Evolutionary Applications. 2017;1–15.  | 1wileyonlinelibrary.com/journal/eva

Received:17April2017  |  Accepted:26June2017DOI: 10.1111/eva.12514

O R I G I N A L A R T I C L E

Profiling the immunome of little brown myotis provides a yardstick for measuring the genetic response to white- nose syndrome

Michael E. Donaldson1,*  | Christina M. Davy1,2,*  | Craig K. R. Willis3 |  Scott McBurney4 | Allysia Park4 | Christopher J. Kyle5

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.©2017TheAuthors.EvolutionaryApplicationspublishedbyJohnWiley&SonsLtd

*Theseauthorscontributedequallytothiswork.

1EnvironmentalandLifeSciencesGraduateProgram,TrentUniversity,Peterborough,ON,Canada2WildlifeResearchandMonitoringSection,OntarioMinistryofNaturalResourcesandForestry,Peterborough,ON,Canada3DepartmentofBiologyandCentreforForestInterdisciplinaryResearch(C-FIR),UniversityofWinnipeg,Winnipeg,MB,Canada4CanadianWildlifeHealthCooperative,AtlanticRegion,AtlanticVeterinaryCollege, UniversityofPrinceEdwardIsland,Charlottetown,PEI,Canada5ForensicScienceDepartment,TrentUniversity,Peterborough,ON,Canada

CorrespondenceChristopherJ.Kyle,ForensicScienceDepartment,TrentUniversity,Peterborough,ON,Canada.Email:christopherkyle@trentu.caand MichaelDonaldson,EnvironmentalandLifeSciencesGraduateProgram,TrentUniversity,Peterborough,ON, Canada.Email:michaeldonaldson@trentu.ca

Funding informationSpeciesatRiskResearchFundforOntario;NaturalSciencesandEngineeringResearchCouncilofCanada,TheCanadianWildlifeHealthCooperative;LiberEroFellowshipProgram

AbstractWhite-nosesyndrome(WNS)hasdevastatedpopulationsofhibernatingbatsineasternNorthAmerica,leadingtoemergencyconservationlistingsforseveralspeciesincludingthepreviouslyubiquitouslittlebrownmyotis(Myotis lucifugus).However,somebatpop-ulationsneartheepicenteroftheWNSpanzooticappeartobestabilizingafterinitialprecipitousdeclines,whichcouldreflectaselectiveimmunogeneticsweep.Toinvesti-gatethehypothesisthatWNSexertssignificantselectionontheimmunomeofaffectedbatpopulations,wedevelopedanovel,high-throughputsequencecaptureassaytarget-ing138adaptive,intrinsic,andinnateimmunitygenesofputativeadaptivesignificance,aswellastheirrespectiveregulatoryregions(~370kbpofgenomicsequence/individ-ual).WeusedtheassaytoexplorebaselineimmunogeneticvariationinM. lucifugus and toinvestigatewhetherparticularimmunegenes/variantsareassociatedwithWNSsus-ceptibility.Wealsousedourassaytodetect1,038putativelyneutralsinglenucleotidepolymorphismsandcharacterizecontemporarypopulationstructure,providingcontextfortheidentificationoflocalimmunogeneticadaptation.Sequencecaptureprovidedacost-effective,“all-in-one”assaytotestforneutralgeneticandimmunogeneticstruc-tureandrevealedfine-scale,baselineimmunogeneticdifferentiationbetweensamplingsites<600kmapart.We identified functional immunogeneticvariants inM. lucifugus associatedwithWNSsusceptibility.Thisstudylaysthefoundationsforfutureinvestiga-tionsofrangewide immunogeneticadaptationtoWNSinM. lucifugus and provides a blueprintforstudiesofevolutionaryrescueinotherhost–pathogensystems.

K E Y W O R D S

genotype-by-sequencing,immunogenetics,Myotis lucifugus,white-nosesyndrome

1  | INTRODUCTION

Host–pathogendynamicsarechangingatanunprecedented rateasclimatechangeandhuman-mediated transportexpand the rangeof

pathogens into previously inhospitable/inaccessible environments(Fisher etal., 2012).As pathogen ranges shift, disease-related pop-ulationdeclines innaïvewildlifepopulationsoften threatenpopula-tionpersistence, asevidencedby several emergingwildlifediseases

2  |     DONALDSON et AL.

(Gallana,Ryser-Degiorgis,Wahli,&Segner,2013;Smithetal.,2012).Selective forcesexertedby infectiousdiseasescan rapidly influencethedistributionofadaptivegeneticvariantsassociatedwithdiseasesusceptibility over short timescales (Gallana etal., 2013). For in-fectious diseases of conservation significance, this process of localadaptation can result in evolutionary rescueof a population,wheredisease-resistantanimals survivea strongselectivesweep fromdis-easeandpasstheirresistancetotheiroffspring(Carlson,Cunningham,&Westley,2016;Maslo&Fefferman,2015).Spatialpatternsoflocaladaptation to strong selective sweeps may be linked to particulargenevariantsfavoredinlocalinteractions(Hansen,Olivieri,Waller,&Nielsen,2012;Kyleetal.,2014;Rico,Morris-Pocock,Zigouris,Nocera,&Kyle,2015;Schovilleetal.,2012).Determininghowthesevariantsarespreadorlocalizedamongpopulationsisessentialtounderstand-ingandmanagingtheemergenceofnewselectivepressures,suchasemerginginfectiousdiseases(Eizaguirre,Lenz,Kalbe,&Milinski,2012;Kyleetal.,2014).

White-nose syndrome (WNS) is a recently emerged disease in hibernating bats caused by the fungal pathogen Pseudogymnoascus destructans. The fungus was introduced from Eurasia to NorthAmerica,where itwas first documented in Schoharie County,NewYork, in 2006 (Blehert etal., 2009; Leopardi, Blake, & Puechmaille,2015).WNShasspreadrapidlyacrossNorthAmerica,causing>80%declines in some eastern bat populations (Frick etal., 2010, 2015;Langwigetal.,2012;Lorchetal.,2016).WhileseveralNorthAmericabatsarehighlysusceptibletoWNS,Europeanbatsdonotexperiencemortality from infection with P. destructans (Puechmaille, Fuller, &Teeling,2011;Puechmaille,Wibbelt, etal., 2011).Controlledexper-imentswithcaptivebatsshowthat identicalstrainsofP. destructans cause mortality in NorthAmerican little brownmyotis (Myotis luci-fugus)butnot inEuropeangreatermouse-earedbats (Myotis myotis; Davyetal.,inpress),suggestingageneticbasisforimmunotoleranceorimmunoprotection.

There are several promising leads for thedevelopmentof treat-ments for WNS (e.g., Cheng etal., 2016; Cornelison, Gabriel,Barlament, & Crow, 2014;Wilcox &Willis, 2016), but no effectivemitigation or treatment protocols are currently available. However,somepopulationsneartheepicenterofWNSmaybestabilizingfol-lowingtheirinitial,precipitousdeclines(Dobonyetal.,2011;Langwigetal.,2012,2017).Persistenceof thesepopulationsdoesnot seemto be associated with immigration (Maslo, Valent, Gumbs, & Frick,2015), butmay indicate evolution of resistance or tolerance to thedisease(Langwigetal.,2017).Thus,adaptationandevolutionaryres-cuemaybethebesthopeforrecoveryofbatpopulationsaffectedbyWNS (Maslo&Fefferman,2015).Understandingpatternsof immu-nogeneticadaptationtoWNSisthereforecriticaltodeterminingdis-easemanagementstrategiesandrecoveryprogramsfortheaffectedpopulations.

Immunegenesmediatetheinitialresponseofindividualstopatho-gensandinmanycases,theacquisitionofimmunity.Atthepopulationlevel,geneticdiversityofimmunegenesinfluencesresistanceortoler-ancetodiseaseviapathogen-mediatedbalancingselection(Eizaguirreetal.,2012;Ricoetal.,2015).Studiesofwildlifepopulationsgenerally

focusonadaptiveimmunity,whichhasoftenbeenassessedbyusinggenetic diversity in the major histocompatibility complex (MHC).DiversityattheMHCprovidesaproxyforpotentialtoadapttoshiftingpathogenpressures,duetotheroleofMHCinpathogenrecognitionand pathogen susceptibility (Acevedo-Whitehouse & Cunningham,2006;Eizaguirreetal.,2012;Kyleetal.,2014).Somestudiesofimmu-nogeneticdiversityalsoincludereceptorgenesassociatedwithinnateimmunity(e.g.,Toll-likereceptorsandinterleukins)andthesemarkershave revealed spatial patterns of resistance to emerging infectiousdiseases such as chytridiomycosis and mycoplasmosis (Bonneaud,Balenger,Zhang,Edwards,&Hill,2012;Savage&Zamudio,2011).

TheMHCDrb1locusinM. lucifugusisamongthemostpolymorphicrecordedinmammalstodate(Palmeretal.,2016).Pyrosequencingof160individualssampledacrossCanadasuggeststhatbalancingselec-tionhasmaintained similarMHCdiversity amonggeneticallydiffer-entiatedsubpopulations,whichmaybedisruptedbyWNS-mediatedimmunogenetic selection (Davy etal., in press). However, the extremeobservedpolymorphismoftheDrb1locus inM. lucifugus is duepartlytomultiplegeneduplications,whichlimitstheuseofthesedata.Furthermore,susceptiblebatsinfectedwithP. destructansupreg-ulatemultiple,complementaryimmuneresponses(Fieldetal.,2015;Lilleyetal.,2017;Mooreetal.,2013;Rapinetal.,2014),soimmuno-geneticselectionbyWNScannotbe fullycapturedbyexperimentaldesignsthattargetsingle,candidategenes.Nootherpopulation-levelimmunogeneticanalysesexistforM. lucifugus,orforanyotherNorthAmericanspeciesofbatsthreatenedbyWNS.Fortunately,newmolec-ulartoolsallowmorecomprehensiveinvestigationofimmunogeneticadaptation(Harrisson,Pavlova,Telonis-Scott,&Sunnucks,2014).

Genotype-by-sequencing (GBS) assays have emerged as a cost-effectivemethodforobtainingpopulation-levelassessmentsofneu-tralandfunctionalgeneticvariation,andidentifyinglocaladaptation(Tiffin&Ross-Ibarra,2014).GBSassaysinvolveenrichingforgenomicsubsetsofDNAviarestrictionenzyme-,amplicon-,orhybridization-basedmethods (Jones & Good, 2016), conducting high-throughputsequencingand identifying singlenucleotidepolymorphisms (SNPs).Targeted approaches, including amplicon- and hybridization-basedGBS,havebeenused inwildlife studies to identifySNPs in specificcodingandregulatoryregionsofimmunegenes,collectivelycalledthe“immunome.”TargetedGBScanidentifypopulation-levelimmunoge-neticshiftsinresponsetopathogens,andhasbeenappliedtoarangeofspecies, includingtheTasmaniandevil (Sarcophilus harrisii;Morris,Wright, Grueber,Hogg, & Belov, 2015), turkey (Meleagris gallopavo; Reed,Mendoza,&Settlage,2016),graywolf (Canis lupus;Schweizeretal.,2016), thinhornsheep (Ovis dalli;Roffleretal.,2016),andredfox (Vulpes vulpes; Donaldson etal., unpublished). GBS is an attrac-tiveoptionforunderstandingtheimpactsofWNSonimmunogeneticdiversity in bat populations, because it allows accurate characteri-zationofdiversity atduplicated loci, andcost-effective targetingofmultiple,relevantgenes.Regardlessofthegenomiccoverageofhigh-throughputsequencingmethods,populationgeneticanalysesstillrelyonadequatesamplesizestodetectthegeneticsignatureofselectionbypathogensorotherselectivepressures,reinforcingtheimportanceofacost-effectiveapproach.

     |  3DONALDSON et AL.

Wedevelopedanovelhybridization-basedGBSassaytocharac-terizethegeneticdiversityoftheM. lucifugus immunome. Our assay includes170 loci, including120 immunegenesand their regulatoryareas,18Drb1-likeexon2regions,and32neutrallocitoallowchar-acterizationofneutralpopulationstructure,againstwhichhypothesesof local adaptationcanbe tested.Weapplied thisassay to test thehypothesis thatWNSexerts significant selectionon the immunomeof affected bat populations. Controlling for neutral genetic popula-tionstructure,wepredictedimmunogeneticdivergencewouldbede-tectable betweenWNS-naïve populations and populations affectedbyWNS.This study provides a foundation for future investigationsofrangewideimmunogeneticadaptationtoWNSinM. lucifugus and otheraffectedspeciesofbats.

2  | MATERIALS AND METHODS

2.1 | Microsatellite markers, immune genes and probe development

Wedevelopedour assay for primary application toM. lucifugus be-cause this species’ genome ispubliclyavailable (Myoluc2.0genomeassembly,Ensemblreleaseversion81;Cunninghametal.,2015),andrecentresearchhasidentifiedputative“WNS-response”genesforthisspecies (Rapin etal., 2014),which informed our selection of targetgenesforsequencecapture.

Toassessfunctionalimmunogeneticvariation,weassembledalistof120candidategenes related to immune systemprocessesbasedon(i)theHumanInnate&AdaptiveImmuneResponsesRT2ProfilerPCRArray(Qiagen);(ii)areviewofinnateandadaptiveimmunity,de-velopment,andsignaling(Knight,2013); (iii)astudyofgeneexpres-sion in M. lucifugusfollowinginfectionwithP. destructans(Rapinetal.,2014);and (iv)ageneontology (GO)termsearch intheM. lucifugus EnsembldatabaseforGOrecordsrelatedtofungi (includingcellularresponsetomoleculeoffungalorigin,defenseresponsetofungus,andneutrophil-mediatedkillingof fungus).Weused thiscandidategenelisttoquerytheM. lucifugusMyoluc2.0genomeassemblyandcreatedaBED-formattedfilecontainingcoordinatesforallexons.Additionally,we targeted potential regulatory regions by including coordinatesforthe1,500-bpregionupstreamfromthefirstexonforeachgene.Finally,weaddedexon2coordinatesfor18Drb1-likegenesidentifiedinEnsemblthatputativelyencodefunctionalfull-lengthproteins.

To target putative neutral markers for the detection of geneticpopulationstructure,weselected32microsatellitemarkersforM. lu-cifugusfromthepublishedliterature(Burns,Broders,&Frasier,2012;Castella&Ruedi,2000;Johnsonetal.,2014;Oyler-McCance&Fike,2011; Piaggio, Figueroa,&Perkins, 2009;Trujillo&Amelon, 2009).Usingtheseprimersets,weaddedcoordinatesforthesemarkerstothe BED-formatted file. In total, the final BED-formatted file con-tainedcoordinates for170 loci.Descriptions forprotein-codingandmicrosatellite regions are provided in the Supporting Information(TablesS1–S2).

CustomNimbleGenSeqCapEZprobes(Roche)wereproducedfor“primary targets” using the BED-formatted file and theM. lucifugus

Myoluc2.0 genome assembly as a reference. We added 100-bp“padding” toeach target to increase theefficiencyof the sequencecapture,andweuseda“relaxed”probedesignthatallowedupto20closematches to theM. lucifugus reference genome.We comparedourprobes to theM. lucifugus referencegenometoensure thatourassayhadalowlikelihoodfor“off-target”sequencecapture:91%oftheprobesmatchedonlytheirtargetsequence,and99%hadfiveorfewermatchestotheM. lucifugusreferencegenome.

2.2 | Sample collection, DNA extraction, and quantification

Allworkwasconductedunderapprovedanimalcareprotocolsfromthe University of Winnipeg and the Ontario Ministry of NaturalResources andForestry.To test the relative impactsof geographiclocation and exposure to P. destructans on neutral and immunoge-netic population structure inM. lucifugus, we assigned bat samplescollected in eastern Canada to three post hoc groups (Table S3).The first group includedbats thatwerenonharmfully sampledat ahibernaculum inManitoba, Canada (MB, n=28), that did not con-tainP. destructans at the time of sampling. The second group con-tainedbatsfromtwohibernaculanearThunderBay,Ontario,whichwerealso sampledbefore thearrivalofP. destructans (ON,n=36).WingbiopsiesfromthesebatswereimmediatelystoredinRNAlater (Qiagen) following sampling. These twogroups representour “pre-WNS treatment.” The third group came from populations of batsinAtlanticCanadathathadbeenexposedtoWNSforat leastonewinter,butwere foundmoribundordead in thewinterof2014 intheAtlanticprovincesofNovaScotiaandPrinceEdwardIsland(ATL,“post-WNS treatment,” n=28). These bats were submitted to theCanadianWildlifeHealthCooperative (CWHC),AtlanticRegion fornecropsy.Ofthesepost-WNSbats,15werediagnosedaspositiveforWNS,12werediagnosedassuspectDforWNS,and1wasnegativeforWNSusingtheapproveddiagnosticcategoriesforWNSfoundintheCanadianBatWNSNecropsyProtocol(CWHC,2014),andtheywereassumedtonotbe immunotolerantnor immunocompetent toWNS.Wingtissuewascollectedfromtheleftdactylopatagiummajorduring thesenecropsiesandstored in lysisbuffer (4Murea,0.2MNaCl, 0.5%n-lauroyl sarcosine, 10mM1,2-cyclohexanediaminetetraacetic acid, 0.1mMTris–HCl pH8.0) until analysis.We dissolvedalltissuesamplesinlysisbuffercontaining600U/mlproteinaseKat56°Cfor2hr.WeextractedDNAusingeithertheautomated96-wellMagneSilBloodGenomicMaxYieldSystem(Promega)ortheDNeasyBloodandTissueKit (Qiagen).We thenquantifiedallDNAextrac-tionsusingtheQuant-iTPicoGreendsDNAAssayKit(ThermoFisherScientific).

Toinvestigatethepossibilitythatourassaycouldalsobeusedtoinvestigate immunogeneticvariationandadaptationinotherspeciesaffectedbyWNS,wealso isolatedDNAfrom“post-WNS”Eptesicus fuscus(n=2)fromNewBrunswick,bothsuspectBforWNS(CWHC,2014) and M. septentrionalis (n=2) from Nova Scotia and PrinceEdwardIsland,positiveandsuspectDforWNS,respectively(CWHC,2014),andincludedthesesamplesintheassay.

4  |     DONALDSON et AL.

2.3 | DNA library preparation, sequence capture, and high- throughput sequencing

WepreparedDNAlibrariesusingtheKAPAHTPLibPrepKit(Roche)andperformed the sequence captureusing theNimbleGenSeqCapEZDeveloper Library kit v5.1 (Roche)with the followingmodifica-tions to themanufacturer’s protocol. EachDNA library preparationused150ngtotalDNA.TruSeqHTDual-IndexAdapters(IntegratedDNA Technologies) resuspended in Nuclease Free Duplex Buffer(IntegratedDNAtechnologies)wereusedatafinalconcentrationof600nMinsteadoftheSeqCapAdapterKitsAandB(Roche)duringadapter ligation.We performed 11 cycles during the LM-PCR, andinitialDNAlibraryqualitywasassessedbyethidiumbromide-stainedgel electrophoresis using a 2%E-Gel (ThermoFisher Scientific).Weused 1 μlofthexGenUniversalBlockingOligoTSHT-i5(IntegratedDNATechnologies)and1μlxGenUniversalBlockingOligoTSHT-i7(IntegratedDNATechnologies) insteadof theNimbleGenMultiplexHybridizationEnhancingOligoPool(Roche),andweusedNimbleGenSeqCap EZDeveloper Reagent (Roche) instead ofNimbleGenCOTHuman DNA (Roche) during hybridization sample preparation. Thehybridization was carried out at 47°C for 72hr. We assessed thepooled target-enriched DNA quality using a bioanalyzer (AgilentTechnologies)andperformedhigh-throughputsequencingonaHiSeq2500rapidrunusing2×100-bpreadsonasingleflowcell(Illumina).

2.4 | Sequence alignment, variant annotation, and SNP/INDEL analysis

We used the bwa-mem command in the burrows-wheeler aligner v0.7.12(bwa;Li,2013)toalignpaired-endreadstotheMyoluc2.0ge-nomesequenceandcompiledsequencealignmentmetricsusingsam-toolsv1.2(Lietal.,2009).Weusedthegenome analysis toolkit v3.5 (gatk;McKennaetal.,2010)forbasequalityscorerecalibration,rea-lignmentofinsertions/deletions(INDELs),duplicateremoval,depthofcoveragecalculations,SNP/INDELdiscovery,andgenotypingacrossallsamples,usingstandardhardfilteringparametersorvariantqualityscorerecalibrationaccordingtogatkbestpracticesrecommendations(DePristoetal.,2011;VanderAuweraetal.,2013).

2.5 | Analysis of targeted microsatellites

We used two different approaches to assign microsatellite geno-types. The firstmethod (gatk) relied on sequence alignment to theM. lucifugusgenome.WeidentifiedasingleINDELtorepresenteachmicrosatellitebyselectingtheshort tandemrepeat thatyieldedthehighest:(i)percentageofheterozygotes;(ii)gatk“quality”score;or(iii)numberofalleles.Foreachofthesethreescenarios,weusedthegatk tocalculatethenumberofheterozygotesforeachmarkerusingasub-setofourdatathatincludedonlythe36ONsamplesandthe28MBsamples.These64sampleswerepreviouslygenotypedbasedontra-ditionalPCRamplificationandsequencingof11microsatellitemark-ers(Davyetal.,inpress).Wecalculatedthenumberofheterozygotesforeachof thesemarkers, toassesswhetheroursequencecapture

assaycouldbeused tobuildonpreviousmicrosatellite-basedstud-ies.OursecondgenotypingmethodusedtheGalaxyplatform(Afganetal., 2016) to run STR-FM (Galaxy Version 1.0.0; Fungtammasanetal.,2015)andidentifydi-andtetra-nucleotideSTRsfromtherawIllumina.fastqdata,withoutgenomealignment.

2.6 | Analysis of functional loci and identification of novel, putatively neutral SNPs

Weusedgatktoassembleamastervariantcallformatfile(.vcf)thatincludedSNPswith amaximummissinggenotype frequencyof5%andaminimumminorallelefrequencyof2%.WethenusedgatktogeneratesubdatasetsofSNPsfromspecificcategories(exon,intron,regulatory region,andDrb1-likeexon2).For the “off-target”SNPs,weusedtheEnsemblvarianteffectpredictor tool todeterminethebpdistancebetweenaSNPandtheclosestgeneandgeneratedalistofputativelyneutralSNPsthatwereatleast100,000bpfromagene(e.g., Kawakami etal., 2014),whichwe considered to be in linkagedisequilibrium.We“binned”theseSNPsbasedontheminorallelefre-quency,andtestedforgeneticstructure(seebelow)usingtheSNPswithminorallelefrequencyvaluesof2%and25%.All.vcffileswerereformatted using pgdspider v2.0.9.2 (Lischer& Excoffier, 2012) fordownstreamanalyses.

Toexplorevariationinfunctionalregions,werantwolositan anal-yses(Antao,Lopes,Lopes,Beja-Pereira,&Luikart,2008;Beaumont&Nichols,1996)toidentifyFSToutliersthatareputativelyunderselec-tion.lositanparameters included1,000,000iterations,a99.5%con-fidence interval, a falsediscovery rate (FDR) thresholdof0.05, andastepwisemutationmodel.Weenabledthe“NeutralmeanFST”andthe“ForcemeanFST”options.Thefirstanalysisusedpopulationpriorsbasedongeography (MB,ON,andATL)and thesecondconsideredexposuretoWNS(pre-WNS,post-WNS).WeextractedthesubsetofdirectionalFSToutliersidentifiedineachanalysiswithvcftools v0.1.14 (Daneceketal.,2011)andusedthemtoexploreimmunogeneticpop-ulationstructure(seebelow).

SNPs that alter amino acids or affect splicing regions can havemajoreffectsonthefunctionofencodedproteins.WeconsideredFST outliersthathadtheseparticularconsequencesasthemostlikelysig-nalsofeitherlocaladaptationtopre-occurringpathogens(inthegeo-graphiccomparison)orallelesdisproportionatelyselectedagainstbyWNS(inthepre-andpost-WNScomparison).Mutationsinregulatoryregionscanalsoinfluencegeneexpressionandultimatelyaffectdis-easeoutcome(Fraser,2013)sowealsoidentifiedSNPswithinregula-toryregions,althoughthefunctionalresultsofthesemutationscannotbeinferredfromsequencecapturedataalone.

2.7 | Characterization of neutral and immunogenetic population structure

Weusedtwoapriorigroupingstotestforneutralgeneticandimmu-nogeneticpopulationstructure:(i)geographicgrouping(MN,ON,andATL),or (ii)groupingbyexposuretoWNS(pre-andpost-WNS).WeexploredgeneticstructurebasedonthedifferentSNPdatasetsusing

     |  5DONALDSON et AL.

structure v2.3.4(Pritchard,Stephens,&Donnel ly,2000)andparal lel izedtherunsusingthestrautov0.3.1script(Chhatre&Emerson,2017).Weran structurewithaburn-inlengthof50,000followedby200,000it-erationsforK=1through4,andeachrunwasperformed20times.Weused structure harvester webv0.6.94(Earl&VonHoldt,2012)tocalcu-latetheΔKstatistic(Evanno,Regnaut,&Goudet,2005).Multiplestruc-turerunswerecombinedwithclumppv1.1.2(Jakobsson&Rosenberg,2007) using the Greedy option (10,000 repeats), and we visualizedtheresultsusingdistructv1.1(Rosenberg,2004).Wealsoperformedprincipal componentanalysis (PCA)usingadegenet v2.0.0 (Jombart&Ahmed,2011).Weobtainedtherequired“genlight”objectsfortheade-genetanalysisusingacombinationofvcftools and plinkv1.07(Purcelletal.,2007)toreformatthe.vcffilestoplink-formattedfiles(.raw).

3  | RESULTS

3.1 | High- throughput sequencing, sequence alignment, and depth of coverage

NimbleGen sequence capture and high-throughput sequencingyielded 717 million paired-end reads for 96 libraries. We mapped712 million of these reads to theM. lucifugus genome (Table S4).Sequencing alignment anddepthof coveragemetrics (Tables1 and2)indicatedthattheM. lucifugusprobesweresuccessfulincapturingthetargetedlociinE. fuscus and M. septentrionalis.Primarytargeten-richmentwas42.1%,41.4%,and37.3%forM. lucifugus, E. fuscus, and M. septentrionalis,respectively(Table1)andcoverageforsequencedsamples from E. fuscus (59.5X, 78.2X) andM. septentrionalis (66.5X,171.0X)fellwithintheobservedrangeforM. lucifugus(26.2X–463.3X;Table2).Tovisualizethevariationindepthofcoverageacrosssam-plesandtheprimarytargets,weplottedthemeandepthofcoverageforprimarytargetsacrossallsamples(Figure1)andthedepthofcov-erageobtainedfromeachsample for theprimary targets (Figure2).Overall,wedeterminedaveragecoveragewashighformicrosatellitemarkers (135X), Drb1-like exon 2 targets (121X), and targeted im-munegenes(145X).

3.2 | Microsatellite genotyping via INDEL detection

When processing the 32 microsatellite loci included in our assay,gatk analysis identified 400 INDELs for the 32 loci, demonstratingthat unique microsatellite regions contained multiple INDEL calls.However,therelativelyshortreadlengthobtainedwithoursequenc-ingmethodfailedtoreliablycaptureentireshorttandemrepeat(STR)regions.Thus,microsatellitegenotypescouldnotberecoveredforallsamples.Asaresult,heterozygousgenotypesscoredfromourhigh-throughput sequencing differed from the previous results obtainedusingtraditionalPCRmethods(Davyetal.,inpress)by−25%to−42%.The STR-FManalysis,which does not rely on aligning reads to thegenome,wasalsounabletogenerategenotypesformorethantwomicrosatellitemarkersusingasubsetofoursamples(datanotshown).Therefore,wedidnotconductfurtheranalyseswiththemicrosatel-litedata. TABLE 1 High-throughputsequencingandreadalignmentsummarystatistics

Sam

ple

cate

gory

Tota

l map

ped

read

s

Tota

l map

ped

re

ads f

ilter

ed

(%)

Dup

licat

es (%

)M

appi

ng q

ualit

y

(%)

Mul

timap

ped

read

s (%

)To

tal m

appe

d

read

s [pa

ss fi

lter]

Read

s map

ped

to

prim

ary

targ

ets

[pas

s filt

er]

Prim

ary

targ

et

enric

hmen

t [p

ass f

ilter

] (%

)

Myo

tis lu

cifu

gus(

n=92)

Mean

7,459,167

80.39

73.7

06.60

0.09

1,411,920

656,230

42.1

Minimum

4,203,530

54.4

547

.70

5.95

0.06

524,146

118,002

15.5

Maximum

11,483,159

91.95

85.37

9.43

0.14

3,061,461

2,023,112

66.3

Epte

sicus

fusc

us(n=2)

Minimum

3,935,930

83.44

67.23

15.58

0.22

651,945

277,998

40.2

Maximum

6,080,356

84.98

69.17

15.98

0.23

913,531

367,312

42.6

M. s

epte

ntrio

nalis(n=2)

Minimum

6,548,133

80.67

70.80

9.62

0.19

913,750

300,166

32.8

Maximum

9,467,611

86.05

76.22

9.68

0.21

1,830,053

765,227

41.8

6  |     DONALDSON et AL.

3.3 | Analysis of neutral genetic structure

The gatkanalysisidentified16,115“off-target”SNPs.TheEnsemblvarianteffectpredictortoolfoundthat1,038oftheseSNPswerelocated >100,000bp from a neighboring gene (Table3; TableS5). The putatively neutral SNPs map to 111 different clusters(>100,000bpfromthenextcluster)on88differentscaffoldsoftheM. lucifugusgenomesequenceassembly.WefoundnoevidenceforgeneticstructurebasedontheseputativelyneutralSNPsintheade-genet-orstructure-derivedplotsbasedongeography(Figure3a),orbasedonthepresenceofWNSinthoseareas(Figure3b),regard-lessoftheminorallelefrequencycutoffusedintheanalysis(datanotshown).

3.4 | Immunome SNP detection and analyses

Weidentified17,495SNPswithintheprimarytargetloci,locatedin exons (3,536 SNPs), introns (5,482 SNPs), and regulatory re-gions (5,482 SNPs). lositan identified 328 and 299 directionaloutlierSNPsinthegeography-andWNS-basedanalyses,respec-tively,32ofwhichweredetectedinbothanalyses.Weacknowl-edgethatfalsepositivesforSNPsunderselectionarecommoninoutliersdetectedusingFST-basedmethods(Narum&Hess2011);therefore, the candidate SNPs identified in this experimentwillrequirefurthervalidationinfuturestudies.Thepredictedimpactsofeachofthose595directionaloutlierSNPsaresummarizedinTables5 and S6. Focusing on SNPs most likely to cause majorfunctional changes,we found that 23 outlier SNPs in 19 genesin thegeographic comparison resulted inanaminoacidchange,as did 28 SNPs in 21 genes in theWNS comparison (Table6).In theWNS comparison, an outlier SNP in the intron region ofHLA-DPB1resultedinlikelymodificationofthesplicedonorse-quence.Wealsodetected194SNPSintheregulatoryregionsof78genes(TableS6),ofwhich11wereidentifiedinbothcompari-sons(Table4).

Analysesofgeneticstructure inadegenet and structuredidnotidentifygeography-orWNS-associatedgeneticstructureusingtheentireprimary target locus,exon, intron,or regulatory regionSNPdatasets (data not shown). However, using the lositan-predictedoutlier SNPs,we observed subtle immunogenetic structure based

on geography or the presence of a brief period of co-occurrencewithP. destructans(Figure4).Theanalysisbasedongeographyiden-tified immunogenetic differentiation betweenM. lucifugus in MBand conspecifics in ON andATL (Figure4a),while analysis basedonco-occurrencewithP. destructansgroupedONandMBtogether(pre-WNS), differentiated from the post-WNS samples from ATL(Figure4b).

4  | DISCUSSION

4.1 | Sequence capture and high- throughput sequencing

Reduced representation genomic profiling strategies have emergedas valuable alternatives to whole-genome sequencing (Narum,Buerkle,Davey,Miller,&Hohenlohe,2013)wherepopulation-levelassessmentsarenotyetfeasiblefornonmodelorganismswithlargergenomes. Reduced representation approaches can include bothtranscriptomestudies (allexpressedgenes)orGBSthatcan includerestrictionsiteassociationDNA(RAD)marker,targetcapture,andam-pliconsequencing.WhileRADsequencinghasmanyadvantageswhengenomicresourcesforthespeciesaresparse,ithasmanylimitationsin identifying patterns of local adaptation (Andrews, Good, Miller,Luikart,&Hohenlohe, 2016).Amplicon sequencingof a largenum-berof locihasmanyadvantages inelucidatingthegeneticvariationfromknowntargets;however,inthisinstance,wechoseasequencecaptureapproachtoalsopulldownlargesegmentsoftheimmunomethat included upstream regulatory regions of the genes of interest.As such, the candidate geneGBS approach employed in this studyprovided several advantages over other means in obtaining immu-nogeneticinformationthatislikelytobeinfluencedbytheselectivepressuresfromdiseasesuchasthatcausedbyP. destructans(Table6;Figure4).

Here,wefoundtargetenrichment ledtoevensequencinguni-formity/coverage,whichhasbeennotedbyother researchgroups(Powell, Amish, Haynes, Luikart, & Latch, 2016; Samorodnitskyetal.,2015;Schweizeretal.,2016).Theassaywedevelopedpro-videdahighontargetmeanstoobtainmoderatetohighcoverageof each target (26–463X;Table1) thatwas relatively even acrosssamples and loci (Figures1 and 2). The assay alsoworked acrossother species (M. septentrionalis and E. fuscus) that are known toalso be impacted byWNS to varying degrees (Frank etal., 2014;Fricketal.,2015;Langwigetal.,2012).Assuch,thisassaysetsthestage for cross-species analyses to further our understanding ofthevariableimmuneresponsestothisdisease.Theassay,however,yieldedfartoohighapercentageofduplicates(54%–92%;Table1)thatcompromisedthelevelofcoverage.TheduplicateswerelikelyamatteroftoomanyPCRcyclesattheadapterligationstageduringDNA library preparations. In the future,wewould decrease from11to6–8cyclesintheLM-PCRstep.Oneaspectoftheassaythatdid notmeet expectationswas the amplification ofmicrosatelliteloci, largelyasamatterofthesequencingtechnologyused(HiSeq2500 rapid runusing2×100-bp readson a single flow cell).The

TABLE  2 Primarytargetdepthofcoveragesummary

Sample category N Mean Minimum Maximum

Myotis lucifugus(total) 92 148.1 26.2 463.3

Ontario (Hibernaculum1)

12 164.9 108.2 220.9

Ontario (Hibernaculum2)

24 44.4 26.2 61.1

Manitoba 28 159.8 78.1 294.6

AtlanticCanada 28 218.2 62.9 463.3

Eptesicus fuscus 2 – 59.5 78.2

M. septentrionalis 2 – 66.5 171.0

     |  7DONALDSON et AL.

F IGURE  1 Meandepthofcoveragefortargetloci,sortedbysampleIDandsamplinglocation.Blackbarsrepresenttheprimarytargetspecies,Myotis lucifugus.Orangebars:M. septentrionalis; yellow bars: Eptesicus fuscus

8  |     DONALDSON et AL.

F IGURE  2 Primarytargetdepthofcoveragepersample,sortedbylocus.Meandepthofcoverageforeachlocusisindicatedbyaredcolored“x.”MicrosatellitemarkersandDrb1-likeexon2targetsaremarkedinthetoppanel.WecondensedthepresenteddatabyabbreviatingEnsembl-derivedMyotis lucifugusgene(MLG)identifiers,where“MLG-”=“ENSMLUG000000,”fortheDrb1-likegenesthatdidnothaveinformativeEnsemblorGenBankgenenames

Dep

th o

f cov

erag

e (p

er s

ampl

e)

DRB1-like exon 2Microsatellite marker

     |  9DONALDSON et AL.

hybridization-based method we employed to capture microsatel-litelociwassuccessful;however,wewereunabletogenerategen-otypesusing the reads containing short tandem repeats.To avoidtheseexperimentaldesignandtechnicalproblems,werecommendusing sequencing technologies thatoffer longer read lengths.TheIlluminaMiSeqandLifeTechnologiesPGMSystemcurrentlyyield300-bpto400-bpreadlengths,whichmayallowforthemicrosat-elliteandflankingregionstobesequenced,andshouldhelpmicro-satelliteanalysisinnonmodelorganisms.Overall,theassayprovided

a relatively high number of variable neutral SNPswith frequencydifferencesamenable topopulationgeneticanalyses (Table3)andalargenumberofFSToutlierSNPsinexons,introns,andregulatoryregions(Table4),severalofwhichwerepredictedtohaveimportantvarianteffects(Table5).

4.2 | Immunogenetic diversity and structure

We developed a GBS sequence capture assay to cost-effectivelyandrapidlyrevealgeneticdiversityintheimmunomeofendangeredM. lucifugusthreatenedbymassdie-offsfromWNS.Theassaychar-acterizedneutralpopulationstructuretocontrolforstochasticimmu-nogeneticdifferentiationamongsampledareas (TableS5;Figure3),andalsoelucidatedgeneticvariationandstructureofimmunegenesvia hundreds of SNPswithin the exons, introns, and regulatory re-gionsof thosegenes (TableS6;Figure4).Preliminaryapplicationoftheassay toE. fuscus and M. septentrionalis indicates itmayalsobeaneffectivetoolforthesespecies.Bytargetingtheassaytoaddressspecificresearchquestions,ourGBSapproachcanbeusedacrosstherangeofM. lucifugustoinvestigatedriversofgenetic,morphological,andbehavioralvariation.

Ourassayrevealedsubtleimmunogeneticvariationandstruc-ture on a relatively small geographic scale, suggestive of local

TABLE  3 Summaryofthenumberofdetected,putativelyneutralsinglenucleotidepolymorphisms(SNPs)binnedbyminorallelefrequency(MAF)

MAF (%) SNPs (nr)

2 1038(111)

5 544(90)

10 343(77)

15 236(68)

20 169(58)

25 142(53)

nr=“nonredundant” number of neutral SNP clusterswith long-distance(>100-Kbp)SNPs.

F IGURE  3 Visualizinglackofgeneticstructureusing1,038putativelyneutralSNPs(>100kpbfromnearestgene)withmax-missinggenotypeof5%andminorallelefrequencyof2%forMyotis lucifugus(n=92).Samplesweregroupedbasedon(a)geographiclocationor(b)previousexposuretoWNS.Principalcomponentanalysisplotswereproducedusingadegenet,andthepercentageofvariationforeachaxisandascatterplotofeigenvaluesareincludedforeachanalysis;barplotshowsresultsofstructureanalysis(K=2).MB=Manitoba(black);ON=Ontario(gray);ATL/post=AtlanticCanada/post-WNS(red);pre=pre-WNS(blue)

(a)

Pre-WNS Post-WNSAtlanticOntarioManitoba

PC

2 (2

.6%

)

PC1 (2.8%)

PC

2 (2

.6%

)

(b)

PC1 (2.8%)

10  |     DONALDSON et AL.

immunogenetic adaptation within an otherwise panmictic pop-ulation (Figures3a and 4a; Davy etal., in press). Comparisonsof samples taken before and after the arrival of WNS suggesta nonrandom removal of genetic variants in the immunome byP. destructans(Figures3band4b).Ifsimilarselectionisoccurringin M. lucifugus that are surviving inWNS-impacted areas, theremay be potential for rapid local adaptation toWNS, raising thepossibility of evolutionary rescue (Carlson etal., 2016;Maslo&Fefferman,2015).Conversely, immunogeneticselectionbyWNSmaydisruptpreviouslyadaptivepatternsof immunogeneticvari-ation asP. destructans continues to spread, further complicatingthe recovery ofM. lucifugus. Our interpretation of this data iseffected by the possibility that the 28M. lucifugus we sampled fromAtlantic Canadawere not exposed toP. destructans duringthepreviousyear,andwhile27oftheseindividualsdiedofWNSin2014,thismighthavebeentheirfirstexposuretoaninfectionwithP. destructans.Topartiallyaddressthisconcern,wenote14of 28M. lucifuguswith sample IDs “X2393-14-N” (whereNvar-ies;TableS3)camefromahibernaculuminPrinceEdwardIslandwhereWNSmortalitywasidentifiedinthepreviouswinter,1yearprior to these individuals dyingofWNSandbeing collected forthisstudy.

Immunogenetic diversity in M. lucifugus is extremely high.Previousattemptstoquantifyvariationwerecomplicatedbydu-plicationof loci intheMHCofM. lucifugus,whichexhibitsupto

24Drb1-like loci (Davyetal., inpress;Palmeretal., 2016).Ourtargeted sequence capture assay controls for this gene duplica-tionandallowsgenotypestobeunambiguouslyassignedtoeachindividual.Wedetectedfunctionallysignificantdifferentiation inseveralDrb1-likelociassociatedwithbothgeographyandprevi-ousexposuretoWNS(Table6).ExposuretoWNSisalsoassoci-atedwithashift ingeneticvariationat interleukinsandToll-likereceptors (Davy etal., in press; Field etal., 2015; Lilley etal.,2017; Rapin etal., 2014), consistent with the hypothesis thatWNS exerts immunogenetic selective pressure onM. lucifugus. Our research on the interactions betweenM. lucifugus geneticsand P. destructans continues to reinforce the need to take bothinterindividual and inter-regional variation of both the host andpathogen into account when interpreting genetic data. In thisstudy, bats collected from sites<600kmapart inManitoba andOntario belong to a panmictic population based on neutralmo-lecularmarkers,butexhibitlocalvariationintheimmunomethatmay result indifferentexpressionof immunegenesamongsites(Table6).Forexample,itispossiblethatlocalimmunogeneticdif-ferentiationbetweenthesesitesresult indifferentsurvivalratesfollowingtheintroductionofWNS.Variationintheregulatoryre-gions(TableS6)couldalsoaltertheexpressionofintegralimmunegenesamongsites.

HighimmunogeneticvariationinM. lucifugushasimplicationsforthe interpretationofgeneexpressionstudiesaswell.Bats fromdif-ferent samplingsitesmay responddifferently to immunechallengesdue to variation in exon and regulatory regions of the immunome.Therefore,experimentalgeneexpressionstudiesrelatedtoP. destruc-tans or other pathogens should explicitly control for potential geo-graphicvariation.Otherwise,observeddifferencesingeneexpressioncannotbeunambiguouslyattributedtotheeffectsofthepathogen(orothertreatmentsofinterest).

TheGBSapproachusedhereprovidesabasisforreal-timeinves-tigations of evolutionary rescue in populations of bats that persistfollowing initialdeclinesfromWNS(Maslo&Fefferman,2015).Theresultsofourstudywerebasedonanalysisofasmallnumberofpopu-lations,andinthepost-WNSpopulation,theM. lucifugusweexamineddidnotsurviveinfection.Ideally,futurestudieswillcompareimmuno-geneticvariationnotonlyamongexposedandunexposedsites,butalsoamong time-series samples taken frombats thathave survivedmultipleselectivesweepsfromone,two,ormorewintersinhibernac-ulacontainingP. destructans.

TABLE  5 Summaryofconsequencespredictedbythevarianteffectpredictor,fordirectionalFSToutliersdetectedbylositanfromimmunomesequencecaptureofMyotis lucifugussamples(n=92)

Feature Consequence Geography WNS

Exon Synonymousvariant 36 43

Exon Missensevariant 21 26

Exon Missensevariant,spliceregionvariant

2 2

Exon Spliceregionvariant,synonymousvariant

1 1

Intron Intronvariant 157 122

Intron Spliceregionvariant,intronvariant

2 8

Intron Splicedonorvariant 0 1

RegulatoryRegion Upstreamgenevariant 109 96

Feature type

Geography WNS

Directional outliers (nr)

Balancing outliers (nr)

Directional outliers (nr)

Balancing outliers (nr)

Exon 60(35) 89(45) 72(43) 416(94)

Intron 159(62) 249(71) 131(54) 1,050(99)

RegulatoryRegion 109(54) 141(74) 96(50) 666(116)

Total 328(151) 479(190) 299(147) 2,132(309)

nr=“nonredundant”numberofgeneswithFSToutliers.

TABLE  4 Summaryoflositan FST outliers(FDR<0.05)intargetedimmunomefeaturesofMyotis lucifugus (n=92).Directionaloutlierswereusedforstructure and adegenetanalyses(Figure4)

     |  11DONALDSON et AL.

TABLE  6  lositan-detectedFSToutliersfromSNPanalysesbasedonapriorigroupingbygeographiclocation(Manitoba,Ontario,andAtlantic)orbyWNSexposurehistory(pre-WNS,post-WNS).OnlytheSNPsthataremostlikelytohaveafunctionalimpactbyalteringaminoacidsoraffectingsplicesequencesarelisted(seeMethodsandTableS6fordetails)

Comparison (#SNPs) Gene name Ensembl transcript ID Brief description Amino acids

Geographic CCL3 ENSMLUT00000002888 C-Cmotifchemokine A/V

Geographic CCR4 ENSMLUT00000027956 Chemokine(C-Cmotif)receptor4 S/F

Geographic CD40 ENSMLUT00000006008 CD40molecule,TNFreceptorsuperfamilymember5 S/N

Geographic Drb1e2-like-e ENSMLUT00000027881 DLAclassIIhistocompatibilityantigen N/H

Geographic Drb1e2-like-f ENSMLUT00000028450 DLAclassIIhistocompatibilityantigen T/M

Geographic Drb1e2-like-l ENSMLUT00000029278 DLAclassIIhistocompatibilityantigen L/R

Geographic Drb1e2-like-n ENSMLUT00000030076 DLAclassIIhistocompatibilityantigen E/D

Geographic Drb1e2-like-r ENSMLUT00000027745 DLAclassIIhistocompatibilityantigen Q/La

Geographic(2) HRG ENSMLUT00000013351 Histidine-richglycoprotein K/R,H/Q

Geographic IFNGR1 ENSMLUT00000008611 Interferongammareceptor1 D/E

Geographic(2) IL12RB1 ENSMLUT00000013802 Interleukin12receptor,beta1 K/R,T/I

Geographic(2) IL1R1 ENSMLUT00000011035 Interleukin1receptor,typeI R/K,E/K

Geographic IL23A ENSMLUT00000006770 Interleukin23,alphasubunitp19 R/T

Geographic IRF6 ENSMLUT00000004509 Interferonregulatoryfactor6 K/N

Geographic(2) MPO ENSMLUT00000006099 Myeloperoxidase Q/La,G/R

Geographic NLRP10 ENSMLUT00000000818 NLRfamily,pyrindomaincontaining10 S/C

Geographic NOS2 ENSMLUT00000015896 Nitricoxidesynthase G/D

Geographic RAG1 ENSMLUT00000000542 Recombinationactivatinggene1 S/N

Geographic SPON2 ENSMLUT00000017687 Spondin2,extracellularmatrixprotein T/M

WNS CCR4 ENSMLUT00000027956 Chemokine(C-Cmotif)receptor4 I/N

WNS DDX58 ENSMLUT00000003044 DEAD(Asp-Glu-Ala-Asp)boxpolypeptide58 V/I

WNS DLA-DRA1 ENSMLUT00000027968 DLAclassIIhistocompatibilityantigen,DRalphachain-like

P/T

WNS Drb1e2-like-i ENSMLUT00000031273 DLAclassIIhistocompatibilityantigen E/V

WNS(2) Drb1e2-like-k ENSMLUT00000023434 DLAclassIIhistocompatibilityantigen D/N,D/E

WNS Drb1e2-like-p ENSMLUT00000022698 DLAclassIIhistocompatibilityantigen S/N

WNS(2) Drb1e2-like-r ENSMLUT00000027745 DLAclassIIhistocompatibilityantigen Q/La,R/H

WNS HLA-DPB1 ENSMLUT00000016285 Majorhistocompatibilitycomplex,classII,DPbeta1 b

WNS IFNAR1 ENSMLUT00000025403 Interferon(alpha,betaandomega)receptor1 S/P

WNS IL12RB1 ENSMLUT00000013802 Interleukin12receptor,beta1 I/L

WNS IL12RB2 ENSMLUT00000001415 Interleukin12receptor,beta2 I/V

WNS(2) IL1R1 ENSMLUT00000011035 Interleukin1receptor,typeI L/M,D/G

WNS IL5 ENSMLUT00000016553 Interleukin5 K/E

WNS(2) ITGAM ENSMLUT00000011332 Integrin,alphaX(complementcomponent3receptor4subunit)

Q/R,V/L

WNS MPO ENSMLUT00000006099 Myeloperoxidase Q/La

WNS(3) NOD2 ENSMLUT00000015164 Nucleotide-bindingoligomerizationdomaincontaining2

L/V,S/R,S/A

WNS NOS2 ENSMLUT00000015896 Nitricoxidesynthase A/V

WNS TBX21 ENSMLUT00000014543 T-box21 Q/P

WNS TLR1 ENSMLUT00000008406 Toll-likereceptor1 V/I

WNS TLR2 ENSMLUT00000012815 Toll-likereceptor2 S/P

WNS(2) TLR6 ENSMLUT00000008414 Toll-likereceptor6 H/L,I/V

WNS TLR9 ENSMLUT00000015105 Toll-likereceptor9 A/V

aIndicatesoutlierSNPswereidentifiedinboththegeographicandWNS-basedcomparisons.bIndicatesaSNPpredictedtohaveahighimpactbyalteringasplicedonorsequenceinanintron.TheotherSNPslistedhereareinexonsandarepredictedhavemoderateimpactsbyalteringtheaminoacidsequence.

12  |     DONALDSON et AL.

5  | CONCLUSION

Wedeveloped a cost-effective high-throughput sequence captureassaytotestforimmunogeneticshiftsinM. lucifuguspopulationsfol-lowingexposuretoP. destructans.Sequenceanalysisfrom92M. lu-cifugus identifiedsequencevariationin138immune-relatedgenes,theirupstreamregulatoryregions,and111putativelyneutralregionsofthegenome.The“one-pot”assaywedevelopedtotestforgeneticstructureand immunogeneticvariation identified functional immu-nogenetic variants inM. lucifugus putatively associatedwithWNSsusceptibility, demonstrated a shift in immunogenetic diversity of

populationspre-andpost-WNSexposure,andprovidedpreliminarysupportforapotentialevolutionaryrescueofM. lucifugusinAtlanticCanadagivenanonrandompurgingofimmunogeneticvariantsintheWNS-susceptiblebats.Wecanusethegeneticvariantsidentifiedinthis studyas abaseline for future investigationsof rangewide im-munogeneticadaptationtoWNSinlittlebrownmyotis.Ultimately,understandingthepotentialforevolutionaryrescueinaspeciescanguidemoreeffectiveandtargetedmanagementactionstomitigatethe impacts ofWNS onNorth American bat populations.Overall,thisstudysetsthestageforfurtherresearchwithlargersamplesizesand increasedpopulationreplicatesunderdifferenttypesofselec-tivepressure to furtherunderstandpatternsof local adaptation inthisbatspecies,mostimportantlyincontextofWNSexposureandsurvival.

ACKNOWLEDGEMENTS

This research was funded by a Discovery Grant from the NaturalSciencesandEngineeringResearchCouncilofCanada(CJK,CKRW),the Liber Ero Fellowship Program (CMD), and the Species at RiskResearchFundforOntario(CJK,CMD,CKRW).TheCanadianWildlifeHealthCooperativeprovidedfundingfortheWNSsurveillancepro-gramthatenabledthediagnosisofthisdisease inthebats includedfromthepost-WNSendemicareaaswellasthecollectionandship-pingoftissuesfromtheircarcasses.WethankKatieDogantzis,KatelynJackson (Trent University), and Matt Harnden (Natural ResourcesDNAProfilingandForensicsCentre) for technical assistance;MenaFarag (Roche) for helping revise themultiplexed sequence captureprotocol;AaronGoldman,GrahamCromar,RogerShi,andSiweiChen(TheClinicalGenomicsCentreatMountSinaiHospital,Toronto)forperforming the DNA library preparation and Illumina sequencing; Dr.BarrySaville(TrentUniversity)forassistanceinanalyzingthehigh-throughputsequencedata;andtwoanonymousreviewersforcritiqueleadingtoanimprovedversionofourmanuscript.

CONFLICTS OF INTEREST

Theauthorsdeclarenoconflictofinterest.

AUTHOR CONTRIBUTIONS

MED, CMD, CKRW, and CJK conceived and designed the experi-ments.SM,JS,andCKRWcollectedbattissue.SMperformedpost-mortem diagnosis of WNS. MED performed the experiments andanalyzedthedata.CJKcontributedreagents/materials/analysistools.MED,CMD,CKRW,CJK,andSMwroteandrevisedthemanuscript.

DATA ARCHIVING STATEMENT

All high-throughput sequencing.fastq files have been archived inthe NCBI Sequence Read Archive database (accession numberSRP100885).TheM. lucifugusSNPdatafile (.vcf) isavailableontheDyradDigitalRepository(https://doi.org/10.5061/dryad.2r4c7).

F IGURE  4 Analysisofimmunogeneticpopulationstructurebased on lositan-detectedFSToutlierswithmax-missinggenotypeof5%andminorallelefrequencyof2%forMyotis lucifugus(n=92).Samplesweregroupedbasedon(a)geographiclocationor(b)previousexposuretoWNS.Principalcomponentanalysisplotswereproducedusingadegenet,andthepercentageofvariationforeachaxisandascatterplotofeigenvaluesareincludedforeachanalysis;barplotshowsresultsofstructureanalysis(K=3).MB=Manitoba(black);ON=Ontario(gray);ATL/post=AtlanticCanada/post-WNS(red);pre=pre-WNS(blue)

Pre-WNS Post-WNS

PC1 (6.1%)

PC

2 (3

.5%

)P

C2

(4.2

%)

PC1 (7.2%)

(A) F S

Tou

tlier

s ba

sed

on g

eogr

aphy

F ST

outli

ers

base

d on

exp

osur

e to

WN

S(a)

(b)

Manitoba Ontario Atlantic

     |  13DONALDSON et AL.

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How to cite this article:DonaldsonME,DavyCM,WillisCKR,McBurneyS,ParkA,KyleCJ.Profilingtheimmunomeoflittlebrownmyotisprovidesayardstickformeasuringthegeneticresponsetowhite-nosesyndrome.Evol Appl. 2017;00:1–15. https://doi.org/10.1111/eva.12514