotgenotypeindividualsfromthreeadditionalsiteseastoftheRockyMountains(ON2,WV,andNJ-Sa).Thetotalnumberofindividualsgenotypedwas510.Amplificationswere carriedoutinfourmultiplexreactionsandtwosingle-locusamplifications(see S2Table ),and weresubsequentlypooledintothreedifferentloadsforfragmentanalysisonanABI3130 sequencer.Thebasiccyclingconditionsconsistedof1minat94C,threecyclesof30secat 94C,20secatTa(54or60C),and5secat72C,33cyclesof15secat94C,20secatTa,and 10secat72C,followedbyafinalextensionat72Cfor30min.Someamplificationsrequired additionalcyclesortheremovalofthefinalextensionstep( S2Table ).Fragmentswereanalyzed andscoredusingGeneMarkersoftware(SoftGeneticsLLC,StateCollege,PA).MitochondrialDNAanalysisTodescribeoveralllevelsofmtDNAdiversitywithinpopulations,wecalculatedhaplotype( h ) andnucleotide( )diversitiesinDNASPv.5.10.1[ 56 ],anddeterminedthenumberofprivate haplotypesineachsiteaftercollapsingsequencesfromtheentiredatasettouniquehaplotypes usingFABOX[ 57 ].Weusedseveralpopulationgeneticapproachestoestablishwhethercurrentpatternsofvariationareindicativeofthepresenceofdistinctgeneticclusters.Wecalculatedpairwise FSTvaluesbetweensitesandtestedforsignificancewith10,000permutationsin Arlequinv.3.11[ 58 ]toidentifypairsofsitesthatweregeneticallydistinct.Wealsoperformed ananalysisofmolecularvariance(AMOVA[ 59 ])todescribetherelativeamountofgenetic variationwithinandamongsites.Basedoninitialpairwise FSTresults,wethenperformed nestedAMOVAstoidentifynaturalgroupsofsites.Siteswereinitiallygroupedtogetherifthey hadlowpairwise FSTvalues,andtheanalysiswasrerun.Anyambiguoussites(sitesthathad low FSTvalueswithsitesinmorethanonegroup)weresequentiallymovedbetweengroups andtheanalysiswasrerun.Alllogicalcombinationsweretestedtoidentifythegroupingthat minimizedamong-site/within-groupvariationandmaximizedbetween-groupvariation. Totestthesignificanceofdefinedsubspecificlineageswithin M lucifugus usingournationwidedataset,weusedamaximumlikelihoodphylogeneticapproachimplementedinPHYMLV.3.0[ 60 ].WesequencedCOIforotherNorthAmerican Myotis spp.( M californicus M ciliolabrum M evotis M keenii M leibii M sodalis M thysanodes ,and M volans ;cf[ 61 ]),anda memberoftheNeotropical Myotis clade( M austroriparius ),whichwasusedastheoutgroup (see S3Table forlistofspecimens).Weusedthebestfitmodelofsequenceevolution(HKY +G)asdeterminedusingMegav.5.0[ 62 ],withthegammadistributionofvariabilityofrates amongsitescalculatedempiricallyfromthedata,SPRmovestoexploretreespace,andSH-Like proceduretoassessbranchsupports[ 60 ].Theproportionofeachsampledpopulationfalling withineachsubspecificcladewasthencalculatedandplottedonamapproducedinARC-GIS v.10.1tovisualizethegeographicdistributionoftheclades. Range-WidePopulationGeneticAnalysisofLittleBrownBats PLOSONE|DOI:10.1371/journal.pone.0128713July8,2015 8/23

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MicrosatelliteDNAanalysisDeviationsfromHardy-Weinbergequilibrium(HWE)wereestimatedforeachlocus,andloci wereconfirmedtobeinlinkageequilibriumusingFSTATv.2.9.3[ 63 ].Totestfordifferences inlevelsofgeneticdiversityamongsitesandregions,severalindicesofnucleargeneticdiversity wereestimated,includingnumberofallelesperlocus,allelicrichness,andtheinbreedingcoefficient( FIS)usingFSTAT,privateallelicrichnessusingHP-RAREv.1.0[ 64 ],andobservedand expectedheterozygosityusingGENODIVE[ 65 ].Wethentestedfordifferencesamongsites (orgroupsofsites)inallelicrichnessand FISinFSTAT,andexpectedheterozygosityinGENODIVE,using10,000permutations.Testswereperformedamongclustersofsitesidentified usingclusteringtechniques(seebelow),andamongsitesfallingwithinstatesorprovincesthat wereWNS-positive(KY,OH,TN,WV,ON-2,PA,MD,NY,NJ-Mo,NJ-Sa,QB; Table1 )or WNS-negative(allothersites)asof2012,althoughitshouldbenotedthattissueswerecollectedpriortotheemergenceofWNSattheselocalitiesinallcases. Weappliedthreedifferentapproachestodeterminethemostlikelynumberofdistinct geneticclustersindependentoforiginalsamplinglocations,asdifferentclusteringalgorithms canproducedifferentsolutionsandconcordanceamongmultipletechniquesissuggestiveof thepresenceofastronggeneticsignal[ 66 ].First,weutilizedthemodel-basedBayesianclusteringapproachinSTRUCTUREv.2.3.3[ 67 68 ]withpopulationmembershipasaprior[ 69 ].To determinetheoptimalnumberofclusters( K ),weran10runsper K ,for K =1 – 10,witha 100,000MCMCiterationburn-infollowedby400,000iterationsusingtheadmixturemodel withcorrelatedallelefrequencies.Themostlikelynumberofclusterswasdeterminedusingthe Evannoetal.[ 70 ]methodimplementedintheprogramSTRUCTUREHARVESTER[ 71 ].The Evannoetal.[ 70 ]methodisnotinformativeforthehighestandlowest K ;therefore,ifthehighestloglikelihoodvaluewasobservedfor K =1or10acrossallreplicates,weacceptedthatas the K withthehighestprobability.Forthebestvalueof K weusedCLUMPP[ 72 ]toharmonize individualassignmentstoclusters. Second,weappliedthe K meansclusteringapproachinGENODIVEv.2.0asoutlinedby Meirmans[ 65 ].ThisapproachisbasedonanAMOVAframeworkandusesasimulated annealingalgorithmtominimizetheamong-populations/within-groupssumofsquares throughmaximizationof FCT,thevarianceamongclustersrelativetothetotalvariance.We determinedthemostlikelynumberofclustersusingthePseudoF summarystatistic,which performsbetterthanthealternativeBayesianInformationCriterion(BIC)whenmigration ratesarehighandmatingisrandom[ 65 ]. ThethirdapproachwasthatofDuchesneandTurgeon[ 73 ]implementedinthesoftware FLOCK.Samplesarerandomlypartitionedinto K clusters( 2),allelefrequenciesareestimatedforeachofthe K clusters,andeachgenotypeisthenreallocatedtotheclusterwiththe highestlikelihoodscore.Repeatedreallocationbasedonlikelihoodscores(20iterationsper run)resultedingeneticallyhomogeneousclusterswithinarun.Fiftyrunswerecarriedoutfor each K ,andattheendofeachrunthesoftwarecalculatedtheloglikelihooddifference(LLOD) scoreforeachgenotype(thedifferencebetweentheloglikelihoodofthemostlikelyclusterfor thegenotypeandthatofitssecondmostlikelycluster)andthemeanLLODoverallgenotypes. Strongconsistencyamongruns(resultingin ‘ plateaus ’ ofidenticalmeanLLODscores)isused toindicatethemostlikelynumberofclusters[ 73 ].Basedonthisanalysis,were-rerantheiterativereallocationprocedureforthemostlikely K andplottedthemeanLLODscoreagainstgeographicallyorderedsitesasameansofidentifyingadmixturelevelsbetweengeneticclusters. Forallthreemethodsofgeneticclusteridentification,wetestedwhetherclusterassignment wasvalidbyquantifyingthenumberofindividualswithineachsamplethatwereallocatedto eachcluster,andthenbuildingan r c contingencytablewhere r isthenumberofgenetic Range-WidePopulationGeneticAnalysisofLittleBrownBats PLOSONE|DOI:10.1371/journal.pone.0128713July8,2015 9/23

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clustersand c isthenumberofsamplesites.Wethentestedforrandomallocationtothe geneticclustersacrossempiricalsamplesusingalikelihood-ratiotestwithWilliams ’ correction withthenullhypothesisthatclusterassignmentswererandomacrosssampledsites.Arejection ofthenullhypothesisindicatedthatthatclustercompositionwasunlikelytoberandomacross thesamples,andthatclusterassignmentswerethereforevalid[ 74 ].Inaddition,giventhat mostclusteringtechniquesassumethatgenotypicproportionswithineachclusterareinHWE andatlinkageequilibrium,wetestedidentifiedclustersforcompliancewiththeseassumptions assuggestedbyGuillotetal.[ 66 ].TotestwhetherclusterassignmentwasindependentofsubspecificmtDNAclademembershipwecouldnotsimplytestforanassociation,ascluster assignmentwasconfoundedbyspatialvariationinthedistributionofmtDNAclades.Therefore,wecompiledclusterandclademembershipforindividualsineachofthefoursitesthat containedmembersofmorethanonemtDNAclade(see Results Fig1a ),andperformedalikelihood-ratiotesttodeterminewhetherclusterassignmentswereindependentofsubspecific clademembershipwithinheterogeneouspopulations. Thelevelofgeneticdifferentiationamongpre-definedsitesandanalternativegrouping basedonsubspecificclademembership,whereindividualswereclassifiedasbelongingtothe M l alascensis M l carissima ,or M l lucifugus cladesbasedonthemtDNAphylogenetic analysis,wasdeterminedbycalculatingpairwisedistancemeasures,including FST[ 75 ],and twomeasuresindependentoftheamountofwithin-populationdiversity:Jost ’ sD[ 76 ],and G ”ST[ 77 ].Differencesinthemagnitudeofpairwisedistancemeasuresamonggroupsofsites wastestedusing10,000permutationsinGENODIVE( G ”STandJost ’ sD)andFSTAT( FST).As withmtDNA,weperformedanAMOVAonmicrosatellitegenotypesusingARLEQUIN,and subsequentlyperformednestedAMOVAsbygroupingsiteswithlow FSTvaluestoidentifythe groupingthatmaximizedamong-groupvariationandminimizedamong-site/within-group variation.IsolationbydistanceThereisconsiderableevidencetosuggestthat,regardlessofthealgorithmemployed,clustering methodsareconfoundedbythepresenceofisolationbydistance(IBD),suchthatconsistent clinalgeneticvariationmaybemisinterpretedbyclusteringalgorithmsasthepresenceofdistinctclusterseventhoughthereisnotruebarriertogeneflow[ 66 78 – 80 ].Wethereforetested forIBDinmitochondrialandnuclearDNAbothglobally(includingallsampledlocations)and withinidentifiedclusters(formicrosatellitedataonly).WeconductedaManteltestcomparing standardizedgeneticdistance[ FST/(1FST)]andthenaturallogofgeographicdistance[ 81 ] usingtheIBDWebService[ 82 ].Tocalculatebetween-sitegeographicdistances,polylineswere constructedfromX,YcoordinatesinArcGIS10.1.Thegeodesicdistanceofthesepolylineswas calculatedusingthe “ Shape.length@meters ” command.Forthemicrosatellites,wefollowedthe recommendationsofGuillotetal.[ 66 ]:weplottedgeneticdistance( FST)againstgeographic distancewhiledifferentiatingbetweendatapointsforsitepairsthatbelongedtothesame geneticclusteranddatapointsforsitepairsbelongingtodifferentclusters.Ifclustersarereal, thenforanygivengeographicdistance,geneticdistancebetweensitepairsindifferentclusters shouldconsistentlybegreaterthandistancebetweensitepairsfallingwithinthesamecluster (e.g.[ 83 84 ]).Inaddition,assuggestedbyMeirmans[ 80 ],weperformedapartialManteltest toinvestigatetheassociationbetweenthematrixofgeneticdistances( FSTand G ”ST)anda matrixofclustermembershipforthemicrosatellitedata,withthematrixofgeographicaldistancesasacovariate.Ifthereisnotarelationshipbetweengeneticdistanceandclustermembershipaftercontrollingforgeographicdistance,thenclustermembershipislikelyconfounded byIBD.Giventwomainclusters(see Results ),wecodedclustermembershipintwoways:a) Range-WidePopulationGeneticAnalysisofLittleBrownBats PLOSONE|DOI:10.1371/journal.pone.0128713July8,2015 10/23

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bothmembersofasamplingsitepairbelongingtothesamecluster(1)ornot(2);orb)both membersofasamplingsitepairbelongingtoawesterncluster(1),bothbelongingtoaneastern cluster(2),orsamplesitesbelongindifferentclusters(3).PartialManteltestswerecarriedout inPASSaGEsoftwareandsignificancewasassessedwith10,000permutations[ 85 ].Results GeneticdiversityWeobserved148uniquehaplotypescharacterizedby104segregatingsitesamongthe617individualssequenced.Thenumberofhaplotypespersiterangedfrom2 – 13(mean:7.8),andthe numberofhaplotypesuniquetoasiterangedfrom1 – 11(mean:4.5; Table1 ).Therewasnosignificantdifferenceinnucleotidediversity( )betweensiteseastversuswestoftheGreatPlainsRockyMountainstransition(Mann-WhitneyU-Test;MeanEast:0.00,West:0.01, P =0.829), orbetweensitesinstatesthatwerepositiveornegativeforWNSasofthe2012 – 2013winter season(Mann-WhitneyU-Test;WNS-Neg:0.01,WNS-Pos:0.00, P =0.132).However,sites westoftheGreatPlains-RockyMountainstransitionhadsignificantlylowerhaplotypediversitythanthoseeastoftheboundary(Mann-WhitneyU-Test;East:0.80,West:0.56, P =0.002), andWNS-freesitesasof2012alsohadsignificantlylowerhaplotypediversitythanWNSaffectedsites(Mann-WhitneyU-Test;WNS-Neg:0.65,WNS-Pos:0.81, P =0.015),although thislatterresultislikelyconfoundedbythehighproportionofwesternsitesintheWNS-Neg group. Althoughweoriginallytyped11microsatelliteloci,twoloci(E24andCOTO_G02_H10) hadhighnullallelefrequenciesandweredroppedfromfurtheranalyses.Theremainingnine lociallmetHWEexpectationsandwereunlinked.Meanobservedandexpectedheterozygositieswerehigh(0.876and0.897,respectively),aswasthemeannumberofallelesperlocus (13.9)andallelicrichness(11.9),althoughprivateallelicrichnesswaslow(0.23; Table1 ;see S4 Table fordiversitystatisticsforeachlocus).Comparingsample-levelmeasuresofgeneticdiversityamongidentifiedclustersandamongsamplesinWNS-positiveandWNS-negativestates orprovincesrevealednosignificantdifferencesinobservedorexpectedheterozygosity,allelic richness,or FIS( P > 0.05inallcasesbasedonpermutationtests).Differentiationof M lucifugus subspeciesPhylogeneticanalysisofthemtDNACOIsequencesconfirmedtheexistenceofthreeclades withinlittlebrownbatsroughlycorrespondingtopreviouslydefinedsubspecies( Fig2 ).FollowingCarstensandDewey[ 32 ],werefertothesecladesas M l lucifugus M l carissima ,and M l alascensis (hereafterlucifugus,carissima,andalascensis,respectively).Thecarissimaclade hadthreeotherspecies( M evotis M keenii M thysanodes )nestedwithinit,aspreviously described[ 32 ].Wehavefocusedon M lucifugussensustricto here.Addressingthetaxonomic relationshipsamong M lucifugus M evotis M keenii ,and M thysanodes isbeyondthescope ofthispaper;thereforeweignoredthepresenceoftheseadditionaltaxaintherestofouranalyses.Forthemostpart,cladesweregeographicallyrestrictedandfollowedpreviouslydefined geographicranges( Fig1a ).AlmostallofthesampledsiteseastoftheGreatPlains-Rocky Mountainstransition(withtheexceptionoftheAlberta-Southsite)werecomposedentirelyof individualsclassifiedaslucifugus.Thecarissimacladewasrestrictedtomountainousbutnot coastalregionsofthewestandaportionofthewesternplains(insouthernAlberta).ThealascensiscladewasfoundalongthePacificCoastandcoastalmountainranges( Fig1a ).However, individualsinthelucifuguscladewerealsosampledatlocationswestoftheGreatPlains-Rocky Mountainstransition,includingtheBritishColumbia-South(BC-S),Wyoming(WY),and Washington(WA)samples.TheAlberta-South(AB-S)sampleeastoftheGreatPlains-Rocky Range-WidePopulationGeneticAnalysisofLittleBrownBats PLOSONE|DOI:10.1371/journal.pone.0128713July8,2015 11/23

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Mountainstransitionalsocontainedbothlucifugusandcarissimahaplotypes(seealso[ 33 ]). NoalascensishaplotypeswerefoundattheBC-Ssite,althoughrangemapsplacethissitein thatsubspecies ’ range;furthermore,theAlaska(AK)sampleclusteredwiththealascensis clade,eventhoughitfellwithinthedescribedrangeofthelucifugusclade.SpatialpatternsofpopulationgeneticstructureMitochondrialDNA. AMOVAanalysisconsideringallsamplesasasinglegrouprevealed highlevelsofdifferentiation( FST=0.721).Weiterativelygroupedsiteswithlowpairwise FSTvaluestodeterminethebestarrangementsofsitesthatmaximized ‘ among-group ’ andminimized ‘ among-site/within-group ’ variationintheAMOVAframework.Mostsampleswere highlydivergentfromallothers(76%ofpairwisecomparisonshad FST> 0.2,and62% were > 0.5; S5Table and Fig1b ),butweidentifiedtwogroupsofsites(oneinthecentral UnitedStatesandCanadaeastoftheRockyMountains,andoneineasternNorthAmerica) withinwhichdivergencewaslow( FST=-0.019 – 0.130; Fig1b ).Aftergroupingthesesites togetherinanAMOVAanalysis,among-groupvariation( FCT)accountedfor73.5%ofvariationinhaplotypefrequencies,andamong-site/within-groupvariationaccountedfor1.4%. MicrosatelliteDNA. Allclusteringmethodsemployed[Bayesianclustering(STRUCTURE),repeatedreallocation(FLOCK),and K meansclustering(GENODIVE)]identified K =2asthemostlikelynumberofgeneticclusters,roughlycorrespondingtoclusterseastversuswestoftheGreatPlains-RockyMountainstransition,andnotcorrespondingtosubspecies affiliation(Figs 3 and 4 ).However,foursitesinthetransition,namelyBritishColumbia-North (BC-N),Alberta-North(AB-N),AB-S,andWYhadintermediateQ(STRUCTURE)orLLOD (FLOCK)valuesindicativeofadmixture,andtherewasacleargradationinthisregionbetween geneticclusters(Figs 3 and 4 ).Onaverage,theBC-NandAB-Nsiteshadhigherproportional membershipwiththeeasterncluster,andAB-SandWYsiteswiththewesterncluster,and thereforetheyweregroupedaccordinglyinallsubsequentanalyses.Totestthevalidityofthe mostlikelynumberofclustersidentifiedusingeachofthethreealgorithms,weperformedcontingencytableanalyseswiththenullhypothesisthatclusterassignmentswererandomwith respecttosamplingsites.Inallcaseswerejectedthenullhypothesis(STRUCTURE: G =521.5; FLOCK: G =422.6;GENODIVE: G =77.1;df=20and P < 0.001inallcases)andconcluded thatclusterswerevalid.However,allclustersidentifiedbythethreealgorithmsfailedtomeet HWEexpectations( P < 0.05inallcases).Acrossthefoursites(WA,BC-S,AB-S,andWY) containingbothcarissimaandlucifugusmtDNAhaplotypes(settingasidethe3alacensis-type individualsinWA),clustermembershipwasindependentofsubspeciesaffiliationfortheclustersdefinedbyFLOCK( 2=1.65, P =0.1990)andGENODIVE( 2=0.22, P =0.6390),but wasnotforSTRUCTURE( 2=5.65, P =0.0175). AMOVAanalysisofmicrosatellitegenotypesindicatedweakbutsignificantpopulation structure(global FST=0.0161, P < 0.001;proportionofvariationwithinsites=0.984).The groupingofsitesthatmaximizedamong-groupvariationandminimizedamong-site/withingroupvariationincludedagroupcontainingAKonly,awesterngroupofsamples(CaliforniaSiskiyou(CA-Si),WA,BC-S,Idaho(ID),andWY),andaneasterngroupcontainingallother samples(variationamonggroups=2.72%, P < 0.001;variationamongsiteswithin groups=0.41%, P < 0.001).Generally, FSTvaluesbetweenAlaskaandallothersiteswerehigh andsignificant(0.049 – 0.089; S6Table ). FSTvaluesbetweensitesinthewesternandeastern groupsrangedfrom0.004 – 0.045,valuesbetweensiteswithinthewesterngrouprangedfrom 0.002 – 0.018,andvaluesbetweensiteswithintheeasterngrouprangedfrom-0.005 – 0.013( S6 Table ).AnAMOVAgroupingindividualsbasedontheirsubspecificmembershipsimilarly indicatedweakbutsignificantpopulationstructure(global FST=0.022, P < 0.001,proportion Range-WidePopulationGeneticAnalysisofLittleBrownBats PLOSONE|DOI:10.1371/journal.pone.0128713July8,2015 12/23

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ofvariationwithinsubspecies=0.978).Themaximumpairwise FSTwasbetweenalascensis andlucifugus(0.0270, P < 0.001),theminimumwasbetweenalascensisandcarissima(0.0141, P < 0.001),anddifferentiationbetweencarissimaandlucifuguswasintermediate(0.0200, P < 0.001). Geneticdistancemeasureswerehigheramongsiteswithinthewesterngroupthanamong siteswithintheeasterngroup,andpermutationtestsapproachedsignificanceatthe P < 0.05 level( FST:West:0.023,East:0.002, P =0.0610; G ”ST:West:0.220,East:0.016, P =0.057;Jost ’ s D:West:0.202,East:0.014, P =0.057).Theseresultswerelargelydrivenbytheinclusionofthe AKsiteinthewesterngroup,andifthissitewasremoved,geneticdistancemeasureswerestill consistentlyhigherwithinthewestbutnotsignificantlydifferentbetweengroups( FST:West: 0.007,East:0.002, P =0.361; G ”ST:West:0.088,East:0.016, P =0.273;Jost ’ sD:West:0.081, East:0.014, P =0.274). Isolationbydistance. TotestforisolationbydistanceweperformedManteltestsonthe logarithmofgeographicdistanceandstandardizedgeneticdistance[ FST/(1FST)].Therewere clearsignalsofIBDforbothmitochondrial( r =0.346, P < 0.0001; Fig5a )andmicrosatellite DNA( r =0.537, P < 0.0001; Fig5b )acrosstherangeoflittlebrownbats.Withinidentified clusters,therewasnosignalofIBDintheeast( r =-0.307, P =0.9925; Fig5c ),buttherewas withinthewest( r =0.913, P =0.0411; Fig5d ).TotestifIBDinthewestwasdisproportionately drivenbytheAlaskapopulation,were-rantheanalysiswiththatsiteremoved,andthesignal ofIDBremained( r =0.880, P =0.0069; S1Fig ). Toassessthevalidityofclustersgiventhepatternofisolationbydistance,weplottedgeographicandgeneticdistance( FST)basedonmicrosatellitesaccordingtoclustermembership (pointsidentifiedseparatelyforcomparisonswithinthesameclustervs.indifferentclusters; Fig6 ).Therewasnoclearseparationbetweensitepairsinthesamevs.differentclusters,and lowgeneticdistancesforagivengeographicdistancewereobservedregularlyforsitepairsin differentclusters.However,partialMantelteststoexaminetheassociationbetweenthematrix Fig3.Proportionalmembership(Q)of M lucifugus togeneticclustersfor K =2estimatedusingSTRUCTUREwithsamplinglocationasprior information. Eachbarisasingleindividual,sampledpopulationsaredelineatedbyblacklinesandareorderedbygeographicalsamplinglocationfromwest toeast.Colorsdistinguishgeneticclusters(blueforproportionalmembershipinthewesterncluster,orangeforproportionalmembershipintheea stern cluster). doi:10.1371/journal.pone.0128713.g003 Range-WidePopulationGeneticAnalysisofLittleBrownBats PLOSONE|DOI:10.1371/journal.pone.0128713July8,2015 13/23

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ofgeneticdistancesandamatrixofclustermembershipforthemicrosatellitedata,withthe matrixofgeographicaldistancesasacovariate,weresignificant( P < 0.05inallcases)regardlessofcodingscheme(see Methods )orgeneticdistancemeasure( FSTor G ”ST)used.DiscussionTheemergenceandspreadofWNShasdecimatedbatpopulationsinaffectedareasandraised thespecterofextinctionfor Myotislucifugus M septentrionalis ,andotherhighly-affectedbat species.Therefore,understandingpopulationconnectivityandpossiblebarrierstodisease transmissionisvitaltotheongoingmanagementandconservationofaffectedspeciesandthe developmentofmitigationstrategiestolimitdiseasespreadandassociatedmortality.Here, usingacombineddatasetofmtDNAsequencesandnuclearmicrosatellitegenotypesforlittle brownbatsfromacrosstheirrange,wedemonstrateconsiderablespatialvariationinpatterns offemaledispersalandsignificantgeneticvariationbetweensitesineasternversuswestern portionsoftherangeoflittlebrownbats.Whethertheobservedvariationisrepresentativeof discretegeneticclustersratherthanisolationbydistanceisdebatable(seebelow),butoverall,it isclearthatlevelsofnucleargeneticdifferentiationarelow,andthereisnoevidenceforany majorbarrierstonucleargeneflowacrosstherangeoflittlebrownbats.However,somekey Fig4.Meanlog-likelihooddifference(LLOD)betweentwogeneticclustersobtainedbyFLOCKalongaseriesofgeographicallyorderedsitesfrom westtoeast. Forpresentation,populationsintheGreatPlains-RockyMountainstransitionzone(BC-NAB-N,AB-S,andWY)areorderedbyLLODto demonstratethetransitionamongclusters. doi:10.1371/journal.pone.0128713.g004 Range-WidePopulationGeneticAnalysisofLittleBrownBats PLOSONE|DOI:10.1371/journal.pone.0128713July8,2015 14/23

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spatialpatternsemergefromouranalyses,namely(1)patternsofmtDNAdifferentiationare highlyvariable,withhigh FSTvaluesbetweenmostsamplepairs(includingbetweenallwestern samples,betweenwesternandeasternsamples,andbetweensomeeasternsamples),whilelow mitochondrialdifferentiationwasobservedwithintwogroupsofsamplesfoundincentraland easternregionsofNorthAmerica(showninAMOVAandpairwise FSTanalyses; Fig1b );(2) thesitefromAlaskaishighlydifferentiatedfromallothersinourstudy(showninAMOVA andpairwise FSTanalyses);and(3)westernsitesarecharacterizedbysignificantisolationby distancebasedonmicrosatellites,whilethoseintheeastarenot.Thesedataraisethepossibility thatthecurrentpatternsofspreadof Pd observedineasternNorthAmericamaynotapplyto theentirerangeofthelittlebrownbat,andthattheremaybebroad-scalespatialvariationin theriskofWNStransmissionandoccurrenceifthediseasecontinuestospreadwest. Thepresenceofisolationbydistanceisamajorconfoundingfactorwhenexamininglevels ofgeneticstructureamongpopulationsofwidespreadspeciessuchaslittlebrownbats,because clinalvariationmaybeinterpretedasthepresenceofdiscreteclustersevenintheabsenceof barrierstogeneflow[ 66 78 80 ].Allclusteringmethodsweemployedonourmicrosatellitedata identifiedthepresenceoftwogeneticclusters,roughlydividingsiteseastvs.westoftheGreat Plains-RockyMountainstransition.However,wealsoobservedastrongpatternofisolationby distance,indicatingthattheseobservedclustersmaybeanartifactofdispersallimitationand clinalvariationacrosstheverylargerangeofthisspecies.Additionalanalysestotestthe Fig5. Standardizedgeneticdistance[ FST/(1FST)]plottedagainstthelogarithmofgeographicdistanceincludingallsampledpopulationsformtDNA(a),and microsatellites(b),andfortheeastern(c)andwestern(d)populationclustersbasedonmicrosatellites. doi:10.1371/journal.pone.0128713.g005 Range-WidePopulationGeneticAnalysisofLittleBrownBats PLOSONE|DOI:10.1371/journal.pone.0128713July8,2015 15/23

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validityofclustersprovidedmixedresults.Ontheonehand,geneticdistancesbetweeneastern andwesternsiteswererelativelylow(thebestgroupingintheAMOVAexplainedonly2.7%of thevariationinmicrosatelliteallelefrequencies),aManteltestshowednoclearseparation betweensitepairsinthesameversusdifferentclusters( Fig6 ),andidentifiedclustersfailedto meetHWexpectations,suggestingthatclustersdidnotrepresentgeneticallypanmicticpopulations(cf.[ 66 ]).Ontheotherhand,partialManteltestscontrollingforgeographicdistance revealedasignificantcorrelationbetweenclustermembershipandgeneticdistance,suggesting thatwesternandeasternclustersweredifferentiateddespitethesignalofisolationbydistance (cf.[ 80 ]). Whatisclearfromthesedataisthatthereissignificantgeneticvariationamongsamples fromeasttowestacrosstherangeoflittlebrownbats(althoughwhethertheyrepresentdiscrete geneticclustersratherthanisolationbydistanceisdebatable),butlevelsofnucDNAgenetic differentiationwererelativelylow,andtherewasnounambiguousevidenceforanymajorbarrierstogeneflowthatmightseverelyrestrictthespreadofWNS.Geneflowintemperatebats ismediatedthroughthepermanentdispersalofindividualsandtheexchangeofgenesatmatingcongregationsduringswarmingandhibernation.Thelackofisolationbydistanceandlow levelsofnucDNAdifferentiationamongsitesineasternNorthAmericaisconcordantwiththe continuousspreadofWNSfromitsorigininNewYork,andindicatesthatgeneflowviamatinghasoccurredoverwidegeographicareas.Furthermore,thediseasehaspassed,oriscurrentlypassing,throughregionsinwhichtherearelowlevelsofmtDNAdifferentiationamong Fig6.Pairwisegenetic( FST)basedonmicrosatellitesandgeographicdistancevalueshighlightingpopulationpairswithinthesamecluster(blue dots)andindifferentclusters(orangedots). doi:10.1371/journal.pone.0128713.g006 Range-WidePopulationGeneticAnalysisofLittleBrownBats PLOSONE|DOI:10.1371/journal.pone.0128713July8,2015 16/23

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sites(onegroupintheeasternUnitedStatesandonegroupinthecentralUnitedStatesand Canadianprovinces; Fig1b ).Mosttemperatebatsarecharacterizedbyrelativelyhighlevelsof femalephilopatryandmale-biaseddispersal,resultinginsignificantmatrilinealgeneticstructuringofpopulations(e.g.[ 35 86 – 88 ]).However,ourdatasuggestthattheexchangeoffemales amongpopulationsacrosslargeportionsoftherangeoflittlebrownbatsisanon-trivialsource ofgeneflowthatmaybecontributingtothespreadofWNS,andareconsistentwithsimilar inferencesoffemaledispersalamongpopulationsoversmallerspatialscalesinlittlebrownbats [ 6 33 35 42 ]andotherbatspecies[ 38 89 ]basedonmtDNA.Inaddition,thesedataareconsistentwithbandingdatashowingextensivemovementsbyindividuallittlebrownbatsofboth sexesoverhundredsofkilometersbetweensummerroosts,swarmingsites,andhibernacula withinandbetweenyearsincentralCanada[ 27 42 ].Bandingstudiesoflittlebrownbatsin otherpartsoftheirrangewouldhelptoresolvewhethertheobserveddifferencesarebetter explainedbyhistoricaldemographyorcurrentgeneflow.ThehighlevelsofobservedmtDNA differentiationinotherportionsoftherange,particularlyinthewest,suggestimportantspatial variationinfemaledispersalpatterns,andhighlighttheneedtoconsiderpermanentmovementsofbothmalesandfemalesandincorporateregionalvariationindispersalratesanddistancesinmodelsofWNStransmissiondynamics. GiventhelackofmajorphysiographicbarrierseastoftheRockyMountainsandthehigh levelsofgeneflowweinferred,itislikelythatWNSwillcontinuetoexpanditsrangeacross easternNorthAmerica.CurrentmodelsofdiseasespreadindicatethatWNSexhibitscharacteristicsofanexpandingepizooticwhereinrelativelydistantsiteshavelowerinfectionrisk,but overtimeinfectionratesincreaseandtheeffectofdistancediminishesasthedisease ‘ fillsin ’ behindtheexpansionfront[ 19 20 ].However,thesemodelsarebasedondatafromeasternportionsoftherangewheregeneflowishigh.Evenwithinthisregiontherateofspreadmaybe restrictedbyconsistentspatialvariationinabove-groundorcavemicroclimatesthatmaylimit thesurvivalandgrowthof Pd (cf.[ 20 21 ]),orbyspatialvariationintopographyorlanduse thatlimitsmovementsanddispersalbybats[ 6 ].InPennsylvania,forexample,topographical featuressuchastheAppalachianhighplateauandtheAlleghenyFrontescarpmentmayhave influencedseasonalmigrationpatternsoffemalebats,therebylimitingmatrilinealgeneflow anddiseasetransmissionratesamongpopulations[ 6 ].Indeed,twogeneticallydistinctpopulationsofwinteringcolonieswereobservedoneithersideoftheAlleghenyFront[ 6 ];hibernating coloniesoflittlebrownbatslocatedonthewesternAppalachianhighplateauwereinfected withWNS1 – 2yearslaterthancoloniesinthecentralmountainousandeasternlowland regionsofthestate[ 16 ].Thus,topographicorclimaticvariationmayslowthespreadofWNS throughsomeareasbylimitingpopulationconnectivityofthehostorthesurvivalandgrowth of Pd ,andmayexplainsomeoftheobservedspatialvariationtodateintherateanddirection ofWNSspreadthrougheasternNorthAmerica. Ourgeneticdataindicatinglowerlevelsofpopulationconnectivityinthewestsuggestthat ifWNSreacheswesternpopulations,therateofdiseasespreadmaydecline.ThehighmtDNA FSTvaluesamongwesternpopulations( S5Table )indicatethatfemalemovementsarehighly restrictedrelativetoeasternpopulations.OveralllevelsofnucDNAgeneflowamongwestern siteswerereducedrelativetotheeast,andwesternsiteswerecharacterizedbyisolationbydistancebasedonmicrosatelliteswhileeasternsiteswerenot.Theseresultsmay,inpart,berelated tothegreatertopographicalandecologicalheterogeneityinthewest,whichincludesmultiple mountainranges,plateaus,basins,andcoastallowlands,andwhichhasbeenimplicatedin recurrentphylogeographicpatternsinawidevarietyofothertaxa[ 90 ].Hibernationbehavior ispoorlycharacterizedforwesternNorthAmericanlittlebrownbatpopulations.Allknown largehibernatingpopulations( > 10,000individuals)aredescribedfromeasternNorthAmerica,andidentifiedhibernaculainthemountainouswesttypicallyhavelowercensussizesthan Range-WidePopulationGeneticAnalysisofLittleBrownBats PLOSONE|DOI:10.1371/journal.pone.0128713July8,2015 17/23

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manyhibernaculaintheeast.Thehighphysiographicvariationinthemountainouswestmay limitpopulationconnectivityandthescaleofbatmovements,andthehighdensityofmines andcavesinmanyregionsinthewestmayresultinsmallerandmorediffusehibernatingcoloniesrelativetoeasternNorthAmerica.Comparativedataonconnectivitybetweensummer andwintersites(asin[ 27 ])areurgentlyrequiredtoquantifyspatialandtemporalpatternsof movementoflittlebrownbatsinthewesternportionoftheirrangeandtopredictpotential ratesofWNStransmission.Further,themostdistantpopulationwesampled(inAlaska)was byfarthemostdivergentfromallotherpopulations,andwerequiremuchmoredensesamplinginthewesternportionoftherangeoflittlebrownbatstodetermineifanyotherpopulationsareequallyormoreisolatedandhencemayhavereducedcontactrateswithother regionalpopulations. Thespatialvariationinpopulationconnectivityweobservedwaslargelyindependentof subspecificaffiliation.Phylogeneticanalysisrevealedthepresenceofthreedivergentlineages basedonmtDNA(correspondingtopreviouslydefinedsubspecies M l alascensis M l carissima and M l lucifugus ;asin[ 32 ]),withthenotablefindingofmultiplelineagesatthesame samplinglocationsinsouthernBritishColumbia,southernAlberta(asin[ 33 ]),andWyoming. AlthoughCarstensandDewey[ 32 ]providedsomesupportfrommtDNAandnuclearintrons fordiscreteevolutionarylineageswithin M lucifugus ,wefoundthatclustermembershipbased onmicrosatelliteswasindependentofsubspecificaffiliation,andweestimatedlowlevelsof nucDNAdifferentiationamongsubspecies( FST=0.022inAMOVAanalysis).Ourobserved discrepancybetweenmtDNAandnucDNAsignalsmaybedueinparttohomoplasy,particularlyatrapidly-evolvingmicrosatelliteloci.Alternatively,incompletelineagesortingmightbe producingdiscordantpatternsamongloci,particularlyforaspecieswitharelativelylarge effectivepopulationsize( Ne 400,000basedonCarstensandDewey ’ s[ 32 ]estimateof for M lucifugus )andrelativelyrecentdivergence(divergencefromwestern Myotis sp.approximately1 – 1.5Mya[ 32 ]).Athirdalternativeisthatourfindingoflowerdifferentiationat nucDNAcomparedtomtDNAisthatpatternsatmtDNAmayreflectgeneticdifferentiation thatevolvedandwerereinforcedaspopulationsuseddistinctglacialrefuges(asin[ 34 ]),while patternsatnucDNAreflectsecondarycontactparticularlymediatedviamalegeneflow. Ourdatashowextensivespatialvariationinlevelsofconnectivityamonglittlebrownbat populationsandprovidevaluableinformationforunderstandingpastandfuturepatternsof WNSspread.However,theuseofgeneticmethodstoinferpatternsoftransmissionassumes thatpatternsofgeneflowareindicativeofthemovementofinfectiousindividuals,andwe mustrecognizethattheriskofdiseasetransmissionmaybehigherthangeneticdatamayindicatebecausetheremaybemorecontactsamonginfectedandsusceptibleindividuals,including amongmembersofmultiplespecies,thanjustthosethatleadtogeneflow.Urgentresearchis requiredtodeterminehowandwhenindividualbatsmaybeexposedto Pd spores,andhow contactsofvaryingdurationsandseasonaltimingsinfluencetheriskofWNStransmission. Ultimatelyweneedtolearnwhetherbriefcontactsduringmatingcanresultintransferof sporesleadingtoinfectionorwhetherpermanentdispersalsaredrivingtransmission.Theusefulnessofourgeneticdataonlittlebrownbatsalsorestsontheassumptionthatintraspecific transmissiondynamicsoutweightheimpactofcross-speciestransmission,giventhatmultiple sympatricbatspeciesareaffectedbyWNS.Thisassumptionmaybejustified,aspost-WNS populationdeclinesofaffectedbatspeciesarenotinfluencedbypopulationsizesofother affected,cohabitingbatspecies[ 21 ],butresearchisrequiredtoassesshowoftencross-species transmissionmaytakeplaceandhowtherateofintroductionofinfectivepropagulestoenvironmentalreservoirsisinfluencedbymultiplespeciescohabitingthesamehibernaculum. Inconclusion,thisstudyidentifiedhighlevelsofgeneticvariationamongpopulationsoflittlebrownbatsacrosstheirrange,andmitochondrialDNAsequencesrevealedconsiderable Range-WidePopulationGeneticAnalysisofLittleBrownBats PLOSONE|DOI:10.1371/journal.pone.0128713July8,2015 18/23

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spatialvariationinpatternsoffemaledispersal.Overalllevelsofnucleargeneticdifferentiation among M lucifugus populationsarelow,andwedidnotidentifyanymajorbarrierstogene flowacrosstheirrange.However,levelsofgeneticdifferentiationatbothmtDNAandmicrosatellitesaresignificantlyhigheramongpopulationstothewestoftheGreatPlains-Rocky Mountainstransition,suggestingthatthecurrentpatternofspreadofWNSandriskoftransmissionof Pd observedineasternNorthAmericamaynotapplytotheentirerangeofthelittle brownbat.SupportingInformationS1Fig.Standardizedgeneticdistance[ FST/(1 – FST)]formicrosatellitesplottedagainstthe logarithmofgeographicdistanceforthewesternpopulationclusterwiththeAlaskapopulationremoved. (DOCX) S1File.Datasourcesandpermissionsusedtoconstruct Fig1 (PDF) S1Table.Listof Myotislucifugus specimensincludedinanalyses. (DOCX) S2Table.Locusinformationfor11microsatellitesusedtoamplify M lucifugus (DOCX) S3Table.Listofspecimens(allinthegenus Myotis ),yearofcollection,samplinglocalities, andGenbankaccessionnumbersforCOIsequencesusedinphylogeneticanalysis. (DOCX) S4Table.Diversityofmicrosatelliteloci,includingobserved( HO)andexpectedheterozygosity( HE),numberofalleles( NA),allelicrichness( AR )andtheinbreedingcoefficient ( FIS). (DOCX) S5Table.Pairwise STvaluesamongpopulationsbasedonmtDNACOIsequences. (DOCX) S6Table.Pairwise FST(lowerdiagonal)andJost ’ s D (upperdiagonal)basedonnucDNA microsatellites. (DOCX)AcknowledgmentsWearegratefultonumerousfieldresearchersforprovidingsamples:E.Adams,E.Britzke,C. Butchkoski,R.Christophersen,M.Delorme,C.Dobony,E.Gates,K.Getz,J.Gruver,M.Gumbert,D.Hobson,J.Johnson,H.Kaarakka,C.Lausen,S.Loeb,F.Martinez,H.Niederriter,G. Nordquist,D.Pierson,W.Rainey,D.Redell,D.Solick,C.Stihler,K.Stroebel,M.Valent,J.Van deVenter,andC.Willis.Wealsothankthefollowingmuseumsforprovidingtissuesamples: AngeloStateNaturalHistoryCollection,CarnegieMuseumofNaturalHistory,Centrode InvestigacionesBiolgicasdelNoroesteS.C.,MuseumofVertebrateZoologyatBerkeley, NationalMuseumofNaturalHistory,andtheUniversityofAlaskaMuseumoftheNorth.We thankE.ClareforgenerouslysharingunpublishedmtDNAsequences.WealsothankJ.Rosensternforassistancewithlabwork,J.Glatzforhelpwithproducingmaps,andS.Gillforprovidingvaluablecommentsonearlierversionsofthismanuscript. Range-WidePopulationGeneticAnalysisofLittleBrownBats PLOSONE|DOI:10.1371/journal.pone.0128713July8,2015 19/23

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