Temporal Fluctuation in North East Baltic Sea Region Cattle Population Revealed by Mitochondrial

16 Pages·2015·0.34 MB·English

RESEARCHARTICLE
Temporal Fluctuation in North East Baltic Sea
Region Cattle Population Revealed by
Mitochondrial and Y-Chromosomal DNA
Analyses
MariannaNiemi1,2*,AuliBläuer1,3,TerhiIso-Touru1,JanneHarjula1,3,VeronicaNyström
Edmark4¤,EveRannamäe5,LembiLõugas6,AnttiSajantila2,KerstinLidén7,
Jussi-PekkaTaavitsainen3
1 BiotechnologyandFoodResearch,MTTAgrifoodResearchFinland,Jokioinen,Finland,2 Universityof
Helsinki,DepartmentofForensicMedicine,Helsinki,Finland,3 DepartmentofArchaeology,Universityof
Turku,Turku,Finland,4 DepartmentofZoology,StockholmUniversity,Stockholm,Sweden,5 Instituteof
HistoryandArchaeology,UniversityofTartu,Tartu,Estonia,6 Instituteofhistory,TallinnUniversity,Tallinn,
Estonia,7 ArchaeologicalResearchLaboratory,StockholmUniversity,Stockholm,Sweden
¤ Currentaddress:DepartmentofBioinformaticsandGenetics,SwedishMuseumofNaturalHistory,
Stockholm,Sweden
OPENACCESS
* [email protected]
Citation:NiemiM,BläuerA,Iso-TouruT,HarjulaJ,
NyströmEdmarkV,RannamäeE,etal.(2015)
TemporalFluctuationinNorthEastBalticSeaRegion Abstract
CattlePopulationRevealedbyMitochondrialandY-
ChromosomalDNAAnalyses.PLoSONE10(5):
e0123821.doi:10.1371/journal.pone.0123821
AcademicEditor:Yong-GangYao,Kunming Background
InstituteofZoology,ChineseAcademyofSciences,
AncientDNAanalysisoffersawaytodetectchangesinpopulationsovertime.Todate,
CHINA
moststudiesofancientcattlehavefocusedontheirdomesticationinprehistory,whileonly
Received:August29,2014
alimitednumberofstudieshaveanalysedlaterperiods.Conversely,thegeneticstructure
Accepted:March7,2015
ofmoderncattlepopulationsiswellknowngiventheundertakingofseveralmolecularand
Published:May20,2015 populationgeneticstudies.
Copyright:©2015Niemietal.Thisisanopen
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,whichpermits
Results
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare BonesandteethfromancientcattlepopulationsfromtheNorth-EastBalticSearegion
credited.
datedtothePrehistoric(LateBronzeandIronAge,5samples),Medieval(14),andPost-
DataAvailabilityStatement:Theanalysed Medieval(26)periodswereinvestigatedbysequencing667basepairs(bp)fromthemito-
sequencesareavailableintheGenBank,accession
chondrialDNA(mtDNA)and155bpofintron19intheY-chromosomalUTYgene.Compari-
numbersKF233429-KF233528.Allrelevantdataare
withinthepaperanditsSupportingInformationfiles. sonofmaternal(mtDNAhaplotypes)geneticdiversityinancientcattle(45samples)with
moderncattlepopulationsinEuropeandAsia(2094samples)revealed30ancientmtDNA
Funding:ThisresearchwasfundedbyAcademyof
Finland(theprojectdecisionnumber128451),http:// haplotypes,24ofwhichweresharedwithmodernbreeds,while6wereuniquetothean-
www.aka.fi/en-GB/A/.WorkofMNwasalsofunded cientsamples.OfsevenY-chromosomalsequencesdeterminedfromancientsamples,six
byNationalPopulationGeneticsDoctoral
wereY2andoneY1haplotype.CombineddataincludingSwedishsamplesfromthesame
Programme,fundedbytheMinistryforEducationand
periods(64samples)wascomparedwiththeoccurrenceofY-chromosomalhaplotypesin
CultureandtheAcademyofFinland,http://www.oulu.
fi/biology/PopGenSchool/.Cattlebonesamplesfrom moderncattle(1614samples).
PLOSONE|DOI:10.1371/journal.pone.0123821 May20,2015 1/16
MitochondrialandY-ChromosomalDNAAnalysesofN-EBalticSeaCattle
Estoniawerepartofthegrantprojectno8156of Conclusions
EstonianScienceFoundation,http://www.etag.ee/
ThediversityofhaplogroupswashighestinthePrehistoricsamples,wheremanyhaplo-
rahastamine/etfgrandid/.Thefundershadnorolein
studydesign,datacollectionandanalysis,decisionto typeswereunique.TheMedievalandPost-Medievalsamplesalsoshowahighdiversity
publish,orpreparationofthemanuscript. withnewhaplotypes.Someofthesehaplotypeshavebecomefrequentinmodernbreedsin
CompetingInterests:Theauthorshavedeclared theNordicCountriesandNorth-WesternRussiawhileotherhaplotypeshaveremainedin
thatnocompetinginterestsexist. onlyafewlocalbreedsorseemtohavebeenlost.AtemporalshiftinY-chromosomalhaplo-
typesfromY2toY1wasdetectedthatcorrespondswiththeappearanceofnewmtDNA
haplotypesintheMedievalandPost-Medievalperiod.Thissuggestsareplacementofthe
PrehistoricmtDNAandYchromosomalhaplotypesbynewtypesofcattle.
Introduction
ArchaeologicalandmitochondrialDNAevidenceindicatethatcattleweredomesticatedfrom
theauroch(Bosprimigenius)[1–5],about10,000yearsagointheFertileCrescent[6].From
theFertileCrescent,domesticcattlespreadtoSouthEasternEuropearound8,800BeforePres-
ent(BP),toCentralEuropearound7,000BP,andtoNorthCentralEuropeafter6,700BP[7].
DomesticcattlereachedsouthernScandinaviaby6,000BP[8],Estoniaby4,100BP[7]andfi-
nallyFinlandinthenorthernBalticSearegionby3,000BP[9].Theoldestradiocarbondated
remainsofcattleinFinlanddatebackto3086±30BP[9].
MolecularanalysesofmitochondrialDNAandtheY-chromosomecanbeusedtotrace
bovinematernalandpaternallineages,respectively[10,11].Variationinthehypervariablere-
gionofthemithochondrialD-loopdefinesthemajorityoftaurinecattle,aswellassomemi-
tochondriallineagesofNearEasternaurochsandmanyItalianaurochs[12],tobelongtothe
Tmega-haplogroup,includingthehaplogroupsT,T1,T2,T3,andT4[1,5,13–15].Astudyof
thewholemitochondrialDNAhassuggestedanadditionalhaplogroup,T5,definedbysites
outsideoftheD-loopregion[15].Threeotherhaplogroupshavebeenidentifiedintaurine
cattle,wheretheclosestinphylogenytohaplogroupTishaplogroupQ[10],differingbyone
diagnosticSNPsiteinthehypervariableregion(position15953inV00654)[15].Haplogroup
QhasbeenfoundatlowfrequencyinmodernSouthEuropeancattlebreeds[10,15].Thedis-
tributionofhaplogroupQhasbeenhypothesisedtoindicateaparallelNeareasternoriginfor
haplogroupsTandQ,whereQrepresentsaminordomesticatedlineage[16].HaplogroupP
thathasonlybeenidentifiedinnorthernandcentralEuropeanaurochs,andinacoupleof
scatteredtaurinesamples,divergedfromTandQpriortotheirsplit[15,17].Theoldestdi-
vergingbranchinthemtDNAphylogenyistheveryrarehaplogroupRthathasonlybeen
identifiedinlocalItaliancattlebreeds[16].
ThegeneticdiversityoftheThaplogroupishighestintheNearandMiddleEastcattlepop-
ulations,wherefourhaplogroupsT,T1,T2,andT3exist[1,14],indicatingaNearEasternori-
ginoftaurinecattle,whichisalsosupportedbynuclearmarkeranalysesthatshowhigher
variabilityintheNearEastthaninotherregions[1,18].Europeandomesticcattlecarrythe
samefourhaplogroupsasNearEastcattle,butwithT3predominatinginEuropeatleastfrom
theNeolithicperiodonwards[11,14,17,19,20].HaplogroupT1isquitefrequentacrossthe
Mediterraneancountries[3,21],andpredominantandalmostfixedinAfrica[14].Haplogroup
T4derivesfromT3andhasthusfaronlybeendetectedinAsianandYakutiancattlefromRus-
sianSiberia[11].Thestar-likepatternsoftheT3-centeredhaplotypesdetectedinmodernand
PLOSONE|DOI:10.1371/journal.pone.0123821 May20,2015 2/16
MitochondrialandY-ChromosomalDNAAnalysesofN-EBalticSeaCattle
NeolithicEuropeancattlepopulationshavebeensuggestedtoresultfrompost-domesticaccu-
mulationofmutations[14,19].
Anorth—southgradientofgeneticdiversityhasbeendetectedinmodernEuropeancattle
(Bostaurus),[11,18],includingtheY-chromosome[11].Asinglenucleotidepolymorphismin
intron19oftheUTYgene(UTY19)canbeusedtodistinguishbetweenthetwoY-chromosom-
alhaplotypes,Y1andY2[22].WhereasY1isthedominatinghaplotypeinmodernWestern
andNorthernEuropeanbreeds,haplotypeY2dominatesinSouthEuropeanbreeds[22],with
acleardividingzoneincentralEurope[23].Apartfromthegeographicalvariation,atemporal
fluctuationinY1andY2haplotypefrequencieshasbeendetected,mainlyfromSwedishan-
cientbullsandaurochs,suggestingthatvariationinpresent-dayfrequenciesofY1andY2hap-
lotypesislikelyduetorecentdemographicevents[24].
Theaimofthisstudywastoexploretemporalpopulationvariationbymaternallyandpater-
nallyinheritedmarkersincattlefromtheNorthEastBalticSearegion(N-EBSR),andtocom-
pareancientpopulationswithmodernbreeds.Haplotypedatafrom45ancientmtDNAand7
Y-chromosomesampleswasusedtogetherwithcontemporarydatafrom2094mtDNA
[10,11,15,16,20]and1614modern[22–24]and71ancientY-chromosomes[24–26]samples.
ThedataindicatesclearchangesintheN-EBSRcattlepopulationsfromlateBronze/IronAge
tomoderntimes.
MaterialsandMethods
Ancientcattlebones
Atotalof77cattleboneswereselectedforaDNAanalysisfromdifferentsitesacrossFinland
andEstoniaandinthetownofVyborgintheLeningradRegioninnorth-westernRussia
(FigAinS1File).Thesamplesforthisstudywerefrommuseumcollectionsheldat1.)The
NationalBoardofAntiquities,2.)MuseumofRaisio(Harkko),3.)TheMuseumCentreof
Turku,4.)ÅlandsMuseum,5.)MuseumofViljandi,6.)PärnuMuseum,7.)SaaremaaMuse-
um,8.)UniversityofTurku,9.)St.Petersburg,InstitutefortheMaterialCultureHistory,Rus-
sianAcademyofSciences,10.)TallinnUniversity,and11.)UniversityofTartu(TableAin
S1File).Allnecessarypermitswereobtainedforthedescribedstudy,whichcompliedwith
allrelevantregulations.
ThesamplesfromVyborgderivefromtheMedievalandPost-Medievalperiods,duringwhich
VyborgwaspartofFinland.Theearliestbones(2samples)availableforthisstudyderivefrom
theLateBronzeAge(700–500BC)fromtheislandofSaaremaa,Estonia.TherestofthePrehis-
toricsamplesdatestotheLateIronAge(800–1200AD).Toverifythateachindividualwithin
onesiteandperiodwassampledonlyonce,samplesderivingfromthesamesideoftheanimal
wereselected,orthesizeandageoftheindividualwasusedtoseparateindividuals.Whenever
possible,metacarpalswerepreferredasmetacarpalsareusedtoosteologicallydeterminethesex
oftheanimal[27,28].From77samplesinitiallyselected,atotalof18bonesorteethfromthePre-
historicperiod(700BC-1200AD),24fromtheMedievalperiod(1200–1550AD),and34from
thePost-Medievalperiod(1550–1800AD)wereusedforaDNAanalyses.Onesamplethatwas
radiocarbondatedasmodernwasomittedfromfurtheraDNAanalysis.Atotalof21skeletal
sampleswereradiocarbon-datedattheLaboratoryofChronologyoftheFinnishMuseumofNat-
uralHistory(LUOMUS),UniversityofHelsinki(TableAinS1File).Radiocarbondatedsamples
coveredallbonesandteethfromnon-distinctculturallayersthatwereusedforaDNAanalyses.
DNAmarkersandlaboratorymethods
TodeterminethemtDNAhaplogroupsT,T1,T2,T3,T4,andT5[15],acombinationofthree
fragmentsyielding486bpofsequencecoveringthemtDNAD-loopfromposition16031to
PLOSONE|DOI:10.1371/journal.pone.0123821 May20,2015 3/16
MitochondrialandY-ChromosomalDNAAnalysesofN-EBalticSeaCattle
178[GenBank:V00654]anda181bpsequencefromtheND5gene(position12911to13091
[GenBank:V00654])wereanalysed.Anadditional77bpD-loopfragment(positions15936–
16012inV00654),determininghaplotypeQ,wasanalysedfromonesample(H01,BtTor4).As
aY-chromosomalhaplotypemarker,a155bpsequencefromintron19intheUTYgenewas
analysed(thetransversionG>Tatposition423in[GenBank:AY936543],defininghaplotypes
Y1orY2)[22].DNAextraction[29],PCRmethodsandsequencingofPCRproductswereas
describedin[30].Briefly,0.2–0.5mlofbonepowderwassuspendedin900μl0.5MEDTA,
100μl10Mureaand5μlproteinaseK(20mg/ml),andincubatedwithconstantshakingat
55°Covernight.DNAfromtheconcentratedsupernatant(Amicon-430Kcentrifugalfilter
units,MerckMillipore)wasextractedwithaQIAquickPCRPurificationKit(Qiagen,Sweden)
accordingtomanufacturer’sinstructions.Approximately5–10μLofDNAextractwasusedin
thePCRperformedwiththeHotStarTaqDNApolymeraseKit(Qiagen,Sweden)withaninclu-
sionof0.4mMdNTP,0.2μMofeachprimerand0.25units(U)ofUracilDNAGlycosylase
(UNG,Sigma-Aldrich).ThePCRprogramincludedinitialstepsof37°Cfor10minand95°C
for15minfollowedby55three-stepcyclesof94°Cfor30s,AT°Cfor40sand72°Cfor1min
and10minat72°,whereATstandsforaspecificannealingtemperatureforeachprimerpair
(TableBinS1File).PrimersandsuccessratesofaDNAanalyses(TextAinS1File,TableBin
S1File)areprovidedintheSupportingInformation.
AuthenticityofancientcattleDNA
TheauthenticityofaDNAanalyseswascontrolledinvariousstepsofthelaboratorywork-flow
andtheanalyseswererepeatedinindependentancientDNAlaboratories.All45ancientsam-
plesincludedinthestatisticalanalyseswereextractedatleasttwice(MTTAgrifoodResearch
Finland,Jokioinen,Finland,StockholmUniversity,Stockholm,SwedenandDepartmentofFo-
rensicMedicine,UniversityofHelsinki,Helsinki,Finland).
EachparticipatingancientDNAlaboratoryfollowedgeneralguidelinesforancientDNA
worksuchasseparatespaceforsamplepreparationandancientDNAwork,separatepre-and
post-PCRareas,air-controlledsterileaDNAworkspace,wearingofprotectiveclothing,using
disposabletools,pipetteswithaerosolresistantfiltertipsandtreatingequipmentandworking
surfaceswithbleachandultra-violetirradiationfrequently.
ToensuretheauthenticityofthemtDNAandY-chromosomalsequences,andtodetectpos-
siblePCRerrors,eachDNAfragmentofeachsamplewassequencedfromatleasttwodifferent
PCRreactionswithDNAderivedfromdifferentextractions.Thesamplewasconsideredtobe
reproduciblewhenconsistentsequencesofeachDNAfragmentwereobtainedfromatleast
threeamplifications.Theconsistentsequenceswereverifiedfromtwoextractionsinanalyses
doneatleastintwoindependentaDNAlaboratories.Overlappingprimersspecifictocattle
DNAweredesignedtopreventcrossreactivitywithhumanDNA(TextAinS1File,TableBin
S1File).NegativecontrolswereappliedforallstepsintheaDNAextractionandamplification.
Apreviouslyanalysedmammothsample[31]wasusedasapositivecontrolwhenthefirstfive
sampleswereextracted.Themammothsamplewassuitableasapositivecontrolasitisancient
anditssequenceclearlydiffersfromcattle.
Forfurtheranalyses,sequencesfromaDNAsamplesobtainedfromdifferentextractions
andamplifications,provenidenticalbyatleasttwoindependentaDNAlaboratorieswereused.
Onesamplewasnotrepeatableandwasthusexcludedfromanalyses(TableAinS1File).For
sixsamplesonlypartialmtDNAwassuccessfullyamplified.Consequently,theywereomitted
fromthestatisticalanalyses.Asamplificationfrom25samples(includingonemodernsample,
TableAinS1File)yieldednoDNA,atotalof45samplesremainedforstatisticalanalyses
(TableAinS1File).
PLOSONE|DOI:10.1371/journal.pone.0123821 May20,2015 4/16
MitochondrialandY-ChromosomalDNAAnalysesofN-EBalticSeaCattle
Statisticalanalysis
ThemtDNAsequencesfromthe45successfullysequencedancientcattlewerealignedsepa-
ratelyforthe486bpD-loopandthe181bpND5genesequencesusingCLUSTALW[32]
wherepenaltiesusedwere10forgapopening,0.20forgapextension,and5forgapdistances.
Thecombinationofthesequencedregionsisreferredtobelowasthe667bphaplotypere-
gion.ACLUSTALWalignmentwasalsoperformedforthesevensuccessfullyamplifiedY-
chromosomal155bpsequences.TheanalysedsequencesareavailableinGenBank,accession
numbersKF233429-KF233528.
TheReducedMedian-joiningNetwork(RMNtobemostconservativeε=0)wasconstructed
accordingtothealgorithmdescribedbyBandelt,ForsterandRohl[33]withNETWORK4.6.0.0
[33].ThetopologyobtainedinRMNwasconfirmedwiththeMaximumlikelihood(ML)and
BayesianMarkovChainMonteCarlo(MCMC)analysesusingjModeltestv2.1[34],PhyML3.0
[35]andMrBayes3.2[36].BoththeMLandtheMCMCtreealongwiththedetailedstatistical
methodsarepresentedinSupportingInformation(TextAinS1File,FigBinS1File).
DnaSP(version5)[37]wasusedtocalculatethegeneticdiversityestimatesbasedonthe
486bpD-loopsequences.Numberofhaplotypes(h),haplotypicdiversity(Hd),numberofseg-
regatingsites(S),nucleotidediversity(π),Tajima’sD(D),andaveragenumberofnucleotide
differences(K)werecalculatedforeachpopulation.Toapproximatethelevelofbiasinthedi-
versityestimatescausedbyheterochronityinthedatasetwhenpoolingsamplesofdifferent
ages,correctedπhμ[38]wascalculatedwithmutationratesof34and53%permillionyearsand
generationlengthsof5and7years(upperandlowerrangesascalculatedfromNear-Eastern
cattlein[39]).Inordertoprovidedatestothesampleswhencalculatingπhμ,radiocarbondates
wereusedandtheuseddateswererandomlyassignedtocovertherangeofcontextforsamples
datedbycontext.
Inordertocompareancientcattlediversitytomoderncattlepopulations,anumberofaddi-
tionalsequencesfromEurope,NearEastandNorthAsiawereincludedinthepopulationdiver-
sityanalysis.Thesesequenceshavepreviouslybeendescribedandanalysed[10,11,15,16,20].
ThesizeofthecommonalignedmtDNAsequenceinthiscomparisonwas245bpfromatotal
of2139individuals.Thisdatasetwasthenusedintwoapproaches.
First,toexplorethetemporalfluctuationinhaplotypeswithintheN-EBSR,49moderncat-
tlesamplesfromfivenativeN-EBSRbreeds(Northern,Western,andEasternFinncattle,Esto-
nianRedandEstonianNative[11,16]),alongwiththe45ancientcattleanalysedherewere
extractedfromthealigned245bpdataset.These94N-EBSRsamplesweregroupedintothree
temporalcohorts;PrehistoricandMedieval(n=19),Post-Medieval(n=26),andModern
(n=49)andintotwogroups:1)themostfrequent245bphaplotypefoundamongtheentire
2139dataset(563samples)and2)therestofthehaplotypes.
Thesecondapproachwasusedtoexploretheappearanceandfrequencyofancienthaplotypes
among2094moderncattledividedintotengeographicalregions(N-EBSR,Scandinavia,West-
ernEurope,SouthernEurope,South-EasternEurope,EasternEurope,WesternRussia,Central
Russia,Siberia,andNearEast/CentralAsia).Forthisapproach,the2094modernsampleswere
groupedintothreehaplotypegroups:1)themostcommon245bphaplotypeintheentiredataset
(563outof2139samples),2)therestofthehaplotypesfoundamong45ancientN-EBSRcattle,
and3)otherhaplotypesnotfoundinancientdata.Theprocedurewasusedtostudythedistribu-
tionofancienthaplotypesamongcontemporarycattle.NotethatthePrehistorichaplotypeswere
excludedhereasmostofthePrehistorichaplotypeswerenotpresentincontemporarydata.
Pearson’schi-squaretest,asimplementedinSPSSv.11.5.0,wasconductedtotestfordiffer-
encesinfrequenciesofmtDNAhaplotypesinbothapproaches,betweenthetemporalcohorts
andthegeographicalregions.
PLOSONE|DOI:10.1371/journal.pone.0123821 May20,2015 5/16
MitochondrialandY-ChromosomalDNAAnalysesofN-EBalticSeaCattle
Sixty-ninesampleswerefurtheranalysedfortheYchromosomalSNPinUTY19,whichdif-
ferentiatescattleYchromosomesintohaplotypesY1andY2[22].Thesevensamplessuccess-
fullyanalysedfortheY1/Y2markerwereanalysedfortemporalfluctuationwiththeSwedish
ancient(n=64)andFennoscandianmodern(Northern,Western,andEasternFinncattle,
SwedishRed,Redpolled,FjallnaraandMountaincattle,n=41)datagivenin[22–25].The
combineddatafromFennoscandianbullsweredividedintofourtemporalgroups:IronAge
(n=8[25]),Medieval(n=37thisstudyand[24,25]),Post-Medieval(n=19thisstudyand
[24]),andmodern(n=28[22]and[11]asreportedin[23]).Tocomparethetemporalanaly-
sesinFennoscandiatoCentralEurope,datafromMedievalbulls(n=14,[26])fromSwitzer-
landwasanalysedtogetherwithdatafrommodernSwissbreeds(Braunvieh,Ehringer,and
Simmental,n=39,[22]and[11,40]asreportedin[23]).
Inordertomakewidergeographicalcomparisons,Y1/Y2informationfrom127modern
Eurasianbreeds(n=1614[22,24]and[23]combiningthedataof[11,40–42])wereincluded.
Datafromatotalof1692bullswasdividedintoninegeographicalregions(theNordiccoun-
tries,WesternEurope,SouthernandCentralEurope,SouthEasternEurope,EasternEurope,
Near-EastandCentralAsia,WesternRussia,CentralRussiaandSiberia).
APearson’schi-squaretest,asimplementedinSPSSv.11.5.0,wasconductedtotestfordif-
ferencesinfrequenciesofY1andY2betweenthetemporalcohorts(FennoscandiaandSwitzer-
land)andgeographicalregions.Incaseswhere20%ormoreofthegroupshadexpectedcounts
lessthan5,Fisher’sexactprobabilitytwo-tailedtestwasusedinstead.
Results
Radiocarbondating
Atotalof21sampleswereradiocarbondated.Threesamplesappearedtobefromalaterperiod
thanexpectedbasedonthecontextdatingwhileonesamplefromanIronAgecontextturned
outtobemodern(TableAinS1File).
Osteologicalanalysis
Themetricalanalysisofmetacarpalsrevealedthreemalesand12femaleswhiletwometacar-
palswereindeterminableandfivemetacarpalsweretoofragmentedtobeanalysedbyosteo-
logicalmethods(TableAinS1File).TheresultsfromtheY-chromosomalUTY19werein
accordancewiththeosteologicalanalysesasnoneofthesamplestakenfromfemalemetacar-
palsamplifiedwithY-chromosomalprimers.Twomalemetacarpalswereconfirmedandone
indeterminablemetacarpalwasdeterminedasmalebyY-chromosomalamplification
(TableAinS1File).
MtDNAhaplotypes
UsingDnaSP,30haplotypeswerefoundamongtheancientcattle,includingonesamplepro-
vidingonlypartialinformation.Twenty-ninehaplotypes,includingthefull667bpsequence,
wereusedforfurtheranalysis.Whenanalysingthephylogenyofthese29haplotypes,Bayesian
MCMC,MLandRMNanalysesgavesimilartopologies(Fig1,Median-joiningnetworkofthe
29ancientmitochondrialhaplotypes(grey-black)with43modernreferencehaplotypes
(white),andFigBinS1File).Alloftheancienthaplotypeswereassignedtothetaurinehap-
logroupsaccordingtotheknowndiagnosticpositionsofcattlemtDNA[10,11,14,15,20](Text
AinS1File).Onesamplewasassignedtotaurinemacro-haplogroupQwhiletherestofthe
sampleswereassignedtothetaurinemacro-haplogroupT(Fig1,TextAinS1File).The28an-
cienthaplotypesinmacro-haplogroupTwerefurtherdividedintohaplogroupsT2(one
PLOSONE|DOI:10.1371/journal.pone.0123821 May20,2015 6/16
MitochondrialandY-ChromosomalDNAAnalysesofN-EBalticSeaCattle
Fig1.Median-joiningnetworkofthe29ancientmitochondrialhaplotypes(grey-black)with43modern
referencehaplotypes(white).Median-joiningnetwork(ε=0)showsmolecularrelationshipsbetween30
ancienthaplotypes(H01-H03andH05-H30).Majorhaplogroups(T1,T2,T3,T5andQ)andsub-haplogroups
(T1f,T3b)aredefinedbyinclusionof43modernreferencehaplotypesfrom[10,15].Eachcirclerepresents
onemtDNAhaplotypewherethesizeisproportionaltothenumberofindividualsinthathaplotype.Black
diamondsrepresenthypotheticalhaplotypes.Thelengthofthebranchesisproportionaltothenumberof
mutationsbetweenthehaplotypesexceptthebranchbetweenBostaurusandBosindicus(32mutations),
whichisshortenedtofitinthepicture.HaplotypesfromthePrehistoric,Medieval,andPost-Medievalperiods
areindicatedinblack,darkgrey,andlightgrey,respectively.
doi:10.1371/journal.pone.0123821.g001
haplotype)andT3(17haplotypes)andsub-haplogroupsT3b(9haplotypes)andT1f(onehap-
lotype)(Fig1,BandCFigsinS1File,TextAinS1File).Thesampleprovidingpartialinforma-
tionwasassignedtoT2(TextAinS1File,FigCinS1File).
Analysisofpopulationdiversity
ThemtDNAdiversityintheFinnish,Estonian,andVyborgancientcattledataaresummarized
inTable1.Thenucleotidediversityfortheentiredatasetwas0.969.Withineachancienttem-
poralcattlecohortthemitochondrialhaplotypediversityestimates(s,h,Hd,K,andπ)indicate
ahighdiversity(Table1).ThehaplotypediversitywashighestinPrehistoriccattle(Hd=1.000)
andslightlylowerinMedievalandPost-Medievalcattle(Hd=0.956and0.972,respectively).
Nucleotidediversityvariedamongperiodswiththehighestobserveddiversity(π=7.41(cid:1)10–3)
inthePrehistoricpopulation(Table1).Thebiasinnucleotide-diversityestimatecausedbyhet-
erochronitywaslow,lessthan1.5%inalltemporalcohorts(Table1).Tajima’sDvaluewasneg-
ativeforalltemporalcohortswithasignificantlynegativepvalueforthePost-Medievalperiod
andthewholeancientcattledatasetsuggestingapopulationexpansioninFinlandincludingVy-
borgandtheBalticregion(Table1).
HaplogroupT3andsub-haplogroupT3bformedastar-likephylogenyofhaplotypes,with
majorhaplotypesH17andH05forT3andT3b,respectively.Thehighesthaplotypediversity
wasdetectedintheoldestandsmallestsample,fromthePrehistoricperiod.Adifferentsetof
haplotypeswasfoundfromtheMedievalandPost-Medievalsamples(Fig1).
TemporalmtDNAanalyses
SignificanttemporalfluctuationsinthefrequencyofmtDNAhaplotypesintheN-EBSRcattle
weredetected(PearsonChi-Squaretest,n=94,χ2=13.1,df=4,p=0.011).Herethemost
PLOSONE|DOI:10.1371/journal.pone.0123821 May20,2015 7/16
MitochondrialandY-ChromosomalDNAAnalysesofN-EBalticSeaCattle
Table1. SummarystatisticsofmtDNAvariationinancientNorthEastBalticSearegioncattlefromPrehistoric,Medieval,andPost-Medieval
periods.
AncientNorthEastBalticSearegioncattle
Prehistory,700BC-1200AD Medieval,1200–1550AD Post-Medieval,1200–1800AD Total
N 5 14 26 45
S 9 15 22 33
h 5 11 20 29
Hd 1.000 0.956 0.972 0.969
K 3.600 3.055 2.788 2.951
θs 4.320 4.717 5.765 7.736
D -1.184 -1.437 -1.869* -2.067*
π 7.41 6.29 5.74 6.07
πhμa 7.35 6.28 5.73 6.04
Biasa 0.84% 0.13% 0.10% 0.43%
πhμb 7.30 6.28 5.73 6.03
Biasb 1.45% 0.22% 0.17% 0.74%
Nisnumberofindividualssampled;Sisthenumberofsegregatingsites(excludingindels);histhenumberofhaplotypes;Hdisthehaplotypediversity;K
istheaveragenumberofdifferences;θsis‘Theta’derivedfromtheobservednumberofsegregatingsites(S);DisTajima0sDstatisticvaluewhere
statisticalsignificancesP<0.05ismarkedwith*.πisthenucleotidediversity*10–3;ThePrehistoriccohortincludestwosamplesfromLateBronzeAgeand
threesamplesfromLateIronAge.
aBasedongenerationlengthof7yearsandmutationrateof43%permillionyears
bBasedongenerationlengthof5yearsandmutationrateof53%permillionyears
doi:10.1371/journal.pone.0123821.t001
common245bphaplotypeincreasedinfrequencymorethantwicefromMedievaltoPost-Me-
dievalandmorethanthricefromPost-Medievaltomoderntime(greyinFig2binFig2,Distri-
butionofancientN-EBSRcattlemtDNAhaplotypesinmodernEurasiancattlepopulations).
Consequently,theproportionofotherhaplotypesdecreasedthroughtime(colouredandwhite
patternsinFig2b).Nearlyhalfoftheseotherhaplotypesincontemporarycattlewerenot
foundinancientcohorts(whiteinFig2b);andthustheproportionoftheancienthaplotypes
(otherthanthemostcommon)inmodernN-EBSRisapproximately20%(coloredpatternsin
Fig2b).Theproportionofuniqueancienthaplotypes(uniqueamong2139samples)washigh-
estinthePrehistoricsample(blackinFig2b).
GeographicalmtDNAanalyses
Themostcommon245bphaplotype(includingtheancient667bphaplotypesH05,H06,H11,
H17,H24,andH26,TableCinS1File)wasfoundinmostmodernEuropeanandRussian
breedswithafrequencyrangingfrom16to63%withingeographicalregions(Table2).The
otherancienthaplotypeshadmorerestrictedoccurrencesandfrequencies,lessthan1.6%
amongthe2094moderncattledataset(TableCinS1File).
Thereweresignificantdifferencesinappearanceandfrequencyofancienthaplotypes
amongtengeographicalregionsofcontemporarycattle(PearsonChi-Squaretest,n=2094,
χ2=355,df=18,p<0.001).Theproportionofancienthaplotypeswashighestincontemporary
N-EBSRcattleandWesternRussiancattle(Table2),whiletheproportionofhaplotypesnot
foundinourancientsampleincreasedwithgeographicaldistanceshowinghighestproportions
inSouthandSouth-EastEurope,andNearEast/CentralAsia(Table2,indicatedinwhitein
Fig2a).
PLOSONE|DOI:10.1371/journal.pone.0123821 May20,2015 8/16
MitochondrialandY-ChromosomalDNAAnalysesofN-EBalticSeaCattle
Fig2.DistributionofancientN-EBSRcattlemtDNAhaplotypesinmodernEurasiancattle
populations.HaplotypedistributioninancientFinnish,EstonianandWesternRussian(Vyborgattheshore
ofBalticSea)cattlepopulationsfromtheLateBronzeAge,IronAge,Medieval,andPost-Medievalperiodsis
indicatedwithpiechartsattherightsideofthemap(2B,seeTableCinS1File).Seventeenancient
haplotypesfoundinmodernEurasianpopulations(TableCinS1File)areindicatedbypiechartswith
correspondingpatterns(seekey)onthemap(2A).Themodernhaplotypesnotfoundinancientcattleare
countedtogetherandindicatedinwhite.Countsofuniqueancienthaplotypesnotfoundinmodern
populationsareindicatedinblack.
doi:10.1371/journal.pone.0123821.g002
Y-chromosomalanalysis
UTY19allelefrequenciesinFennoscandiancattle(TableDinS1File)differedsignificantlybe-
tweentemporalcohorts(Chi-Squaretest,p<0.001).TypeY2wasdominatinginboththeIron
Age(7/8)andtheMedievalperiod(36/37),withnostatisticaldifferenceinallelefrequencies
betweenthetwoperiods(Fisher’sExacttest,p=0.327).TheproportionofY1increasedsignifi-
cantlyfromtheMedieval(1/37)tothePost-Medievalperiod(9/19,Fisher’sExactTest,
p<0.001)andthenagainfromthePost-Medievalperiod(9/19)toModerntimes(33/41,Fish-
er’sExactTestp=0.015).TheY1typewasfixedinmostcontemporaryFennoscandiannative
breedswithonlyoneexceptionwhereY2wasdominating(8/9),viz.inoneFinnishbreed,the
EasternFinncattle.
Table2. DistributionofN-EBSRancienthaplotypesinmodernEuropeanandAsiancattlebreeds.
N-EBSR Scandinavia Western Southern South- Eastern Western NearEast Central Siberia Total
Europe Europe Eastern Europe Russia andCentral Russia
Europe Asia
CommonH 31 23 93 334 8 13 16 4 12 14 548
63.3% 28.4% 38.1% 21.7% 16.0% 50.0% 61.5% 16.0% 37.5% 58.3% 26.2%
Other 10 27 17 51 7 2 9 1 10 2 136
AncientH 20.4% 33.3% 7.0% 3.3% 14.0% 7.7% 34.6% 4.0% 31.3% 8.3% 6.5%
Hnotfound 8 31 134 1152 35 11 1 20 10 8 1410
inAncient
16.3% 38.3% 54.9% 75.0% 70.0% 42.3% 3.8% 80.0% 31.3% 33.3% 67.3%
data
Total 49 81 244 1537 50 26 26 25 32 24 2094
Figuresrepresentthecountandpercentageofmoderncattledatafromtengeographicalregionsgroupedinthreehaplotype(H)groupsaccordingtothe
appearanceofthehaplotypesinancientN-EBSRdata:Themostcommon245bphaplotype(CommonH),otherancienthaplotypesfoundinPost-
MedievalorMedievalperiodsandhaplotypesnotfound(Hnotfound)inancientNorth-EastBalticSearegioncattle.
doi:10.1371/journal.pone.0123821.t002
PLOSONE|DOI:10.1371/journal.pone.0123821 May20,2015 9/16
MitochondrialandY-ChromosomalDNAAnalysesofN-EBalticSeaCattle
Table3. SummaryofancientandmodernY-haplotypesdistributionacrossEurasia.
Nordic Western Southernand South- Eastern Western NearEastand Central Siberia Total
counties Europe CentralEurope Eastern Europe Russia CentralAsia Russia
Europe
Ancient
Y1 11 1
17% 7%
Y2 53 13
83% 93%
Total 64 14 78
Modern
Y1 101 334 120 53 9 1 24
84% 83% 13% 82% 100% 3% 96%
Y2 19 70 806 10 12 31 1 23
16% 17% 87% 100% 18% 97% 4% 100%
Total 120 404 926 10 65 9 32 25 23 1614
Dataincludes78ancient(fromFinland,SwedenandSwitzerland)and1621modernEurasianbulls.Separatefiguresforeachbreedandancient
populationsaregiveninTableEinS1File.
doi:10.1371/journal.pone.0123821.t003
TherewasnosignificanttemporalchangesdetectedinCentralEurope(Switzerland)from
Medieval(late13thcentury)tomoderntimes(Fisher’sExactTest,p=0.462),whereY2domi-
natedboththeMedieval(13/14)andthemodern(38/39)periods.
Y1andY2haplotypefrequenciesvariedsignificantlybetweengeographicalregions(Chi-
Squaretest,p<0.001,Table3,TableEinS1File).Mostofthemodernbreeds(105from127)in
allregionswerefixedforoneY-haplotype,eitherY1(46)orY2(59),while22displayedboth
Y1andY2(TableEinS1File).
Discussion
MtDNAhaplogroups
Theassignmentofancientsamplesintobovinehaplogroups(Q,T2,T3)orsub-haplogroups
(T1f,T3b),withT3andT3bpredominating,isingoodagreementwithpopulationanalysisof
moderncattle,whereT3isthemajormtDNAhaplogroupinEurasianpopulations[11,14].Itis
alsoinaccordancewithpreviousanalysisofancientEuropeancattlepopulationswhereapre-
dominanceoftheT3haplogrouphasbeenshownfromtheNeolithic[19].
Ararehaplotype,belongingtosub-haplogroupT1f,wasfoundinasampledatedtotheLate
BronzeAgeinEstoniaatafrequencyof1/5inthePrehistoriccohort(Fig2).Inaprevious
study,T1fhasbeenfoundinthreeindividualsfromthemodernItalianbreedPodolian(3/80of
T1haplogroupsequencesfoundinEurope)andinthemodernbreedMenofifromEgypt(fre-
quency1/196ofT1haplogroupsequencesfoundinAfrica)[20].TakingintoaccountthatBon-
figlioetal.[20]analysedmorethantwothousandmtDNAsamplesinordertoobtain54T1
haplotypes,thefrequencyofT1fmustbelessthan4/2000amongEuropean,African,and
Americancattlebreeds.
HaplogroupsQandT2wererareintheancientcattlepopulationsintheN-EBSRjustas
theyareincontemporarypopulations[11].HaplogroupQwasfoundintheNorthernFinnish
Post-Medievalpopulationatafrequencyof1/26;ithaspreviouslybeenfoundinfiveItalianna-
tivecattlebreeds[10,15,16].Mostpreviousstudies,however,failedtodifferentiatehaplogroup
QfromhaplogroupT,astheyoverlookedthesequenceofthediagnosticsiteoutsidetheD-
PLOSONE|DOI:10.1371/journal.pone.0123821 May20,2015 10/16