Large-scale mitochondrial DNA analysis reveals new light on the phylogeography of Central and

20 Pages·2017·7.23 MB·English

RESEARCHARTICLE
Large-scale mitochondrial DNA analysis
reveals new light on the phylogeography of
Central and Eastern-European Brown hare
(Lepus europaeus Pallas, 1778)
MohammadRezaAshrafzadeh1,MihajlaDjan2,La´szlo´ Szendrei3,AlgimantasPaulauskas4,
MassimoScandura5,Zolta´nBagi6,DanielaElenaIlie7,NikolozKerdikoshvili8,
PanekMarek9,Noe´miSoo´s3,SzilviaKuszaID3*
a1111111111 1 DepartmentofFisheriesandEnvironmentalSciences,FacultyofNaturalResourcesandEarthSciences,
ShahrekordUniversity,Shahrekord,Iran,2 DepartmentofBiologyandEcology,FacultyofSciences,
a1111111111
UniversityofNoviSad,NoviSad,Serbia,3 InstituteofAnimalHusbandry,BiotechnologyandNature
a1111111111
Conservation,UniversityofDebrecen,Debrecen,Hungary,4 DepartmentofBiology,FacultyofNatural
a1111111111
Sciences,VytautasMagnusUniversity,Kaunas,Lithuania,5 DepartmentofVeterinaryMedicine,University
a1111111111 ofSassari,Sassari,Italy,6 InstitutesforAgriculturalResearchandEducationalFarm,Universityof
Debrecen,Debrecen,Hungary,7 ResearchandDevelopmentStationforBovineArad,Academyfor
AgriculturalandForestrySciences,Arad,Romania,8 TbilisiZoo,Tbilisi,Georgia,9 PolishHunting
Association,ResearchStation,Czempiń,Poland
*[email protected]
OPENACCESS
Citation:AshrafzadehMR,DjanM,SzendreiL,
PaulauskasA,ScanduraM,BagiZ,etal.(2018) Abstract
Large-scalemitochondrialDNAanalysisreveals
newlightonthephylogeographyofCentraland
Europeanbrownhare,Lepuseuropaeus,fromCentralandEasternEuropeancountries
Eastern-EuropeanBrownhare(Lepuseuropaeus
(Hungary,Poland,Serbia,Lithuania,Romania,GeorgiaandItaly)weresampled,andphylo-
Pallas,1778).PLoSONE13(10):e0204653.
https://doi.org/10.1371/journal.pone.0204653 geneticanalyseswerecarriedoutontwodatasets:1.)137sequences(358bp)ofcontrol
regionmtDNA;and2.)105sequencesofaconcatenatedfragment(916bp),includingthe
Editor:Tzen-YuhChiang,NationalChengKung
University,TAIWAN cytochromeb,tRNA-Thr,tRNA-ProandcontrolregionmitochondrialDNA.Oursequences
werealignedwithadditionalbrownharesequencesfromGenBank.Atotalof52and51hap-
Received:February13,2018
lotypesweredetectedwithinthetwodatasets,respectively,andassignedtotwopreviously
Accepted:September12,2018
describedmajorlineages:Anatolian/MiddleEastern(AME)andEuropean(EUR).Further-
Published:October4,2018
more,theEuropeanlineagewasdividedintotwosubcladesincludingSouthEasternEuro-
Copyright:©2018Ashrafzadehetal.Thisisan pean(SEE)andCentralEuropean(CE).Sympatricdistributionofthelineagesofthebrown
openaccessarticledistributedunderthetermsof
hareinSouth-EasternandEasternEuroperevealedcontactzonesthere.BAPSanalysis
theCreativeCommonsAttributionLicense,which
assignedsequencesfromL.europaeustofivegeneticclusters,whereasCEindividuals
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal wereassignedtoonlyonecluster,andAMEandSEEsequenceswereeachassignedto
authorandsourcearecredited. twoclusters.OurfindingsuncovernumerousnovelhaplotypesofAnatolian/MiddleEastern
DataAvailabilityStatement:Allsequencesare brownhareoutsidetheirmainrange,asevidenceforthecombinedinfluenceofLatePlei-
availablefromtheGenbankdatabase(accession stoceneclimaticfluctuationsandanthropogenicactivitiesinshapingthephylogeographic
number(s)MG865671-MG865724forCRand
structureofthespecies.Ourresultssupportthehypothesisofapostglacialbrownhare
MG841060-MG841112fortheCytb+tRNA-Thr+
expansionfromAnatoliaandtheBalkanPeninsulatoCentralandEasternEurope,andsug-
tRNA-Pro+CRregion.
gestsomeslightintrogressionofindividualhaplotypesfromL.timidustoL.europaeus.
Funding:ThisworkwassupportedbytheJa´nos
BolyaiResearchScholarshipoftheHungarian
AcademyofSciences.Thepublicationissupported
bytheEFOP-3.6.3-VEKOP-16-2017-00008project.
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PhylogeographyofCentral-,Eastern-EuropeanBrownhare
Theprojectisco-financedbytheEuropeanUnion Introduction
andtheEuropeanSocialFund.
Thebrownhare(LepuseuropaeusPallas,1778)isanativespeciestoNorthern,Central,West-
Competinginterests:Theauthorshavedeclared
ernEuropeandtheWesternpartofAsia,anditwasintroducedasagameintoseveralcoun-
thatnocompetinginterestsexist.
tries(Argentina,Australia,Barbados,Brazil,Canada,Chile,FalklandIslands,NewZealand,
RèunionandtheUnitedStates;[1]).
Theeffectoftranslocationonharegenomewasprovedbypreviousgeneticstudiesandthey
suggestedthatthebrownhareandtheCapehare(Lepuscapensis)arethesamespecies[2].How-
ever,laterthesameauthorsperformedmitochondrialDNA(mtDNA)analysisandfoundasig-
nificantdivergencebetweenthem,andthereforetheyarecurrentlyconsideredtobetwo
differentspecies[3].Pierpaolietal.[4]showedthatItalianandEuropeanharesdidnotshare
anymitochondrialhaplotypes,indicatingthelackofinterspecificgeneflowbetweenthetwo
speciesduetoreproductiveisolationinthecourseoftheirlongseparateevolutionaryhistory.
TheyidentifiedtwomaingroupsofEurasianandAfricanharehaplotypes:CladeA(L.grana-
tensis,L.corsicanus,L.timidus)andCladeB(L.c.mediterraneus,L.habessinicus,L.starcki,L.
europaeus).TheseresultssuggestthatthethreespeciesbelongingtoCladeA,withacommon
ancestor,wouldhavecolonizedEuropeindependentlyofL.europaeusandwouldhaveorigi-
natedbyisolationduringthePleistoceneglaciationsinthesouthernornorthernareasofrefuge.
Itisstronglyarguedthatthecurrentgeographicaldistributionoftemperatespeciesand
geneticrelationshipsamongtheirpopulationshavebeeninfluencedbytheclimaticoscillations
duringtheLateQuaternary[5,6].Specifically,differentlineagesrepresentpopulationsrepeat-
edlyisolatedintodistinctglacialrefugiasuchastheIberian,theApennine,theBalkanPeninsu-
lasandTurkey[5,7–10].Furthermore,differenthumanactivities,competitionforfoodor
breedingandhybridizationbetweenspeciesalsoledtoahigherdiversityinthesouthernrefu-
gialareasandthepresentgeneticdiversityofthehares[11–13].Thereisevidenceforhuman-
mediatedtranslocationsthatiswelldocumentedinthesouthernpartofEurope[14].
PreviousstudiesbasedonmitochondrialDNA(mtDNA)analysisonextantbrownhare
populationshasrevealedarelativelyhighdegreeofgeographicpartitioning[6,15–18].These
studiesdistinguishedtwomajorgeographicallydistinctlineages,theEuropean(EUR)andthe
Anatolian/MiddleEastern(AME)clade.TheEURlineageisfurthersubdividedintotwosub-
clades:theCentralEuropean(CE)andtheSouth-EasternEuropean(SEE)[6].TheCEsub-
cladeincludesindividualsfromacrossNorth-CentralEurope,whereastheSEEcomprises
hareslivinginSouth-EasternEurope.Thesecondlineage,AME,includesindividualsfrom
Anatolia,South-EasternEuropeandtheeasternMediterraneanIslands[17].
Arecentstudy[18]foundthattherewerethreemajorhaplogroupsincludingAnatolia/
MiddleEast(AMh),Balkans(BLh),andcentralEurope(cEUh)amongbrownharepopula-
tionsworldwide.Additionally,threesubgroupswererevealedwithintheBLhhaplogroup
includingSouth-EasternBalkans(SEB),SouthernBalkans(SB)andGreekislandsexcluding
thoseharboringA-lineages(GI-B)offtheAnatoliancoast.Moreover,theSouth-Easternand
CentralBalkans(SEB),comprisingnortheasternGreece,southandNorth-Westernaswellas
South-CentralBulgaria,north-easternpartofRepublicofNorthernMacedonia,South-Eeast-
ernandSouth-WesternSerbia,wasidentifiedastheprimarysourceregionformostotherBal-
kanbrownharepopulations[18].
Ontheotherhand,Anatolian/MiddleEasternhaplotypeshavenotbeenobservedinSouth,
CentralandNorth-WesternGreeceandtherestofEurope,withtheexceptionofoneSerbian
haplotype[18].Also,Europeanhaplotypeshavenotbeenreportedacrosstheentirespecies
rangeintheMiddleEast[6,15,19].Further,theexistenceofacontactzonebetweentheEuro-
peanandAnatolian/MiddleEasternlineageswasdetectedinBulgariaandNorth-Eastern
Greece[6,10,15].
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PhylogeographyofCentral-,Eastern-EuropeanBrownhare
DetectionofbrownharelineagesismostlybasedonthemtDNAcontrolregion(CR),and
isusuallywellsupportedbycytochromeb(cytb).ItprovesthatmtDNAgenomicdataareuse-
fulindeterminingphylogeneticrelationshipsbetweencloselyrelatedspeciesandwithinspe-
cies[20–21]andforunderstandingtheextentandnatureofcontactzones[10].
Overall,despitearelativelylargenumberofgeneticstudiesonbrownhares,theirphyloge-
neticrelationshipsstillremainchallenging.Onlyseveralbroad-rangestudiesofphylogeography
ofbrownhareshavebeendone,relyingonmtDNAcontrolregionsequencesfromSerbian,
GreekandBulgarianhares[6,15,18,22–26].Usingwide-rangegeographicsamplingover
sevencountries,weaimedtostudy(i)theextentofmitochondrialgeneticvariabilityanddiver-
sityofthebrownhareinCentralandEasternEurope;(ii)thephylogeographicrelationshipsof
thestudiedpopulations,andfurthermore(iii)toprovidecomprehensiveinformationonthe
geneticcharacteristicsofbrownharesforconservationprogramsandmanagementplans.
Materialsandmethods
Samplecollection
Atotalof137legallyhunted,unprotectedadultbrownharesweresampledinsevencountries
(Hungary,Poland,Serbia,Lithuania,Romania,Georgia,Italy;Fig1,andseeS1Table)between
2010and2015.Also,threemountainhareshavebeenaccidentallycollectedalongwithour
samples.Noanimalswerekilledforthepurposesofthisresearch.
Alltissuesampleswerestoredin96%ethanolat-4˚C.Hairfolliclessampleswerekeptin
individuallyregisterednylonorpaperbagsandstoredat-4˚Cuntilthelaboratoryanalysis.
TotalDNAwasextractedusingtheE.Z.N.A.TissueDNAKit(OmegaBio-Tek,USA),the
HighPurePCRTemplatePreparationKit(Roche,USA)andstandardFAOprotocol.DNA
concentrationswereevaluatedspectrophotometricallyandvisuallybystandardagarosegel
electrophoresis.
DifferentregionsofthemitochondrialDNAwereamplified.PCRprotocolsandprimers
(Le.H-Dloop_F,Le.L-Dloop_R[15]forthecontrolregion(CR)andLepCyb2L_F,LepD2H_R
[4]forcytochromeb(cytb)+tRNA-Thr+tRNA-Pro+controlregion)wereusedtothe
amplification.PCRswerecarriedoutinatotalvolumeof25μl,usingthefollowingsequence
ofsteps:denaturationat94˚Cfor5minutes,followedby35cyclesofamplification94˚Cfor1
minute,60˚Cfor1minuteand72˚Cfor1minute,andafinalstepat72˚Cfor5minutes.The
forwardsequencingreactionwasperformedbyMacrogenEurope(TheNetherlands).
Inaddition,previouslypublishedsequencesofthespeciesweredownloadedfromtheGen-
Bank(S1andS2Tables).
Ethicsstatement
Animalswerenotshotforthepurposeofthisstudy.Thestudydidnotinvolvethecollectionof
samplesfromliveanimals.Anethicsstatementwasnotrequired.Samplesfromthedifferent
countrieswereobtainedfromlicensedcollaboratorsandlicensedhunterswhotooksamples
followingtheirregulationsinbrownharemanagement.
Sequenceanalysis
Twodatasetswerecreatedfromthesequences.Thefirstdatasetcomprised137CRmtDNA
sequenceswithatotallengthof358bp.Theseconddatasetcomprised105concatenated
sequencescytb+tRNA-Thr+tRNA-Pro+CR,withatotallengthof916bpafteralignment.
AlignmentwasperformedusingSeqscape2.6(AppliedBiosystems)andClustalWinMEGA6
[27],respectively.ThealignedsequencesweredepositedinGenBankwiththeAccession
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PhylogeographyofCentral-,Eastern-EuropeanBrownhare
Fig1.SpatialdistributionoftheEuropeanhares’maternallineages,basedonthe358-bpmtDNAcontrolregion,resultingwhencombiningsequencedatafrom
GenBank(S1Table)andthepresentstudy.SquaresandpolygonsindicatetheCentralEuropeanandSouth-EastEuropeansubclades,respectively,intheEuropean
lineage.CirclesandtrianglesindicatetheAnatolian/MiddleEasternlineageandMountainhare(L.timidus),respectively.Ellipsesdepictthetwodiscoveredcontactzone
areasbetweenbrownharelineagesinSouth-EasternandNorth-EasternEurope.Filledgeometricshapesindicatethegeographiclocationofthesamplingsitesinthis
study.Coloursofthegeometricshapesareinaccordwithclades/lineages;lightgreen:CentralEuropean,darkgreen:South-EastEuropean,red:Anatolian/Middle
Eastern,blue:Mountainhare.
https://doi.org/10.1371/journal.pone.0204653.g001
numbers:MG865671-MG865724forCRandMG841060-MG841112forthecytb+tRNA-Thr
+tRNA-Pro+CRregion(S1andS2Tables).TheEuropeanRabbit(Oryctolaguscuniculus)
(GenBank:AJ001588)[28]wasusedasanoutgroupforthephylogeneticanalyses.DAMBE6
[29]wasusedtoanalyzesubstitutionsaturation.
Thenumberofpolymorphicsites,haplotypediversity,nucleotidediversity,averagenumber
ofnucleotidedifferencesforeachlocationandnumberofhaplotypeswereestimatedwith
DnaSP5.10[30].Thebest-fittingpartitioningschemeandnucleotidesubstitutionmodelwere
selectedusingtheBayesianinformationcriterion(BIC)andthecorrectedAkaikeInformation
Criterion(AICc)implementedinPartitionFinder2.1.1[31].
Bayesianinference(BI)wasperformedusingBEASTv2.3[32]with40,000,000generations
ofMonteCarloMarkovchains(MCMC),samplingevery100generations.Maximumlikeli-
hood(ML)analyseswereimplementedinIQ-TREE1.6[33]with10,000bootstrapsteps.Addi-
tionally,MEGA6[27]wasusedtoconstructaneighbour-joining(NJ)phylogenetictree,
applyingthepairwisedistancedataandp-distancemodelwith10,000bootstrapreplications.
Furthermore,median-joiningnetworks[34]amonghaplotypeswereinferredusingPopART
1.7[35].
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PhylogeographyofCentral-,Eastern-EuropeanBrownhare
Fu’sFS[36]andTajima’sD[37],performedinArlequin3.5[38],wereemployedtoassess
thedemographichistoryandtoexaminehypothesesofselectiveneutrality[39].Thesignifi-
canceofthesetestswascalculatedusing10,000permutations.Thehierarchicalanalysisof
molecularvariance(AMOVA)andfixationindexwereimplementedwith10,000iterations
usingArlequin3.5[38]toevaluatelevelsofpopulationstructure.TheaimoftheAMOVA
analysiswastoexaminewhethergeneticvariationwassignificantlystructuredamongdifferent
haplogroups.F canprovideanestimateofthegeneticdifferentiationamongpopulationsin
ST
ordertomakeinferencesofpastdemographicchanges.
Toestimatethepresenceofgeneticclusters(populations)withinthesequencesofL.euro-
paeusandL.timidus(orintrogressedindividuals),weusedBayesianAnalysisofPopulation
Structure(BAPS)v6[40–41]implementingthemethodof“clusteringforlinkedloci”withtwo
independentrunsandK=10repetitions.Toassessintrogressionoccurringwithinthepopula-
tionsofthesetwospecies,weperformedthemethodof“admixturebasedonmixturecluster-
ing”implementedinBAPS.Toprovideacorrectassessmentofpopulationgeneticstructure,it
isrecommendedtousetheadmixturemodels,becausethesemodelsarerobusttoanabsence
ofadmixtureinthesample;reciprocally,modelswithoutadmixturearenotrobusttotheinclu-
sionofadmixedindividualsinthesample[42].
Results
MtDNAcontrolregionsequences(358bp)
Thesubstitutionsaturationtestbasedonbothdatasets(916bpand358bpsequences)revealed
thatthebasesubstitutionsdidnotreachsaturation,andthesedatasetsweresuitableforphylo-
geneticanalyses.
Forthe358bpcontrolregion,137samplesweresequencedfromCentral-EasternEurope
(S1Table).AdditionalsequencesfromEuropeandtheMiddleEastpublishedinGenBank
wereincludedintheanalyses,yieldingadatasetcomprisingatotalof447sequencesand259
haplotypes(S1Table).Atotalof52haplotypeswereidentifiedamongthe137newsequences,
including40novelhaplotypesand12previouslyreportedhaplotypes.
Thephylogeneticanalyses(BI,ML,andNJtrees)yieldedrelativelyidenticaltopologies,
indicatingthatamong137selectedhaplotypesfromthedataset(447individuals)twolineages
wereidentified(Fig2).
TheMJnetworkanalysis(Fig3)alsosupportedtheclustersdistinguishedinthephyloge-
netictrees.Thefirstlineage,European(EUR),wasdividedintotwophylogeographicallydis-
tinctsubclades:CentralEuropean(CE)andSouth-EastEuropean(SEE).
ThesubcladeCEwasmostlydistributedacrossvariousregionsofCentralEurope,Scotland,
England,theNetherlands,France,Germany,Italy,Austria,Switzerland,Poland,Lithuania,
HungaryandNorthernSerbia(Fig1).However,twoindividualsbelongingtothesubclade
werefoundinEasternRomaniaandSouthernSerbia.Also,onebrownharefromCyprus
(Cyprus4inS1Table)clusteredwithinCE(fallingintohaplotypeCR40,S1Table).Haplotype
CR40alongwithhaplotypesCR1andCR10wasthemostcommonhaplotypeinthesubclade
CEandwasshowntoinhabitmorethanoneregioninEurope(Fig3).HaplotypeCR40was
identifiedasthemostabundant(38individuals)andcentralhaplotypeinthesubclade,and
wasobservedacrossNorthernEurope,fromLithuaniatoPoland,Germany,France,England,
andScotland.HaplotypeCR1wasobservedinPoland,Hungary,Romania,Serbia,andItaly,
whereashaplotypeCR10wasobservedinLithuania,Poland,Hungary,Serbia,Austria,Italy
andFrance.ThesubcladeSEEpredominantlyoccurredinSouth-EasternEuropeincluding
Bulgaria,Greece,RepublicofNorthernMacedoniaandSerbia(Fig1).However,individuals
belongingtothissubcladewerealsopresentinHungary,Poland,CentralItalyandFrance
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PhylogeographyofCentral-,Eastern-EuropeanBrownhare
Fig2.PhylogeneticrelationshipsofbrownharefromCentral-EasternEuropewithotherbrownhares,basedonthe358-bpmtDNA
controlregionsequencesandrootedwithOryctolaguscuniculus(AJ001588).Thenumbersonthebranchesareposteriorprobabilitiesin
theBayesianinferenceandbootstrapsupportinmaximumlikelihoodandneighbour-joining.Coloredovalsrepresenthaplotypesidentified
inthecurrentstudy.Thebrancheswithinbluerectangularincludemountainharesequencesorintrogressedhaplotypesofthisspeciesin
otherharespecies.FordetailedinformationonhaplotypesseeS1Table.
https://doi.org/10.1371/journal.pone.0204653.g002
(CorsicaIsland)(Figs1and2,S1Table).HaplotypesinSEEweremostlyspecifictorelatively
limitedspatialdistributions(Fig3).However,threehaplotypesbelongingtothissubcladewere
recordedoveralargergeographicalrange:CR8(HungaryandItaly),CR32(SerbiaandItaly)
andCR62(ItalyandPoland).Phylogeneticanalysesrevealednosharedhaplotypebetweenthe
subcladesinthislineage.
Thesecondcluster,theAnatolian/MiddleEasternlineage(AME)waspredominantlypres-
entinGeorgia,TurkeyandtheMiddleEast,andwasalsofoundinLithuania,Poland,Roma-
nia,North-EasternGreece,RepublicofNorthernMacedonia,ItalyandFrance(Corsica
Island)(Fig1).Haplotypesinthislineageweremostlyrestrictedtosmallgeographicranges.
However,withinAME,haplotypesCR52,CR53,andCR54wererecordedbothinRomania
andItaly,buthaplotypesCR57(ItalyandRepublicofNorthernMacedonia)andCR200(Tur-
keyandCyprus)werealsofoundindistantlocalities(Figs1,2and3).
MtDNAcytochromeb,tRNA-Thr,tRNA-Proandcontrolregion(916bp)
PhylogeneticanalysesofthecontrolregionrevealedtwomajorlineagesinCentral-Eastern
Europe,withcontactzonesdiscoveredinthegeographicrange(Fig1).Toobtainabetter
assessmentofphylogeographicstructure,wesequencedtheadditionalfragmentscytb(426
bp),tRNA-Thr(66bp)andtRNA-Pro(66bp)of105brownharesfromItaly,Hungary,Serbia,
Georgia,Romania,PolandandLithuania(S2Table).Sixteenadditionalsequencesbelonging
tobrownharesfromGermany,Sweden,Poland,Greece,TurkeyandCyprusavailableinGen-
Bankwerealsoaddedtothealignment(S2Table).Finally,atotaldatasetcomprising124
sequences(916bpfragmentofmtDNA),correspondingtoatotalof62haplotypeswasused
forphylogeneticanalysis.Accordingtothislongerfragment,andinaccordancewithcontrol
regionsequences,thebrownharepopulationinCentral-EasternEuropeisdividedintothe
sametwodistinctphylogeographiclineages(EURandAME)(Figs4and5).
Furthermore,brownharesbelongingtothelineageEURfallintotwosubclades,thesame
CEandSEEasinthefirstdataset.Thecontactzonesamongalllineagesandsubcladeswere
identifiedinthesamegeographicrangesasinFig1.
Atotalof51haplotypeswasfoundthroughoutCentral-EasternEurope.Moreover,50novel
haplotypesandonlyonepreviouslyreportedhaplotypeweredetectedamongthem.The
geneticstatisticsforthesequencedbrownharesinthisstudyaredisplayedinTable1.
Highhaplotypediversityvaluesandrelativelylow-moderatenucleotidediversitywere
obtainedforbrownharesofthestudypopulations.ThelineageAME(onlyforFu’sFS)and
boththesubcladesoflineageEURpresentednegativevaluesforTajima’sandFu’sneutrality
tests,butonlytheoutcomefortheCentralEuropeansubcladewasfoundsignificant(D=
-1.455,P=0.045;FS=-15.34,P=0.00)(Table1).Thus,thissubcladeislikelytohaveunder-
gonearecentpopulationexpansion.ResultsoftheAMOVArevealedthattheamong-hap-
logroupscomponentofvariance(67.59%)washigherthanthevariationwithinhaplogroups
(32.41%)(Table2).Accordingtothefixationindexasignificantgeneticstructureamongall
haplogroupswasalsoobserved(F =0.676,P=0.00)(Table2).
ST
TheanalysisperformedwithBAPSv6separatedL.europaeusandL.timidus(andintro-
gressedmountainhareinotherharespecies)withK=6(ln(P)=−8954.5009).Thisanalysis
assignedsequencesfromL.europaeustofivegeneticclusters,andL.timidustoonlyonecluster
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Fig3.MedianjoiningnetworkofbrownharefromCentral-EasternEuropeandotherbrownhares,basedonthe358-bp
mtDNAcontrolregion.Thenumbersonthehaplotypes(1–259)arethesamehaplotypecodes(CR1-CR259)asinFig2andS1
Table.Darkcirclesareconnectingnodes(i.e.putativeundetectedhaplotypes).Bluecirclesincludemountainharesequencesor
introgressedhaplotypesofthisspeciesinotherharespecies.
https://doi.org/10.1371/journal.pone.0204653.g003
(Fig6).WithinL.europaeus,sequencesbelongingtolineageAMEandsubcladeSEE(lineage
EUR)wereeachassignedtotwoclusters,whereasindividualsbelongingtosubcladeCE(line-
ageEUR)fellintoonecluster.
Discussion
Previousstudiesestimatedphylogeneticrelationshipsamongbrownharepopulationsin
EuropeandtheMiddleEast,whereinsufficientsamplingleftarelativelylargegapinseveral
geographicranges,especiallyinCentral-EasternEurope.Thisinformationgaphasprevented
thedelineationofacomprehensivepictureofgeneticdiversityandphylogeographicstructure
ofthespecies.Europeanbrownhareshavebeenclassifiedtotwomajorlineages,European
(EUR)andAnatolian/MiddleEastern(AME)[6,15,17–18]thatco-existinRepublicofNorth-
ernMacedonia,North-EasternGreeceandBulgaria[6,10,15].Inthisstudy,wepresenteda
relativelycomprehensivedatasetonmtDNAcytochromeb,tRNA-Thr,tRNA-Proandcontrol
regionfragments(atotalof916bp)ofbrownharesinCentral-EasternEurope,wheretwo
datasetswereusedinthegeneticanalyses;thefirstdatasetincludeda358-bpcontrolregion
sequence,whereastheseconddatasetcoveredaconcatenatedsequenceofmtDNAfragments
(the916-bpsequence).
Ourfindingsrevealedahighgeneticdiversitywithinthe916-bpmtDNAsequence(105
newsequences,51haplotypes)ofbrownharesfromCentral-EasternEurope,where50haplo-
typeswerereportedforthefirsttime(Table1).Phylogeneticanalysesrevealedtwomajorline-
agesofbrownhareinthestudyarea,basedonacombinationofoursequencesandpreviously
publishedsequences(S1andS2Tables)forbothdatasets:(i)AME,whichcomprisesindividu-
alsfromGeorgia,Anatolia,theMiddleEastandalsoincludessomehareslivinginSouth-East-
ern,North-EasternandCentralEurope,and(ii)EUR,whichincludesharesfromCentral,
South-Eastern,EasternandNorthernEurope.Inaccordancewithothers[6,15],theEURline-
ageissubdividedintotwowell-supportedsubclades,CentralEuropean(CE)andSouth-East
European(SEE).
ThesignificantgeneticstructureamongbrownharehaplogroupsfromCentral-Eastern
EuropewaswellsupportedbyF andAMOVA(Table2).Thefixationindexisastandard
ST
measure,whichgivesanestimateofthedegreeofgeneticdifferentiationamongandwithin
populations/haplogroups[43].Infact,theanalysesdemonstratedthatpartitioningintothe
majorhaplogroupsexplains67.59%oftheoverallmtDNAvariabilityandcorrespondstoa
highlysignificantfixationindex(p<0.000).Thefemalephilopatryofbrownhares[16,44]
couldhaveresultedintheformationofmultigenerationalmatrilinealassemblagesthataregeo-
graphicallystructured[45].
ThepopulationstructuredeterminedbyBAPSv6partiallydescribeddiversityallocation
betweenclustersbasedonthecontrolregionmtDNAsequences.BAPSisknowntoberela-
tivelyhighlyefficientinidentifyinghiddenpopulationstructures[46].Theanalysisrevealed
fivegeneticclusterswithinthepopulationsofL.europaeusandonlyoneclusterwithinL.timi-
dus(andintrogressed)sequences.WithinL.europaeus,individualsbelongingtothemajor
lineageAMEwereassignedtotwoclusters:(i)cluster1,whichincludesbrownharesfrom
Georgia,Turkey,Cyprus,Bulgaria,Romania,RepublicofNorthernMacedonia,CentralItaly,
France(CorsicaIsland),PolandandLithuania;(ii)cluster2,whichcomprisesbrownhares
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