Natural selection in bats with historical exposure to white-nose syndrome


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Natural selection in bats with historical exposure to white-nose syndrome

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Title:
Natural selection in bats with historical exposure to white-nose syndrome
Series Title:
BMC Zoology
Creator:
Harazim, Markéta
Horáček, Ivan
JakeÅ¡ová, Lucie
Luermann,, Kristína
Moravec, JiÅ™í C.
Morgan, Shannon
Pikula, Jiri
Sosík, Petr
VavruÅ¡ová, Zuzana
Zahradníková, Alexandra Jr.
Zukal, Jan
Martínková, Natália
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English

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Skin ( local )
Fungal Infection ( local )
Wound Healing ( local )
Immunity ( local )
Gene Evolution ( local )
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serial ( sobekcm )

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Background Hibernation allows animals to survive periods of resource scarcity by reducing their energy expenditure through decreased metabolism. However, hibernators become susceptible to psychrophilic pathogens if they cannot mount an efficient immune response to infection. While Nearctic bats infected with white-nose syndrome (WNS) suffer high mortality, related Palearctic taxa are better able to survive the disease than their Nearctic counterparts. We hypothesised that WNS exerted historical selective pressure in Palearctic bats, resulting in genomic changes that promote infection tolerance. Results We investigated partial sequences of 23 genes related to water metabolism and skin structure function in nine Palearctic and Nearctic hibernating bat species and one non-hibernating species for phylogenetic signals of natural selection. Using maximum likelihood analysis, we found that eight genes were under positive selection and we successfully identified amino acid sites under selection in five encoded proteins. Branch site models revealed positive selection in three genes. Hibernating bats exhibit signals for positive selection in genes ensuring tissue regeneration, wound healing and modulation of the immune response. Conclusion Our results highlight the importance of skin barrier integrity and healing capacity in hibernating bats. The protective role of skin integrity against both pathophysiology and WNS progression, in synergy with down-regulation of the immune reaction in response to the Pseudogymnoascus destructans infection, improves host survival. Our data also suggest that hibernating bat species have evolved into tolerant hosts by reducing the negative impact of skin infection through a set of adaptations, including those at the genomic level.
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BMC Zoology, Vol. 3, no. 8 (2018-08-29).

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K26-00078 ( USFLDC: LOCAL DOI )
k26.78 ( USFLDC: LOCAL Handle )

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RESEARCHARTICLEOpenAccess Naturalselectioninbatswithhistoricalexposuretowhite-nosesyndromeMarkétaHarazim1,2,IvanHoráek3,LucieJakeová2,KristínaLuermann4,JiíC.Moravec1,ShannonMorgan5,9,JiriPikula6,PetrSosík7,ZuzanaVavruová4,AlexandraZahradníkováJr.1,8,10,JanZukal1,2andNatáliaMartínková1,4* AbstractBackground:Hibernationallowsanimalstosurviveperiodsofresourcescarcitybyreducingtheirenergyexpenditurethroughdecreasedmetabolism.However,hibernatorsbecomesusceptibletopsychrophilicpathogensiftheycannotmountanefficientimmuneresponsetoinfection.WhileNearcticbatsinfectedwithwhite-nosesyndrome(WNS)sufferhighmortality,relatedPalearctictaxaarebetterabletosurvivethediseasethantheirNearcticcounterparts.WehypothesisedthatWNSexertedhistoricalselectivepressureinPalearcticbats,resultingingenomicchangesthatpromoteinfectiontolerance.Results:Weinvestigatedpartialsequencesof23genesrelatedtowatermetabolismandskinstructurefunctioninninePalearcticandNearctichibernatingbatspeciesandonenon-hibernatingspeciesforphylogeneticsignalsofnaturalselection.Usingmaximumlikelihoodanalysis,wefoundthateightgeneswereunderpositiveselectionandwesuccessfullyidentifiedaminoacidsitesunderselectioninfiveencodedproteins.Branchsitemodelsrevealedpositiveselectioninthreegenes.Hibernatingbatsexhibitsignalsforpositiveselectioningenesensuringtissueregeneration,woundhealingandmodulationoftheimmuneresponse.Conclusion:Ourresultshighlighttheimportanceofskinbarrierintegrityandhealingcapacityinhibernatingbats.TheprotectiveroleofskinintegrityagainstbothpathophysiologyandWNSprogression,insynergywithdown-regulationoftheimmunereactioninresponsetothePseudogymnoascusdestructansinfection,improveshostsurvival.Ourdataalsosuggestthathibernatingbatspecieshaveevolvedintotoleranthostsbyreducingthenegativeimpactofskininfectionthroughasetofadaptations,includingthoseatthegenomiclevel.Keywords:Skin,Fungalinfection,Woundhealing,Immunity,GeneevolutionBackgroundEmergenceofanovelinfectiousdiseaseorpathogentransmissiontoanaïvepopulationleadstoallelefre-quencychangesinpopulationsexperiencingdiseaseout-breakswithhighmortality[1,2].Carriersofallelesthatfacilitatelessseriousdiseasemanifestationhaveahigherchanceofsurvival,whilegenevariantscausingincreasedsusceptibilitytotheinfectionormoreseverediseasesaremorelikelytovanishfromthepopulationgenepool.Pathogensserveasaselectiveforceinsusceptiblehosts,directingchangesinhostpopulationgeneticdiversity.AlterationsinhostgeneticstructuredrivenbypathogensaredetectableincodingDNAsequencesassignalsfornaturalselection.Whileequalratesofbothsubstitutiontypesoccurduringneutralevolution,ahigherrateofnon-synonymoussubstitutions(dN)thansynonymoussubstitutions(dS)isasignofpredominantpositivese-lectivepressure(dN/dS>1)and,conversely,higherratesofsynonymoussubstitutionsareasignofpredominantnegativeselectivepressure(dN/dS<1)[3,4].White-nosesyndrome(WNS),afungalinfectionofhi-bernatingbats,potentiallyappliesstrongselectionpressureonbatpopulationsintheNearctic.Sinceitsemergencein2006,WNShascausedthedeathofmillionsofbatsacrosstheeasternpartofNorthAmerica[5].Indeed,WNShasledtothenearextirpationofsomeofthemostcommonhibernatingMyotisspeciesduetolocaldeclinesexceeding90%peryear[6,7].Incomparison,Palearcticbatspecies *Correspondence:martinkova@ivb.cz1InstituteofVertebrateBiology,TheCzechAcademyofSciences,Brno,CzechRepublic4InstituteofBiostatisticsandAnalyses,MasarykUniversity,Brno,CzechRepublicFulllistofauthorinformationisavailableattheendofthearticle BMC Zoolo gy ©TheAuthor(s).2018OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.0InternationalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.Harazimetal.BMCZoology (2018) 3:8 https://doi.org/10.1186/s40850-018-0035-4

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sufferingfromWNSshowgreatersurvival,withnoreportsofpopulationdeclinesattributabletoWNS[8,9]despitesporadicmortality[10,11].ThedifferenceinsurvivalrateofinfectedNearcticandPalearcticbatsismostlikelyconnectedtospecies-specificpathophysiologicimpactsduringhost-pathogeninteraction.Theinfectiveagentofthedisease,thepsychrophilicfungusPseudogymnoascusdestructans,invadesdeepintolivingskinlayersonthehibernatingbat’snose,ears,limbsandmembranes[10–14].Thediseasemaysubsequentlyhaveanegativeeffectonionandbloodgasbalance[15,16],evap-orativelossofwater[17]andoverallhibernationbehaviour[18].Frequentarousalsfromhibernationininfected,mori-bundbats[19]mayalsocontributetolossofenergyre-servesessentialforsurvival.Followingarousalfromtorpor,thehost’sinfectionresponsewillexacerbatetissuedamagebyimmunopathology[20]andreactiontofungalmetaboliteaccumulation.Inparticular,riboflavinproducedbyP.destructans,whichhyperaccumulatesinskinlesionsduringhibernation,causesoxidativetissuedamage[14]andtrig-gersacytotoxicimmuneresponsewhenpresentedtotheinnateimmunesystem[21].TherecentemergenceofWNSintheNearcticresultinginhighmortalityrates,andlaterrecognitionofthepatho-geninthePalearcticwithdifferingdiseasemanifestation,suggestsspatio-temporalvariationinhost-pathogencoevo-lution[22].RecentevidencedemonstratesthatP.destruc-tansinfectionoccurredinPalearcticbatspriortoitsemergenceintheNearctic[23,24].CoupledwithcurrentdiseasetoleranceinthePalearctic,historicalexposuretothepathogenhasgivenrisetothehypothesisthatWNSmayhavecausedmassmortalityeventsinthePalearcticinthepast.Martínkováetal.[8]speculatedthatthelargefossilde-positsofbatspeciesnowrareinundergroundhibernaculamayhaveaccumulatedduetoaWNSepidemicinthePleis-tocene.BasedontheassumptionthatPalearcticbatswereexposedtothelethalskininfectionpriortotheHolocene,wehypothesisedthatPalearcticbatspeciesmayhaveevolvedinheritablemechanismsleadingtotolerancetowardthedisease.Ifso,anyalterationingeneticinformationshouldbedetectableingenesencodingproteinsinteractingwiththepathogeninamannerdependentoninfectionpathophysiology.Identificationofthosegenestargetedbypositiveselectionshouldenhanceourunderstandingofdis-easepathogenesis.Consideringthedisease’setiologyandclosefunctionalconnectiontoacid-baseandelectrolytehomeostasisandskinlayerdamageduringdiseaseprogres-sion,wehypothesisethatgenesinvolvedinwatermetabol-ismandskinfunctioninhibernatingbatspeciesaremostlikelytodisplaysignaturesofpositiveselection.ResultsAnnotatedsequencesof23geneswereretrievedfromtheNCBIdatabaseforspeciesintheVespertilionidaeandMiniopteridaefamilies,namelyacad10,acp5,anxa1,aqp3,aqp4,aqp7,aqp9,bcam,ctnnb1,fads1,fgf10,guca2b,has2,hyal2,hyal3,krt8,lrp4,psen2,ptch2,pxn,sncg,tgm1,andtnfsf13(Additionalfile1).WesequencedfivegenesinsevenbatspeciesusingPacBioSMRTtechnology(GenBankAccessionNumbers:MH178037-MH178081)andsupplementedthedatasetsfromthepublicdatabasewith71phasedpartialcodingsequences.Inotherspecies(Additionalfile2),PacBiosequenceswerenotofsufficientqualitytoprovideatleast10×coverage.Intotal,weana-lysed11batspecieswith1–23genesavailableperspecies,andpartialcodingsequencesof23genesavailablein3–9species(Additionalfile1).Usingamaximumlikelihoodframework[25],weesti-matedtherateratioofnon-synonymoustosynonymoussubstitutions=dN/dSfortheavailablecodingsequences.Weusedthelikelihoodratiotest(LRT)todifferentiatebe-tweennestedmodelsofDNAsequenceevolution,wheretwicethedifferenceinmodellog-likelihoods(2lnL)ap-proximatelyfollowsa2distributionwithdegreesoffree-domequaltothedifferenceinthenumberofmodelparameters.ComparisonofM0andM3nestedmodelsofDNAsequenceevolution(testingforvariabilityofbe-tweensites)revealed13ofthe23genestobesignificantaftercorrectionformultipletestingwithfalsediscoveryrate(FDR)(Table1).ComparisonofM1toM2andM7toM8modelpairs(differentiatingbetweenneutralandposi-tiveselection)revealedsignificanceinsevengenes.Statis-ticallysignificantsupportforpositiveselection(FDRadjustedp<0.05)wasdetectedingenesencodingannexinA1(anxa1),tartrateresistantacidphosphatase5(acp5),aquaporin3(aqp3),thebasalcelladhesionmolecule(bcam),LDLreceptorrelatedprotein4(lrp4),patched2(ptch2)andsynucleingamma(sncg)(Table1).Inthosegenesshowingpositiveselection,weusedBayesempiricalBayes(BEB)toidentifyspecificaminoacidsen-codingsitesundersignificantpositiveselection(Fig.1).Weidentifiedfoursitesunderpositiveselectioninacp5,sevensitesinanxa1(withoneadditionalsiteidentifiedfromacomparisonoftheM7andM8models),onesiteinaqp3,onesiterecognisedfrombothmodelcomparisonsandoneadditionalsiteidentifiedbycomparingM7toM8inptch2.AllsixsitesidentifiedinbcamweresupportedbytheM7toM8comparison(Table2,Fig.1).Aminoacidsitesunderpositiveselectionweremappedontopredicted3Dproteinstructuremodelsoffourgenes(Fig.2).Intartrateresistantacidphosphatase(TRAP;encodedwithintheacp5gene),theselectedsiteswerespreadalongtheproteinsequence,whileinANXA1,theselectedsiteswereaccumulatedintheN-terminalregionoftheprotein(sixoutofeightsitesdetectedoccurredwithintherange17–27;Fig.1).ThesoleAQP3siteunderselectionwaslocatedwithinatransmembranehelix,andfouroutofsixsitesidentifiedunderpositiveselectioninHarazimetal.BMCZoology (2018) 3:8 Page2of13

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Table1MaximumlikelihoodanalysisofpositiveselectioninDNA-encodingproteinslinkedtowatermetabolismandskinstructureGeneProteinnnM0-M3M1-M2M7-M8sp.ind.LRTp(prop.)LRTp(prop.)LRTpacad10ACAD104419.60..4951.01(0%)0.01.0001.36(13.3%)1.40.877acp5TRAP926191.50.0016.16(2.0%)34.40.0016.05(2.0%)38.10.001anxa1ANXA1916157.40.0018.57(15.3%)57.20.0018.03(15.5%)59.10.001aqp3AQP382869.70.0019.22(1.1%)14.80.0049.19(1.1%)17.10.001aqp4AQP4331.31.0001.00(3.0%)0.01.00014.87(2.5%)0.61.000aqp7AQP7330.11.0001.00(5.1%)0.01.0001.00(0%)0.01.000aqp9AQP94412.20.0283.19(15.5%)4.30.2923.19(15.5%)4.60.226bcamBCAM/Lu6873.50.0045.81(5.2%)10.50.0255.32(5.8%)11.10.018ctnnb1CTNNB1440.01.0001.00(0%)0.01.0001.00(0%)0.01.000fads1FADS13318.00.0313.40(1.5%)7.00.08513.46(1.6%)7.20.081fgf10FGF10557.70.1551.00(1.7%)0.01.0001.00(1.8%)0.11.000guca2bGUCA2B71229.10.0013.53(5.1%)3.60.3453.23(6.3%)5.40.172has2HAS2440.01.0001.00(0%)0.01.0001.01(0%)0.01.000hyal2HYAL25517.80.0031.00(2.9%)0.01.0001.00(2.9%)0.01.000hyal3HYAL3440.41.0001.00(8.2%)0.01.0001.01(0%)0.01.000krt8KRT8440.01.0001.00(0%)0.01.0001.00(0%)0.01.000lrp4LRP44414.10.01552.96(1.11%)9.30.03552.95(1.11%)10.10.021psen2PSEN23312.980.00111.39(1.1%)4.10.35110.95(1.2%)3.90.226ptch2PTCH24467.20.00110.75(0.4%)9.1100.03510.45(1.2%)10.70.018pxnPXN446.60.2251.00(0%)0.0001.0001.00(0%)0.01.000sncgSNCG3313.20.02084.38(1.1%)11.5060.01885.32(1.1%)11.80.016tgm1TGM1556.70.6201.00(4.8%)0.0001.0001.00(0%)0.01.000tnfsf13TNFSF13/APRIL4412.10.02814.26(0.6%)3.2860.3708.6(1.1%)3.10.404Thetableshowsvaluesindicatingtheratioofnon-synonymoustosynonymoussubstitutionrates.n=samplesize;sp.-species;ind.-individuals;LRTlikelihoodratioteststatistic,givenas2lnLinthenestedmodelscompared(M0-M3,M1-M2andM7-M8);prop.-proportionofsiteswith>1;andp-values(adjustedformultipletestingwithFDR;significantat=0.05)forthecorrespondingtests.Testsindicatingsignalsofnaturalselectionareinbold Fig.1Aminoacidsitesunderpositiveselectioninbats.Sitesunderselectioninpartialcodingsequencesofthegenesacp5,anxa1,aqp3,bcamandptch2wereidentifiedphylogeneticallyfor1–11individualsofeachspeciesusingamaximumlikelihoodframework.Thepiechartsdisplayedshowtherelativefrequencyofaminoacidsineachsample.Numbersabovethealignmentpositionsrefertotheaminoacid’spositioninthereference(seeTable2).Thephylogenetictreedepictsrelationshipsprunedfromapreviouslypublishedmultilocusphylogeny[70].P=batspeciesdistributedinthePalearctic,N=Nearctic,A=AfrotropicalHarazimetal.BMCZoology (2018) 3:8 Page3of13

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BCAMlayinthe2ndIg-likeC2typedomain(Fig.2).ItwasnotpossibletomodelthestructureofPTCH2,thefifthproteinwithsitesunderpositiveselection.Weas-sumethatthetwositesunderpositiveselectionwerebothlocatedintheintracellularpartitionoftheprotein.Thebranchsitetestofpositiveselectioncomparedthetwonestedmodelsidentifyingthebranchunderpositiveselection(A1andA).AsignificantdifferencebetweenthemodelsindicatedpositiveselectioninEptesicusfuscus(p=0.001)andE.nilssonii(p=0.001)inacp5(Table3).WethentestedforpositiveselectiononspeciesinaPalearcticmonophyleticcladethatincludedMyotisdavi-dii,MyotisemarginatusandMyotismyotis.Threegenesunderselectionwereavailablefortheclade(Fig.1,Additionalfile1).ClademodelCvs.M2a_relrevealedpositiveselectioninacp5forthespecies(p=0.005),butnosignificantevidenceofselectioninanxa1andaqp3(p>0.05).DiscussionHibernationisastrategyusedbytemperate-zonebatstoin-creasesurvivalrateunderconditionsofconstrainedenergyreserves[26,27].Exposureofhibernatingindividualstoadditionalstressors,suchaspathogenpressure,couldresultinhealth-relatedcostsdecreasingtheirabilitytooverwinter.Mountinganimmuneresponseattimesoflowactivity,lowbodytemperatureandreducedpossibilityofregulationandenergyuptakeisariskydefencemechanismagainstpoten-tialmicrobialthreatsasitmaycontributetomortalitythroughdepletionoffatreserves[19,28]oroverwhelminginflammation[20].Frequencychangesinfunctionallyim-portantsinglenucleotidepolymorphismsinhistocompati-bilityantigens,cytokinesandtoll-likereceptorshavebeenrecordedinMyotislucifuguspopulationsthatsurvivedtheinitialP.destructansepidemicfrontassociatedwithpopula-tiondecline[2].Consideringtherisksassociatedwithim-muneresponseinvestmentinhibernators,toleranceofthepathogenatamolecularlevelislikelytobethebestpos-sibleapproachtoinfectioninhibernation.WNSpathophysiologyisdirectlyconnectedtothebat’shibernationability.Assumingthata)PalearcticbatshavebeenhistoricallyexposedtoP.destructans[23,24],b)Nearcticbatsrepresentnaïveanimalswithnohistor-icalinfluenceofinfection,andc)Afrotropicbatsremainhealthyastheyliveinanenvironmenttoowarmforpathogengrowth[29],itshouldbepossibletodetect Table2BataminoacidsitesunderselectionM1-M2M7-M8GeneposaapmeanSEposaapmeanSEacp598S/A0.0036.5421.42498S/A0.00017.2991.384XP_006104612.1125G/R/K0.00016.5591.394125G/R/K0.00017.3041.372145R/Q0.00016.5571.396145R/Q0.00017.3041.373223Q/R0.0176.4541.554223Q/R0.0067.2611.461anxa117E/Q0.0248.2271.71817E/Q0.0027.8411.296XP_014396764.120E/K0.0097.7921.41022T/M/V/I0.00018.4201.29722T/M0.00017.8571.25523K/N0.00018.4201.29923K/N0.00017.8571.25525I/V0.0208.2611.65325I/V0.0027.8421.31827G/A/T0.00018.4201.29727G/A/T0.00017.8571.25534P/S0.0028.4031.34534P/S0.00017.8561.25955I/V0.0128.3251.52655I/V0.0027.8461.285aqp3XP_006758647.1128L/W0.0018.6931.604128L/W0.00018.0271.895bcam365N/D0.0386.8501.934XP_015416692.1407V/I0.0127.0191.678431S/V/P0.0087.0461.628434I/L0.0137.0131.690440F/H0.0356.8661.902523S/P0.0097.0401.641ptch23R/G/H0.0282.8061.006XP_014319265.11155T/S/A0.0464.571.7781155T/S/A2.8420.963AminoacidsitesunderpositiveselectionwereanalysedusingBayesempiricalBayes.Position(pos)referstothepositionofanaminoacid(aa)mappedagainstthereferencesequencestatedinthetable.ThecalculationisbasedonbothM1-M2andM7-M8comparisonsandshowstheM1–M2comparisonasmorestrictHarazimetal.BMCZoology (2018) 3:8 Page4of13

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selectivechangesinthebatgenometestifyingtotheconsequencestoinfection-relatedmortality,thisservingasaselectivepressurefavouringbeneficialmutationsovertheancestralgeneticsequence.InfectionbyP.destructansisdependentonlowtempera-turesasthefungusisunabletogrowattemperaturesgreaterthan20°C[29].Atenvironmentaltemperaturesprevalentinhibernacula,thefungusinvadesthehost’sskinfromtheepi-dermistothedeeperskinlayers[20,30,31].Anabilitytoprotecttheskinfromlesionsandfunctionaldisruptioniscriticaltothebat’ssurvival,andevenmoresoasregardstheskinformingthewing(consideredthebat’slargestorgan).Ofthegenessignallingpositiveselection,manyareinvolvedinmaintainingskinhomeostasisandpromotingwoundhealing(Table1,Fig.3).Whileprovingadirectcausallinkbetweenselectiononparticulargenes(Table1)andhistor-icalP.destructansexposureisimpossiblewithoutexperi-mentalmanipulation,themolecularfunctionofgenesunderselectionmaybeinterpretedinrelationtoaskininfection.SitesunderpositiveselectioninthePatched2protein(PTCH2)werelocatedintheproteinintracellularpartition(Fig.1).PTCH2isfunctionallysimilartoitshomologue,PTCH1,bothofwhichserveasreceptorsintheSonichedgehogpathway,crucialinembryonicdevelopmentandadulttissuehomeostasis[32,33].WhilePTCH2appearstoberedundantinembryonicdevelopment,itpreservesacrucialroleasregardstheadultepidermis[34,35],thoughthemolecularmechanismremainsunclear.Synucleingammaprotein(SNCG)isexpressedinstratumgranulo-sum,bothinembryonicdevelopmentandinadults,whereitfunctionsasakeratinnetworkmodulatorintheepider-mis[36].Thebasalcelladhesionmolecule(BCAM/Lu),amembrane-boundmoleculeexpressedbykeratinocytesininflammatorystates[37],maycontributetoanti-infectionreactionsinthebat’sskin.Theaccumulationofsitesunderpositiveselectioninthe2ndIg-likeC2typedomaininBCAM(Fig.2)mayindicatetheimportanceofthisdomainasregardsmolecularfunctioninginskinpathology,though Fig.2Positivelyselectedsitesinbatproteinstructure.SitesinTRAP,ANXA1,AQP3andBCAMhighlightedinredareunderpositiveselection(estimatedbyBayesempiricalBayes)whileyellowsitesrepresentironbinding(TRAP)andcalciumbinding(ANXA1)sites(UniPROTP29288andP07150,respectively).ThemodelswerecreatedbyPhyre2structurepredictionsoftware[76],usingareferencebatspeciesproteinsequence(XP_006104612.1,XP_014396764.1,XP_006758647.1,XP_015416692.1).ThePTCH2modelisnotincludedaswewereunabletopredictareliablemodelwithoutahighproportionofab-initiomodelledsitesHarazimetal.BMCZoology (2018) 3:8 Page5of13

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themolecularmechanismforBCAMactivityremainsunclear.LDLreceptorprotein4(LRP4)belongstoafamilyofLDLreceptorproteinsthat,indimericform(LRP5/LRP6),serveasactivatingreceptorsfortheWnt/-ca-teninpathway.Apartfromitsroleinembryogenesis,thispathwayisreactivatedinadultsandmediatestissuere-generationfollowinginjury[38],itsrolelyinginnegativeregulationagainstactivationoftheWnt/-cateninpath-way[39,40].WhileLRP4sharesthegeneralstructuralmotifsofthefamily,itlackssomebindingmotifs.LRP4mutationsseverelyalterpathwaysignalling.BatswithskindisruptionsaremostlikelyaffectedbychangesinLRP4structureanditssubsequenteffectontheWnt/-cateninpathwayduringwoundhealing.Aquaporin3(AQP3)isexpressedinkeratinocytes[41]andepidermaltissuesandisinvolvedintheregulationofwaterandglycerolcontentinskin[41],therebyinfluen-cingwoundhealing.Byregulatingwaterflow,AQP3facili-tatesepidermalcellmigrationandproliferation[42].AQP3hasbeenshowntohavearoleinthehealingofcu-taneousburnwounds[43],whicharesimilartotheskindisruptioncausedbyriboflavinaccumulation,tissuene-crosisandsubsequentoxidativestresscontributingtoWNSpathology[14].ThesiteunderselectioninAQP3possiblyaffectstheshape(andthusfunction)oftheporinthroughitspositioninthetransmembranehelix(Fig.2).SkinbarrierintegrityisanimportantfactorinWNSpathologyandsurvival(Fig.3).DiseasedandmoribundbatswithWNSskinlesionsarousefromhibernationmorefrequentlythanhealthyanimals[19,28].Duringarousal,bodytemperaturerisesandisfollowedbyanot-ableincreaseinactivityandmetabolism,followingwhichtheimmunesystemmountsaresponsetothechronicinfection.Thisreactionisoftenuncontrolled,causing Table3BranchsitetestsforproteinsundergoingpositiveselectionGene/Foregroundbranchnforegroundind.2lnLpacp5Eptesicusfuscus1730.001Eptesicusnilssonii127.50.001Myotisdavidii10.71.000Myotismyotis50.71.000Myotisbrandtii10.01.000Barbastellabarbastellus40.01.000Myotisemarginatus80.01.000Myotislucifugus50.71.000anxa1Myotisdavidii18.10.153Myotisemarginatus48.10.153Myotisbrandtii13.11.000Eptesicusfuscus13.11.000Neoromicianana11.91.000Eptesicusnilssonii20.01.000Myotislucifugus10.01.000Myotismyotis40.01.000Pipistrelluspipistrellus11.81.000aqp3Eptesicusfuscus10.91.000Myotismyotis40.81.000Eptesicusnilssoni20.01.000Myotisbrandtii10.01.000Myotisdavidii10.01.000Myotisemarginatus110.01.000Myotislucifugus10.01.000Neoromicianana40.01.000Pipistrelluspipistrellus30.01.000bcamEptesicusfuscus10.11.000Myotisbrandtii10.01.000Myotisdavidii10.01.000Myotisemarginatus10.01.000Myotislucifugus20.01.000Pipistelluspipistrellus20.01.000lrp4Eptesicusfuscus10.71.000Myotislucifugus10.21.000Myotisbrandtii10.01.000Myotisdavidii10.01.000 Table3Branchsitetestsforproteinsundergoingpositiveselection(Continued)Gene/Foregroundbranchnforegroundind.2lnLpptch2Eptesicusfuscus10.71.000Myotislucifugus10.21.000Myotisbrandtii10.01.000Myotisdavidii10.01.000sncgMyotisdavidii10.81.000Eptesicusfuscus10.01.000Myotisbrandtii10.01.000Proteinsweretestedforpositiveselectionusingthelikelihoodratiotest(A-modelasalternativehypothesisandA1-modelwith=1asnullhypothesis).p-values(FDRcorrected;significantat=0.05)indicatingsignalsofnaturalselectionaremarkedinboldHarazimetal.BMCZoology (2018) 3:8 Page6of13

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tissuedamage[20].AnnexinA1(ANXA1),whichispositivelyselectedforinbats,actsasamediatorofglucocorticoidanti-inflammatoryactivity[44],andhencemaybeabletodown-regulatesuchanimmuneresponse.ANXA1,whichisproducedbyinnateimmunitycellssuchasneutrophils,hasanautocrineandparacrineef-fectontheinnateimmunitycellsthroughinhibitionofvascularattachmentandextravasation[45].ANXA1alsoplaysanimportantroleinadaptiveimmunityagainstchronicinfectiousdisease[46],modulatingT-celladaptiveresponseanddirectingtheimmuneresponsetowardstheTh1/Th17response[47,48].Increasedexpressionofil-17andil-6[49]directsproliferationofTh17andTh1/Th17cells,thelatterT-cellsalsobeingaffectedbyifn,whichishighlyvariableinpost-WNSpopulations[2].Theob-serveddecreaseinneutrophiladhesionandinefficientantibody-mediatedimmuneresponse[50]maybeanef-fectofapredominatingprotectiveTh1/Th17responsetothepathogeninlaterstagesoftheinfection.TheoverallcharacteroftheimmunereactionsupportsourproposalforanimportantroleofANXA1ininfectionpathologyandregulationoftheimmuneresponse.Asaregulatorofinflammation,ANXA1alsoplaysaroleintheoutcomeofinflammatoryprocesses,woundrepairandepithelialre-covery[51].TheselectedsitesinANXA1areaccumulatedintheN-terminalregionoftheprotein(Figs.1and2)and,whiletheydonotoverlaythebindingsites,theypointtotheimportanceofthearea.Similarly,TRAPalsoservesasaninnateimmuneregu-lator,participatinginthemacrophageimmuneresponseandimpactingonpathogenclearancefromthehost,mostlikelybyaffectinginnateimmunecellactivityatthesiteofinfection[52]bycatalysingproductionofre-activeoxygenspecies[53].Italsoparticipatesindown-regulationoftheimmunesystemandmodulationoftheTh1response[54],whichmaycontributetoTh1/Th17modulationoftheimmuneresponsebyANXA1.MetalbindingsitesinTRAParespreadalongthepro-teinsequenceanddonotdirectlycoincidewiththese-lectedsites;hence,theeffectofselectedsitesonbinding Fig.3Molecularmechanisticmodelofwhite-nosesyndrome(WNS)toleranceinbats.Duringhibernation,abatÂ’sbodytemperature,metabolicrateandimmunesystemareloweredforuptosixmonths,onlyincreasingforperiodslastinguptoseveralhoursduringperiodicarousalfromtorpor.BathibernationprovidessuitableconditionsforPseudogymnoascusdestructansinfectionanddevelopmentofthefungaldiseaseWNS.ThefungusinitiallygrowsontheskinÂ’ssurfaceandprogressestowardinvasiveinfection,whereuponitdepositslargeamountsofvitaminB2intoskinlesions,leadingtoskinnecrosis.Inthemostseverecases,eitherlargeareasofskinbecomenecroticortheimmunesystemÂ’sresponsetomassiveinfectionoverwhelmstheanimaluponarousal.MolecularmechanismssupportingWNStolerancearelikelytoincludestrengtheningofskinintegritymaintenanceandenhancementofwoundhealing.Surprisingly,theremayalsobenegativemodulationoftheimmuneresponse,whichcouldotherwisedepletethebatÂ’senergyreservesorcausedeaththroughimmunereconstitutioninflammatorysyndrome[20]Harazimetal.BMCZoology (2018) 3:8 Page7of13

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capacitycannotbeevaluatedfromasimplesequencecomparisonandwouldrequirefurtherstudy.Theaminoacidsitesunderpositiveselectiondonotne-cessarilyreflecttheactiveorbindingsitesofproteins.Infact,itismoreprobablethatproteinfunctionismaintainedthroughpurifyingselection(<1),withahigherpropor-tionofsynonymouslymutatedsitesconservingtheactiveaminoacidsite.Whileapositivelyselectedactivesiteisun-likely,itispossible,mostnotablyincasesofproteinco-evolutionwithaligand.Variableregionsinthesequencearelocatedontheprotein’ssurfaceandinfluencestructuralfolding.Proteinstructureinfluencesaffinityandinteractiontopartners,whichisprobablythecaseasregardsthesitesdetectedinthisstudy.Justaswiththeshapeofafoldedprotein,proteininteractionbarriersmayaffecttheirbio-logicalfunction;hence,anabsenceofmechanicalbarriersintheareasurroundingtheactivesiteisessentialforphys-icalinteractionoftheproteins,andpositiveselectionintheseregionsmayfacilitateinter-proteincontact.Thesignalforpositiveselectioninaproteincanbeallo-catedtospecificbranchesonaphylogenyandcanbeiden-tifiedatsitesintheDNAsequence.Nestedbranch-sitemodelsrequiredifferencesinthenaturalselectionsignalatanaprioridefinedbranch,comparedwiththeremainingdiversity,whichispresumedtobeunderpurifyingselec-tioninthemodel.Wedetectedpositiveselectionintheacp5geneinbothNearcticandPalearcticEptesicusandinacladeofPalearcticMyotis.TheNearcticbatspeciesEpte-sicusfuscusshowslimitedlevelsofresistancetoWNSin-fection[55],whilethePalearcticspeciesEptesicusnilssoniirarelydevelopssevereWNSpathology[10].PalearcticM.myotisandM.emarginatusarerepresentativeofthosespeciesdisplayingahighfungalloadandsevereWNSpathology[10,22],thoughtheyareabletotoleratethein-fection[22];informationoninfectionstatusofthethirdspeciesinthePalearcticclade,M.davidii,ispresentlyun-available.Whilethemechanismsexertingselectivepres-sureonacp5maydifferfromP.destructansinfection,itmaynowprovideprotectionagainstWNSprogression.Wewereunabletolocatebranchesofpositiveselec-tioninsomegenes,mostlikelyduetoalackofavailablesequences(nlrp4=4,nptch2=4,nsncg=3)and,therefore,lowsequencevariabilityinthedataset.Ithasalsobeenshownthat,withanincreasedproportionofsynonym-ousmutations(dS),thebranchsitetestforpositivese-lectionismorepronetofalsenegativeresults[56].Lackofselectionsignalheterogeneityinbranchescouldalsobeassignedtohost-unspecificcharacteristicsofthepathogen,wheretheselectivepressurewouldaffectallspeciesintheanalysis.However,asthenumberofsequencesintheana-lysisincreaseswithadditionalsequencingeffort(e.g.[57]),sothechancesofdetectingsignificantsignsofpositivese-lectioninatleastsomebranchesincreases,thusdetectionofalackofpositiveselectionintheanalysisismorelikely.Inabilitytodetectaspecificbranchunderselectionmayalsobeaffectedbytheuseoftwodifferenthaplotypesfromeachindividualduringsequenceanalysis,whiletherelationshipbetweenthetwohaplotypesremainsunevalu-ated.Incaseswhereonehaplotypedominates,presenceofthesecondhaplotypeinthesequenceanalysismaymaskthesignalofapositivelyselectedhaplotypeinthespecies.SincetheproteinproductsofgenesforwhichpositiveselectionhasbeendetectedarerelevanttothepathologyandpathophysiologyofWNS,weexpectthemtoplayaroleinskinintegrity,woundhealingandimmunesup-pression.Hence,wecanhypothesisethattheselectedgenescontributetothemechanismofinfectiontoleranceinPalearcticbatspeciesinfectedwithWNS(Fig.3).ExtrapolatingfromthedevastatingeffectsonNearcticbatpopulationsnotpreviouslyexposedtoskininjuriescausedbyP.destructans[6],WNShastobethehistoricalfactorexertingenormousselectionpressureonPalearcticbats.NotonlycouldWNShaveinfluencedgeneticchangesinskinintegrityithasprobablyalsoinfluencedotherrear-rangementscapableofpreventingthedevastatingeffectsofthedisease.CleardifferencesbetweenNearcticandPalearcticbatsindicateatleasttwosuchrearrangements.Thefirstisamuchhighertolerancetomiteandinsectec-toparasitesinPalearcticbats(typically100%prevalenceinbreedingcolonies)[58–60].Thisresultsinhabituationtothestressofskininjuries,whichmayalsoactasafeedbackfactorreducingneuralsensitivitytosuchstimuliandtheireffectuponarousalfromtorpidity.TheseconddifferencebetweenPalearcticandNearcticbatsrelatestohibernationtactics.MosthibernatingPalearcticbatsdisperseintoalargenumberoflesspopulatedhibernacularatherthanforminggiantclustersinasinglemasshibernaculum,char-acteristicformultipleNearcticspecies[61,62].WhilethisbehaviouralpatternappearstohavedeclinedaftertheWNSinvasionfront[63],repeatedarousalcausedbythegroomingofinfectedindividualscouldstillleadtoanunin-tentionaldominoeffectofmultiplearousals[64].Thisre-sultsinthebreakdownofthecoreadvantageofsuchhibernationtactics,i.e.sociallycontrolledthermalhomeo-stasisreducingdemandsonfatreservesandtheneedofin-dividualbehaviouralskillsforhibernationperformance.Theabovefactors,suggestedbythedifferencesbe-tweenNearcticandPalearcticbats,illustratethecom-plexnatureofskininfectionandtheintricaciesinvolvedinanadaptiveresponsetoaninfectiousagent.Weshowthatpositiveselectionatthegeneticleveliscombinedwiththeeffectsofincreasedtolerancetoaparasiteloadandbehaviouralrearrangements,reducingthebat’scap-acitytoperformadvancedhibernationtactics.ConclusionsDuringhibernation,batsconserveenergybymaintainingalowbodytemperatureandminimisingmetabolism.Harazimetal.BMCZoology 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Whilethisstrategyenablesthemtosurviveperiodsofresourcescarcity,theybecomevulnerabletoinfectionastheirimmunesystemfailstoactivelybattleagainstinfec-tionwhileintorpor.Onceithadinvadedlivingtissue,thepathogencausesmajordamage,forminglesionsanddepositingmetabolitesthatleadtonecrosis.Theaccu-mulationofphysiologicalconsequencesstartsacascadeofadverseeffectsthatculminatesinthedeathofthedis-easedanimal.Wefoundthatgenesinvolvedinthede-velopment,structureandmaintenanceofskinshowsignsofpositiveselection.Withrespecttolethaldermalinfection,theepidermislikelyprotectsheterothermsbyactingasapassivebarrieragainstinfectionduringhiber-nation,atimewhentheanimalcannotinvestenergyintoimmunereactions.Thegenesidentifiedinthisstudymayprovideinspirationindesigningtargetedtreatmentsforskininfectionsandforelucidatingmecha-nismsinvolvedindiseasetoleranceandresistancetootherfungalinfections,suchassnakefungaldiseaseoramphibianchytridiomycosis.MethodsIdentificationofputativegenesWefirstselectedgeneswithwatermetabolismandskinfunctionsintheGeneOntologydatabase[65],thenusedthekeywords‘water’and‘epidermis’tofindgeneswithfunctionsrelatedtothesekeywordsinRattusnorvegicus(223genesfound).WethensearchedUniProtKBfororthologousproteinsintheVespertilionidaeandMini-opteridaefamilies(50genesfound).ToidentifygenesexpressedinVespertilionidaeduringnaturalP.destructansinfection,weanalysedIlluminareadsofM.myotistranscriptome(Accessionnumbers:SRX2270325,SRX2266671)bymappingtheminGeneiousmapperontothereferencenucleotidesequencesofthese-lectedgeneswithhighsensitivity.WemappedthereadstothereferencesequencesusingGeneioussoftwarever-sion6.1.6(BiomattersLtd.,Auckland,NewZealand).Fromthesetof50proteinorthologsinVespertilioni-dae,weidentifiedasubsetofmRNAsequencesfor30selectedgenesexpressedinM.myotis.Sequencesof23ofthe30genesidentifiedwerefoundinmorethantwospeciesofPalearcticandNearcticbatsinfectedbyP.destructansbynameandtaxasearchintheNCBINu-cleotidedatabase,andthesewereusedforfurtherana-lysis.WeavoidedsearchingfororthologswithBLASTduetothenon-negligiblepossibilityofgenetreetospeciestreediscordancebetweenparalogsandorthologsincloselyrelatedspeciescausedbyincompletelineagesorting[66].AssembledcodingDNAsequencesexpressedinM.my-otiswerethenalignedwiththereferencetoidentifycon-servedregionsflankingavariableregioninordertodesignprimerswithinanexon.Primersforamplificationofthese-lectedcodingregionsweredesignedinPrimer3web4.0.0[67,68].Forwardandreversegeneprimersweresupple-mentedwithanM13oligonucleotidetailatthe5endtofacilitatebarcoding,formingPrimerset1.Primerset2con-tainedpairedbarcodesandacomplementarysequencetotheflankingM13tailsofPrimerset1atthe3end.ThepairedbarcodesequencesconformedtothebarcodingprotocolinSMRTAnalysis1.4(PacificBiosciences,MenloPark,CA,USA).SamplecollectionandDNAprocessingSampleswereobtainedfromethanol-storedtissuecol-lectionsattheInstituteofVertebrateBiologyoftheCzechAcademyofSciences,NationalAnimalGeneticBank,Studenec,CzechRepublic.DNAwasextractedusingtheDNeasy®Blood&TissueKit(Qiagen,Halden,Germany)accordingtothemanufacturer’sprotocol,additionalDNAsamplesbeingobtainedfromtheGriffinRabiesLaboratoryattheStateofNewYorkDepartmentofHealth,Wadsworth,NY,USA.Intotal,240samplesrepresenting32speciesfromEurope,NorthAmericaandAfricawereamplifiedwithnestedPCR(Additionalfile2).InthefirstPCR,codingregionsoftheselectedgeneswereamplifiedwiththegene-specificprimers,formingPCRset1.Themastermixforeachgenecontained1×buffer,0.2mMdNTP,0.2Mofforwardandreverseprimers,0.05UPlatinumTaqDNApolymerase(Invitrogen,Carls-bad,CA,USA)and1lofDNA.Eachreactionwassupple-mentedwithMgCl2atfinalconcentrationsgiveninAdditionalfile3.ThePCRwasinitialisedwithahot-startat95°Cfor3min,followedby35cyclesatannealingtemper-aturesandannealingandextension(72°C)timesspecifiedinAdditionalfile3,afterwhichthereactionwasfinalisedat72°Cfor3min.ThePCRproductwasdiluted33×andusedasatemplateforthesecondPCR.InthesecondPCR,Primerset2wasusedforallgenes,takingcarethatindivid-ualsampleswereamplifiedwithauniquebarcodecombin-ation.ThePCRreactionwasidenticaltothefirstPCR,with1.5mMofMgCl2andthe35cyclesusingthe95–53-72°Ctemperatureprofilefor40–40-extseconds,whereextrepre-sentsextensiontimespergene(Additionalfile3).ThePCRproductconcentrationwasestimatedfrom2%agarosegelsstainedwithGoldViewrelativetoa100bpDNALadderstandard(Invitrogen,availablefromLifeTechnologies,Prague,CzechRepublic)intheGenoSoft4.0program(VWRInternationalBVBA,Leuven,Belgium).ThePCRproductconcentrationenabledequimolarpoolingofallsamplesforeachgene.ThePCRproductpooledforallsampleswasseparatedonagel,thebandofexpectedlengthexcisedandDNApurifiedwiththeHighPurePCRPurifica-tionKit(RocheDiagnosticsGmbH,Mannheim,Germany).Thegeneproductswerepooledequimolarlywithafinalconcentrationof29ngl1andtheDNAsamplesweresequencedcommerciallyonaSMRT(singlemoleculereal-time)platform(PacificBiosciences)intwotechnicalHarazimetal.BMCZoology (2018) 3:8 Page9of13

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replicates.TheDNAtemplatelibrarywaspreparedwiththePacBioDNATemplatePrepKit2.0accordingtothePacBioprotocolfor10kbTemplatePreparationandSequencing.TheDNAtemplatelibrarywasboundtotheDNApolymer-asewiththePacBioDNA/PolymeraseBindingKitP4forthefirstreplicate,andP5forthesecondreplicate.Sequen-cingonthePacBioRSIIsequencerwasperformedwiththePacBioDNASequencingKit,usingC2andC3chemistryforthetworeplicates,respectively.Sequencingwasper-formedon2SMRTCellswitha180-minmovietimeperSMRTCell.DataprocessingThereadswerede-multiplexedaspartofthecommercialrawdataanalysisduringsequencing.DataobtainedfromSMRTgenesequencingwereassembledtothereferencesequencesinGeneiouswithcoverage>10×andpercentsimilarityinthealignment>50%.ProcessedsequencedatawerealignedusingMAFFTversion7.307[69]toananno-tatedsequencereferenceobtainedfromtheNCBIdata-base.AlignmentswereeditedinGeneioustocontainonlycodingsequencesofthegenefittingtheappropriateopenreadingframefromthesequencereferenceannotation.DataanalysisAmaximum-likelihoodphylogenyofChiropteranspe-ciesobtainedinapreviousstudy[70]wasusedforphylogeneticanalysisofpositiveselection.Thetreewasunrootedandsubsettocontainspeciespresentineachcorrespondingsequencealignment(Additionalfile1).Ingeneswheremorethanoneindividualperspecieswassequenced,therespectivetreetipwaspopulatedwithapolytomywithzero-lengthbranchestofitthenumberofindividualsperspeciesanalysedinthealignment.Wetestedthecodonsinalignmentforsignsofposi-tiveselection,definedasrateratioofnon-synonymousandsynonymoussubstitutions(=dN/dS).WeusedtheCODEMLprogramfromthePAML4.9package[25]toestimatefortherespectivepartialgenesequences,andvariabilitybetweensitesusingthemaximumlikelihoodmethod.SignalsforpositiveselectionwereestimatedfromacomparisonofnestedmodelsimplementedinPAMLusingthelikelihoodratiotest(LRT).Theoneratiomodel(M0)[71,72]setsoneforallsitesalongthetestedgene.Acorrespondingalternativemodel,thediscretemodel(M3)[4],allowsapredefinednumberofsiteclassestovaryin.Whilethenearlyneutralmodel(M1)[73]doesnotallowtovary,therateofsynonym-ousmutationsmayvaryateachsite,withtherateofnon-synonymousmutationsbeingequaltothesynonym-ousorequalto0.AnalternativetoM1,thepositiveselec-tionmodel(M2)[73],isderivedfromtheneutral(M1)modelandallowstherateofnon-synonymousmutationstoexceedtherateofsynonymousmutations(>1).InthebetaM7model[71],distributioninsitesislimitedtointerval[0,1],meaningthatthesignalforpredominantpositiveselectioncannotbedetected.Thealternativenestedmodel,beta&(M8)[74],allowsthevaluesoftobelargerthan1.Acomparisonoftheoneratio(M0)modelandthediscrete(M3)modelwasusedtotestwhethervariedbetweensites.Totestforpositiveselectionsignalsinthecodonsequencedata,wepairedthenearlyneutral(M1)andpositiveselection(M2)modelsandthebeta(M7)andbeta&(M8)models,withthefirstmodelpairusedasanullmodel.Thenestedmodel’slikelihoodvalueswerecomparedusingtheLRT(twicethedifferencebetweenthelog-likelihoods;2lnL)ofthenullandalternativemodels.The2lnLvalueswerethencomparedto2dis-tributionsforM0-M3,M1-M2andM7-M8compar-isons.Wecorrectedsignificanceofthoseanalyseswithfalsediscoveryrates(FDR)andacceptedtheadjustedlevelsofsignificanceat5%assignificant.Proteinswithsignificantresultsinlocus-levelselectionwereanalysedforsitesunderpositiveselection,identi-fiedbasedontheBayesempiricalBayesmethod(BEB)[75]implementedinPAMLforsitetestsofpositivese-lectionM1–M2andM7-M8.TheBEBmethodincor-poratesuncertaintyinmaximumlikelihoodestimatesofparametersofthedistributionbyintegratingovertheirpriordistribution.Bycorrectingfortheuncertaintyinparameterestimates,BEBiswellsuitedforsmalldata-sets[75].Forvisualisationofsiteswithintheproteinstructure,Phyre2structurepredictionsoftware[76]wasusedtopredictproteinmodelsusingareferencebatspe-ciesproteinsequence(XP_006104612.1,XP_014396764.1,XP_006758647.1,XP_015416692.1).Ingeneswithsitesundergoingpositiveselection,weidentifiedphylogenybranchesunderselectionusingthebranch-sitetestofpositiveselection[75,77].Thebranchsitetestisusedtodetectbranchesunderpositiveselec-tionpre-specifiedinthetestedphylogeny(foregroundbranches),wheretheotherbackgroundbrancheswouldundergopurifyingselection.Ineverytree,wetestedeachindividualbranchasaforegroundbranchforsignsofpositiveselection.ThebranchsitetestisperformedbycomparingmodifiedbranchsitemodelA,allowingtovarybetweenbranches,withnullmodelA1where=1.ThenestedmodelsweretestedbyLRTandcomparedto2distributionwithdf=2andp-valueswereadjustedwiththeFDR.ThemonophyleticcladeofPalearcticspeciesincludingM.davidii,M.emarginatusandM.myotiswasmodelledbyclademodelC[78]asaforegroundtestedclade,whichwascomparedtoanullmodelM2a_rel[79].ThenestedmodelsweretestedbyLRTandcomparedto2distribu-tionwithdf=1,withp-valuesadjustedbytheFDR.Harazimetal.BMCZoology (2018) 3:8 Page10of13

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AdditionalfilesAdditionalfile1:AccessionnumbersofbatDNAsequencesandtheirrespectivephylogeny.Thecodingsequencesoftherespectivegenes(alignmentlengthinparentheses)wereusedformaximumlikelihoodanalysisofnaturalselectioninbats.Theguidetreewasprunedfromapreviouslypublishedmultilocusphylogeny[70].Thescalebarisinsubstitutionsbp1.(PDF189kb)Additionalfile2:Batsamplesamplifiedinthisstudy.Populationswereconsideredashibernating(+)ornon-hibernating()inthecountryofsampleorigin.SpecieswereconsideredinfectedwhenPseudogymnoascusdestructanswasdetectedinatleastoneindividualusingmoleculargeneticorcultureexperimentsandpositiveforWNSwhenP.destructanswasconfirmedanddiagnosticlesionsfoundonskinhistopathology[10,22].(XLSX42kb)Additionalfile3:Primersandamplificationconditionsforgeneswithaskinintegrityorwatermetabolismfunction.PrimerpairsweredesignedforgenesexpressedinMyotismyotiswithwhite-nosesyndrome(Accessionnumbers:SRX2270325,SRX2266671).(XLSX47kb)AbbreviationsBEB:BayesempiricalBayes;FDR:Falsediscoveryrate;LRT:Likelihoodratiotest;WNS:White-nosesyndromeAcknowledgementsTheauthorsacknowledgeAnnaBryjováforstudydesignconsultation;Dagmar—oukalováandPetraRabuicováforlaboratoryassistance;JosefBryjaandAlenaFornskováfromtheInstituteofVertebrateBiology(Brno),PetraHájkováandBarboraZemanováfromtheNationalAnimalGeneticBank,Studenec;andRobertRuddandAprilDavisfromtheGriffinRabiesLaboratoryfortheStateofNewYorkDepartmentofHealth,Wadsworth,NY,forprovidingDNAandtissuematerialusedinthisstudy.FundingThisstudywassupportedbytheCzechScienceFoundation(17-20286S)andbytheMinistryofEducation,YouthandSportsoftheCzechRepublicthroughtheNationalProgrammeofSustainabilityProjectIT4Innovations-ExcellenceinScience(LQ1602).Thefundersplayednoroleinthestudydesign,datacollectionandanalysis,decisiontopublishorpreparationofthemanuscript.AvailabilityofdataandmaterialsThedatasetsgeneratedandanalysedduringthisstudyareavailableintheNCBINucleotiderepository,MH178037-MH178081.Authors’contributionsIH,SM,JP,JZandNMconceivedandconceptualisedtheidea;ZVandNMdesignedthestudy;IH,SM,JP,JZandNMcollectedthematerial;LJ,AZjr.andNMperformedthelaboratoryanalysis;MH,KL,JCM,PSandNManalysedthedata;MHandNMwrotethemanuscript,towhichallauthorscontributed.EthicsapprovalTheauthorsareauthorisedtohandlefree-livingbatsunderCzechCertificateofCompetency(No.CZ01341;§17,ActNo.246/1992Coll.).TheCzechAcademyofScience’sEthicsCommitteehasreviewedandapprovedtheAnimalUseProtocolNo.169/2011incompliancewithActNo.312/2008Coll.onProtectionofAnimalsagainstCruelty,asadoptedbytheParliamentoftheCzechRepublic.Non-lethalbatsamplingcompliedwithActNo.114/1992Coll.onNatureandLandscapeProtection,andwasbasedonpermits01662/MK/2012S/00775/MK/2012,866/JS/2012and00356/KK/2008/AOPK,issuedbytheNatureConservationAgencyoftheCzechRepublic.ConsentforpublicationNotapplicable.CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.Publisher’sNoteSpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.Authordetails1InstituteofVertebrateBiology,TheCzechAcademyofSciences,Brno,CzechRepublic.2DepartmentofBotanyandZoology,FacultyofScience,MasarykUniversity,Brno,CzechRepublic.3DepartmentofZoology,FacultyofScience,CharlesUniversityinPrague,Prague,CzechRepublic.4InstituteofBiostatisticsandAnalyses,MasarykUniversity,Brno,CzechRepublic.5DepartmentofBiologicalSciences,UniversityatAlbany,StateUniversityofNewYork,Albany,NY,USA.6DepartmentofEcologyandDiseasesofGame,FishandBees,UniversityofVeterinaryandPharmaceuticalSciencesBrno,Brno,CzechRepublic.7ResearchInstituteoftheInnovationsCentreofExcellence,SilesianUniversityinOpava,Opava,CzechRepublic.8DepartmentofMuscleCellResearch,CentreofBiosciences,InstituteofMolecularPhysiologyandGenetics,SlovakAcademyofSciences,Bratislava,Slovakia.9Presentaddress:PublicHealthMicrobiologistfortheStateofAlaska,DepartmentofHealthandSocialServicesattheAlaskaStateVirologyLabinFairbanks,Fairbanks,AK,USA.10Presentaddress:DepartmentofCellularCardiology,InstituteofExperimentalEndocrinology,BiomedicalResearchCenter,SlovakAcademyofSciences,Bratislava,Slovakia.Received:16March2018Accepted:16August2018 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