Rethinking the history of common walnut (Juglans regia L.)

24 Pages·2017·1.95 MB·English

RESEARCHARTICLE
Rethinking the history of common walnut
(Juglans regia L.) in Europe: Its origins and
human interactions
PaolaPollegioni1¤*,KeithWoeste2,FrancescaChiocchini1,StefanoDelLungo3,
MarcoCiolfi1,IreneOlimpieri1,VirginiaTortolano1,JoClark4,GabrielE.Hemery5,
SergioMapelli6,MariaEmiliaMalvolti1
1 InstituteofAgro-environmentalandForestBiology,NationalResearchCouncil,Porano,Terni,Italy,2 U.S.
D.A.ForestService,HardwoodTreeImprovementandRegenerationCenter,DepartmentofForestryand
a1111111111
NaturalResources,PurdueUniversity,WestLafayette,Indiana,UnitedStatesofAmerica,3 TheInstituteof
a1111111111
ArchaeologicalandMonumentalHeritage,NationalResearchCouncil,TitoScalo,Potenza,Italy,4 Earth
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Trust,LittleWittenham,Abingdon,Oxfordshire,UnitedKingdom,5 SylvaFoundation,LittleWittenham,
a1111111111 Oxfordshire,UnitedKingdom,6 InstituteofAgriculturalBiologyandBiotechnology,NationalResearch
a1111111111 Council,Milan,Italy
¤ Currentaddress:Genomics,GeneticsandBiologyInnovationPole,Perugia,Italy
*[email protected]
OPENACCESS
Abstract
Citation:PollegioniP,WoesteK,ChiocchiniF,Del
LungoS,CiolfiM,OlimpieriI,etal.(2017)
Commonwalnut(JuglansregiaL)isaneconomicallyimportantspeciescultivatedworldwide
Rethinkingthehistoryofcommonwalnut(Juglans
foritshigh-qualitywoodandnuts.ItisgenerallyacceptedthatafterthelastglaciationJ.
regiaL.)inEurope:Itsoriginsandhuman
interactions.PLoSONE12(3):e0172541. regiasurvivedandgrewinalmostcompletelyisolatedstandsinAsia,andthatancient
doi:10.1371/journal.pone.0172541 humansdispersedwalnutsacrossAsiaandintonewhabitatsviatradeandculturalexpan-
Editor:Tzen-YuhChiang,NationalChengKung sion.ThehistoryofwalnutinEuropeisamatterofdebate,however.Inthisstudy,weesti-
University,TAIWAN matedthegeneticdiversityandstructureof91Eurasianwalnutpopulationsusing14neutral
Received:September22,2016 microsatellites.Byintegratingfossilpollen,cultural,andhistoricaldatawithpopulation
genetics,andapproximateBayesiananalysis,wereconstructedthedemographichistoryof
Accepted:February5,2017
walnutanditsroutesofdispersalacrossEurope.Thegeneticdataconfirmedthepresence
Published:March3,2017
ofwalnutinglacialrefugiaintheBalkansandwesternEurope.Weconcludethathuman-
Copyright:©2017Pollegionietal.Thisisanopen
mediatedadmixturebetweenAnatolianandBalkanwalnutgermplasmstartedintheEarly
accessarticledistributedunderthetermsofthe
BronzeAge,andbetweenwesternEuropeandtheBalkansineasternEuropeduringthe
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and RomanEmpire.Apopulationsizeexpansionandsubsequentdeclineinnortheasternand
reproductioninanymedium,providedtheoriginal westernEuropewasdetectedinthelastfivecenturies.Theactualdistributionofwalnutin
authorandsourcearecredited.
Europeresultedfromthecombinedeffectsofexpansion/contractionfrommultiplerefugia
DataAvailabilityStatement:Thedatasetforthis aftertheLastGlacialMaximumanditshumanexploitationoverthelast5,000years.
studyisavailablefromthefigsharerepository
(https://doi.org/10.6084/m9.figshare.4665235.v1).
ThedatasetincludesgeographicsitesandSSR
geneticdatafor91walnutpopulationssampledin
Eurasia.
Funding:Thisstudywassupportedbythe Introduction
EuropeanCommunityintheframeworkofthe
Commonwalnut(JuglansregiaL.)isaneconomicallyimportanttreespecies,highlyvaluedfor
SeventhFrameworkProgrammeundertheMarie
itstimberandediblenuts.Thisspeciesgrowswellinvirtuallyallpartsoftheworldwithatem-
CurieActionsCOFUNDproject“I-MOVE”(N.
267232)Grantnumber206405. perateclimate[1].Itsancienthistoryofcultivationandwidespreadusethroughoutmuchof
PLOSONE|DOI:10.1371/journal.pone.0172541 March3,2017 1/24
RethinkingthehistoryofwalnutinEurope
Competinginterests:Theauthorshavedeclared Eurasia,fromChinatowesternEurope,andtherelativescarcityofwide-scalemolecularphy-
thatnocompetinginterestsexist. logeographicstudies,madeaccuratedeterminationofitsnativegeographicrangedifficult.
Newdataandresources,however,includingthedevelopmentofhighlyinformativemolecular
markersinJuglansspp.suchasSSRmarkers[2],therecentadventoflandscapegenetics[3],
thepersistenceofcenturies-oldwalnuttreesinnaturalreservesisolatedfromplantations,
accesstoremoteuncultivatedAsianwalnutpopulations[4]andarichfossilpollenrecordin
Europe[5],providetheopportunitytoresolvethecomplexhumanandnaturalinteractions
thatshapedtheevolutionaryhistoryofwalnutsincetheHoloceneinEurasia.
ThehistoryofwalnutinAsiahasemergedasacomplexinteractionofbiogeographicand
humanforces.AftertheLastGlacialMaximum(LGM),J.regiasurvivedandgrewspontane-
ouslyinalmostcompletelyisolatedstandsinAsia,fromXinjiangprovinceofwesternChina
throughcentralAsiatotheCaucasus.Barrierstogeneflow,suchastheTienShanandHima-
layamountains,andtheprogressivedesertificationofcentralAsiaduringtheHolocenepro-
motedthefragmentationandisolationofnaturalJ.regiapopulationsintheseregions[4].
Geneticanalysiscombinedwithethno-linguisticandhistoricaldatarevealedthatwhat
appearedtobenativewalnutstandswereactuallytheresult,atleastinpart,ofancienthuman
effortstomodifytheAsianlandscape[6].Humans,tradingwalnutalong"greencorridors”
suchastheSilkRoadsandthePersianRoyalRoad,overcamegeographicbarriersandfacili-
tatedwalnutexchangesacrossAsia[6–7].Theoriginandhuman-mediatedexpansionofwal-
nutacrossEurope,however,isamatteroflong-standingdebate.
FossilpollendepositsclearlyindicatethatJ.regiawasgrowinginsouthernSpain,Italy,France,
Switzerland(Alps),Bulgaria(RhodopesMountains),Greece(Epirus),southwesternTurkeyand
AlbaniaduringtheUpperPleistocene(126,000–12,000BP)(S1TableandS1Video).Considering
thescarceremainsofwalnutpollenintheEuropeanpost-Eemiansequences,severalauthorspro-
posedthatJ.regiaessentiallydisappearedinEuropeattheonsetoftheHolocene(~(cid:21)11,700BP),
aftertheLastGlacialMaximum[8]anditshuman-mediatedreintroductionintoEuropefrom
westernAsia(e.g.easternTurkeyandTransCaucasus)totheAegeanbasinoccurrednotbefore
themiddleofthefourthmillenniumBP[9].Asecondwaveofmigrationintosouthwestern
EuropefollowedtheGreekcolonizationaroundthe8thto5thcenturiesBCE(2800–2500BP)
[10–11].Thisviewisbeingchallengednow.SubsequentpalynologicalfindsdemonstratedthatJ.
regiamayhavesurvivedthecold,dryglacialintervalsinrefugialocatedinsouthernEurope[12–
14]andtheBalkans[15–18](S1Video).Carpologicalfossilsofwalnuthavebeenfoundinnorth-
easternItaly(Sammardenchia,7,550BP)[19],Switzerland(nearLakeConstance,~6,000–4,350
BP)[20]andSlovenia(Hocevarica,5,600–5,500BP)[21].Assumingwalnutwasharvested/culti-
vatedfromtheearlyNeolithictoBronzeAgeinEurope[13],wecan’truleouttheuseoflocal
stands,formerlyprotectedfortheirfuelandfoodduringtimesofdramaticlandtransformations
duetoglobalclimateoscillationsandgrowingeconomicpressures[22–23].Despiteinformation
fromthefossilrecord,thereisnomolecularevidencethatJ.regiasurvivedtheLGMinEuropeas
nopolymorphismwasdetectedin29demesofwalnutinEurasiausingchloroplastPCR-RFLP
markers[24].
Irrespectiveoftheresolutionoftheconflictbetweenfossilandmolecularevidence,thedis-
tributionofJ.regiainEuropewassurelymodifiedbyhumanmanagementoverthelast2,500
years.SuddenincreasesinJ.regiafossilpollencurveswererecordedinthetimewindow
between2500and1000yearsBP,presumablyreflectingthewidespreadincreaseofwalnutcul-
tivationfromGreekandRomantimesonwards[11](S1Video).AsdetectedinItaly,theselec-
tionandfrequentinter-regionaltransferofwalnutseedsalongancientroutesprobably
affectedthespatialgeneticstructureofJ.regia,decreasingitsdiversitybyselectionandincreas-
ingitsgenetichomogeneitybydispersal[25–26].Nevertheless,despitelong-termscientific
interest,acomparativeandfull-scaleoverviewofthegeneticresourcesofwalnutinEuropehas
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neverbeenundertaken,andneithertheputativedispersalroutesofwalnutacrossEuropenor
thetime-scalesoverwhichsuchprocessesoccurredsincetheLateHolocenehavebeenstudied
usingmolecularphylogeny.
Here,wereportonalarge-scalestudyofthespatialgeneticstructureofwalnutpopulations
inEuropeusingmicrosatellitemarkers,anditscomparisonwithautochthonouswalnutpopu-
lationssampledinAsia.Ourobjectivewastheintegrationoffossilpollen,culturalandhistori-
caldatawithpopulationgenetics(thelandscapegeneticoverlayapproach)[27],andthe
inferenceofdemographichistoryusingapproximateBayesiancomputation(ABC)[28]to
evaluateif(1)ancientreservoirsofwalnutdiversitystillexistinEurope,(2)clinesofgenetic
diversityarepresentbetweenEuropeandAsianregionswherewalnutstillgrowsnaturally,(3)
geneticclinesinJ.regiacanbeexplainedbytherecolonizationofwesternEuropefromrefugia
ofwesternAsiathroughtheBalkans,or(4)whetherwesternAsiaandthesouthernEuropean/
Balkanspeninsulasrepresenttwoseparateregionsofwalnutevolution.Inaddition,weevalu-
atedthehypothesisthatcultural/historicalfactorsplayedakeyroleinshapingthegenetic
resourcesofwalnutinEuropesincetheLateHolocene.
Materialsandmethods
Commonwalnutcollection
Overthelasttwodecades,theCNR-InstituteofAgro-environmentalandForestBiology
(IBAF,Porano,Italy),CNR-InstituteofAgriculturalBiologyandBiotechnology(Milan,Italy)
andtheEarthTrust(Oxfordshire,UK)havemonitoredandsampledcommonwalnuttreesin
Europeandacrossthespecies’rangeinAsia.Althoughwewereunabletoobtainsamplesfrom
allareas,weassembledalargeanduniquecollectionofJ.regia,including40Asianautochtho-
nouswalnutpopulationssampledfromChina(6populations),Kyrgyzstan(9),Uzbekistan
(17),Tajikistan(1),Pakistan(2),Iran(1),Turkey(2)andGeorgia(2)whichhavealreadybeen
genotyped[4,6]and51EuropeanwalnutpopulationssampledfromGreece(4),Romania(1),
Moldova(1),Hungary(10),Slovakia(1),Spain(1),France(4)andItaly(29),foratotalof91
populationsand2,008genotypes(S2Table,S1Fig).Thesewalnutpopulationsspanthirteen
mountainsystemsinEurasia(TienShan,Gissar,Zaamin,Nurata,Pamir,Himalayas,Alborz,
Trans-Caucasus,Balkans,Carpathians,Alps,Pyrenees,andApennines)(S1Fig).Onlycentu-
ries-oldtreesinancientforestsornaturereservesweresampledinAsia.InEurope,wecare-
fullyavoidedcollectingwalnutsamplesfromplantationsornon-protectedareas(S2Table).
Sampledtreesineachpopulationwereseparatedby>100metres.Matureleavesweresampled
fromeachwalnuttree,dehydrated,andthenstoredat−80˚CatCNR-IBAF(Porano,Italy).
Microsatelliteanalysis
Dehydratedleaftissue(60mg)fromeachEuropeanwalnutsamplewashomogenizedina2ml
micro-centrifugetubecontaininga5-mmsteelbeadcooledwithliquidnitrogenusinga
MixerMill300(Qiagen,Hilden,Germany).GenomicDNAwasextractedandpurifiedusinga
DNeasy96PlantKit(Qiagen),andstoredat-20˚C.
Inthisstudy,allEuropeansamplesweregenotypedusing14unlinkednuclear,neutral
microsatellite(SSR)markers(WGA1,WGA4,WGA9,WGA27,WGA32,WGA69,WGA72,
WGA79,WGA89,WGA118,WGA202,WGA276,WGA321,WGA331)alreadyselectedand
usedforgeneticcharacterizationofwalnutinitsAsianrange[4,6].PCRamplificationandthe
visualizationofamplifiedSSRallelesforeachsamplewerecarriedoutasdescribedbyPolle-
gionietal[26].TheallelesizescoringwasperformedusingGeneMapperversion4(Applied
Biosystems,FosterCity,CA,USA).
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RethinkingthehistoryofwalnutinEurope
Dataanalysis
Geneticdiversityofwalnutpopulations. Standardmeasuresofgeneticdiversity,total
numberofobservedalleles(A),effectivenumberofalleles(A ),observed(H )andexpected
e O
(H )heterozygosityandpolymorphicinformationcontent(PIC),thethreeunbiasedestima-
E
torsofWright’sF-statistics,within-populationinbreedingcoefficientf(F ),total-population
IS
inbreedingcoefficientF(F )andamong-populationgeneticdifferentiationcoefficientθ(F )
IT ST
andtheunbiasedestimatorofJost’s(D )[29]werecomputedforeachlocusandoverallloci
est
asdescribedbyPollegionietal[4].Similarly,alleledropoutandnullallelesweretestedfor
eachlocususingFreeNasoftware[30].
Levelsofgeneticdiversitywereestimatedforeachwalnutpopulationacrossalllociintermsof
themeannumberofallelesperlocus(A),observed(H )expected(H )andunbiasedexpected
o E
heterozygosity(UH )usingtheGenAlExsoftware6.3[31].Toaccountfordifferenceinsample
E
size,allelicrichness(Rs)andprivateallelerichness(PAR)werecomputedbytherarefaction
methodwithHP-Raresoftware[32].TheestimatesofRsandPARwerestandardizedonamini-
mumsamplesizeofeightindividuals.FollowingtheprocedureofPollegionietal[4],theInverse
DistanceWeighted(IDW)interpolationfunctionimplementedintheGeographicInformation
System(GIS)softwareArcGIS9.3(ESRI,Redlands,Calif.USA)wasusedtodisplaythegeo-
graphicpatternsofallelicrichness(Rs)andunbiasedexpectedheterozygosity(UH )computed
E
forall91walnutpopulations.Thewithin-populationinbreedingcoefficientF [33]wasestimated
IS
foreachpopulationusinghierarchicallocus-by-locusAMOVAasimplementedinArlequin3.11
software[34].ThestatisticalsignificanceofF wastestedusinganon-parametricapproachwith
IS
1,000permutations.Todeterminewhetherwithin-populationgeneticvariationwascorrelated
withgeographicgradients,weperformedasimplelinearregressionanalysisfollowedbythe
sequentialBonferronicorrectionofallelicrichness(Rs)andunbiasedexpectedheterozygosity
(UH )againstthreegeographicvariables,latitude,longitudeandelevationofsampledsites.
E
EvidenceofrecentpopulationsizedecreasewasinvestigatedusingtheprogramBOTTLE-
NECK1.2.02[35].AsdescribedbyPollegionietal[4],significancewasassessedusingthe‘Wil-
coxon’ssigned-rank’test,whichprovidesrelativelyhighpowerandcanbeusedwithasfewas
fourpolymorphiclociandanynumberofindividuals.Bottlenecktestswereconductedunder
Two-PhaseModel(TPM)ofevolutionwhichmostaccuratelyreflectedthemutationalmecha-
nismofthemicrosatellitelociusedinthisstudy[26].AsrecommendedbyPiryetal[35],we
usedtheTPMwith70%StepwiseMutationsModel(SMM)and30%multistepmutations.For
eachmutationalmodel,10,000replicateswereperformed.Nevertheless,asreportedbyHenry
etal[36],theheterozygosityexcesstestissensitivetoveryrecentdisturbances.Twoadditional
testswereusedtoidentifybottlenecksignaturesfromlargertimescales:shiftedalleledistribu-
tionanalysis[37]andtheM-ratiotest[38]implementedinBOTTLENECKandArlequinsoft-
ware,respectively[4].
Geneticstructureanalysisofcommonwalnutpopulations. Twocomplementarystatis-
ticalapproacheswereusedtodetectthegeneticstructureofwalnutpopulationsinEurasia.
First,aBayesianclusteringapproachimplementedinSTRUCTUREsoftware2.3.3[39]was
appliedasdescribedbyPollegionietal[6].Wereconstructedtheunderlyinggeneticstructure
ofwalnutpopulationsandcomputedtheproportionofmembership(Q-value)foreachprede-
finedpopulationandeachindividualmultilocusgenotypeineachoftheinferredclusters
usingMarkovChainMonteCarlo(MCMC)simulations.STRUCTUREwasrunassuminga
pre-assignednumberofclusters(K)rangingfrom1to90,theadmixturemodelonthewhole
datasetwithoutaprioripopulationinformationandwiththecorrelatedallelefrequencies
betweenpopulationsoption.Basedoninitialresults,aseriesofsixindependentrunswasper-
formedforKbetween1and12withaburn-inperiodof100,000stepsfollowedby106MCMC
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RethinkingthehistoryofwalnutinEurope
replicates.ThefinalposteriorprobabilityofK,LnP(K),andΔK,therateofchangeofLnP(K)
betweensuccessiveKvalues[40],wascalculatedtodetectthemostlikelynumberofpopula-
tions.Therefore,thegroupsinferredbythefirstSTRUCTUREanalysisweresubsequently
reprocessedseparatelyinordertoidentifypossiblesub-structure(sub-clusters).Thesixruns
fromthemostprobablenumberofclusterswereaveragedapplyingtheFullSearchalgorithm
providedbyCLUMPPsoftware1.1.2.[41].ThecorrespondingQ-matricesweregraphically
displayedbyDISTRUCTsoftware[42].Afterdeterminingthemostprobablenumberofclus-
ters,anarbitrarythresholdofQ(cid:21)0.75wasusedtoassignpopulationsand/orgenotypesto
onegroup.Populationswith0.25<Q<0.75wereclassifiedasadmixed.Followingtheproce-
dureofPollegionietal[6],wederivedKcontinuousclusteringsurfacesbyinterpolationofthe
populationmembershipQ-valuesfortheKclustersestimatedfromSTRUCTUREusingthe
IDWfunctionimplementedinArcGIS9.3.Asyntheticmaprepresentingthegeneticstructure
ofwalnutinEurasiawasobtainedbyoverlayingthecomputedKclusteringsurfacemaps.We
combinedmultipleKinterpolatedrasterbandsinasinglemultibandrasterdatasetbythe
CompositeBandsfunctionimplementedinArcGIS9.3.TheintegrateduseoftheComposite
Bands-toolandRGBcolorcodeallowedustodisplaytheinferredgeneticclustersofJ.regia
populations.
Finally,toconfirmthegeneticrepartitionofwalnutpopulationsinferredbySTRUCTURE,
aNeighbor-Joiningphylogenetictreewasconstructedbasedon[43]geneticdistanceusing
POPTREE2software[44]anddrawnusingFigTreesoftware(http://tree.bio.ed.ac.uk/software/
figtree/).
ReconstructionofpostglacialdispersalroutesofwalnutinEurope. Thedemographic
historyofwalnutanditshuman-mediatedpostglacialdispersalroutesinEuropewerefurther
exploredusingtheapproximateBayesiancomputation(ABC)procedureoriginallyintroduced
byBeaumontetal[28]andrecentlyimplementedinDIYABCv.2.0.4software[45].
Duetocomputationallimitationsandthecomplexityofpossiblescenarios,wesplit
DIYABCanalysisintotwostagesandwepooledasubsetofEurasianwalnutsamplesintofour
groupsasinferredbySTRUCTUREandNeighbor-Joiningtreeanalysis(S3Table).Genepool
1consistedof41individualsfromAnatolia(Turkey,Sub-cluster1–1);genepool2comprised
131individualsfromtheBalkans(admixedgenotypesbetweenCluster1andCluster4);gene
pool3comprised279individualsfromnortheasternEurope(Sub-cluster4–2);andgenepool
4included650individualsfromwesternEurope(Sub-cluster4–1).
InthefirststageoftheDIYABCanalysis,wetestedfivecompetingbroad-scalescenarios
(Scenarios1a-5a)basedonhistoricalandfossilpollendata(S4Table).Scenarios1aand2a
assumedafirstintroductionofwalnutfromAnatoliatotheBalkansfollowedbyaexpansion
fromtheBalkansthroughnortheasternEuropetowesternEurope(Scenario1a,LandRoute)
orfromtheBalkansthroughwesternEuropetonortheasternEurope(Scenario2a,Maritime
Route).Then,wetestedthepresenceoftworefugia(AnatoliaandtheBalkans)withsubse-
quentexpansionofwalnutfromtheBalkansthroughnortheasternEuropetowesternEurope
(Scenario3a)orfromtheBalkansthroughwesternEuropetonortheasternEurope(Scenario
4a).Thefinalmodelofstage1(Scenario5a)testedacomplexscenarioofindependenthuman-
induceddispersalofwalnutfromAnatoliaandwesternEuropetotheBalkanswhereadmix-
tureoccurred,asubsequentexpansionfromwesterntonortheasternEuropeandafinalpopu-
lationdeclineofwesternEurope.Alternativescenarios(e.g.westernEuropeasauniquecentre
ofwalnutorigin)wereconsideredunrealisticandthereforewereexcluded(S4Table).
ThesecondstageoftheDIYABCanalysiswasatafinerscale,assumingtworefugia(Anatolia
andwesternEurope;Scenarios1b-3b)orthreerefugia(Anatolia,theBalkansandwesternEurope;
Scenarios4b-6b),withadmixtureintheBalkans,apopulationsizeexpansioninnortheastern
Europe,anddeclineinwesternEurope.Wealsoevaluatedthedispersalofwalnutintonorthern
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RethinkingthehistoryofwalnutinEurope
EuropefromwesternEurope(Scenario1b,4b),fromtheBalkans(Scenario2b,5b)orasaresult
ofadmixturebetweenwesternEuropeandtheBalkangermplasm(Scenario3b,6b)(S4Table).
Thepriorvaluesusedforallthedemographicparametersselectedfortenscenariosare
listedinS3Table.FollowingtheprocedureofBaietal[46]forshorter-livedAsianbutternuts,
weassumed80–110yearsasareasonablegenerationtimeforwalnut(productionofnuts10–
20yearsaftergermination;meanlifespanof~100–200years;[47]).Basedontheseassump-
tions,wechosepriordistributionsforthetimingofevents(ingenerations)ofstage2including
LastGlacialPeriod(100–10,000),postLGMperiod(10–200),BronzeandIronAge(20–60),
Hellenistic-Romanperiod(10–25)andMiddleAges(1–15or1–10).Weassumedageneralized
stepwisemutationmodelforSSRloci,withauniformmutationrateprior(μ)between10−4and
10−3.Theobservedandsimulatedgeneticdatasetsweresummarizedusingthemeannumberof
alleles,meangeneticdiversity,meanallelesizevarianceforeachpopulation,Wright’sF ,mean
ST
individualassignmentlikelihoodsandmeanindexofclassification(DAS)amongpopulations.
Wegeneratedareferencetablecontaining105simulateddatasetsforeachscenario,andsubse-
quently1%ofthesimulateddatasetsclosesttotheobservedgeneticdatasetwereusedtoestimate
posteriorprobabilities(with95%confidenceintervals)foreachscenariousingdirectandlogistic
regressionapproaches[45].Theposteriordistributionofhistoricaldemographicparameterswas
estimatedusingalogisticregressionof1%oftheclosestdatasetssimulatedaccordingtothemost
likelyscenario.Tovalidatetheconfidenceinascenariochoice,wecalculated(1)TypeIError
(Falsenegativerate)astheproportionoftimesthattheselectedscenariodidnotexhibitthehigh-
estposteriorprobabilitycomparedwiththecompetingscenariosfor500pseudo-observeddata-
setsgeneratedunderthebest-supportedmodel,and(2)TypeIIError(Falsepositiverate)asthe
proportionoftimesthattheselectedscenariowasincorrectlyselectedasthemostlikelyscenario
for500pseudo-observeddatasetsgeneratedundereachofthecompetingscenarios.Finally,we
performedamodelcheckinganalysistoevaluateifamodel-posteriorcombinationfittedthe
observeddatacorrectly.Theobservedsummarystatisticswerecomparedwiththosecomputed
for1,000datasetssimulatedfromtheposteriordistributionofparametersobtainedunderthe
selectedscenario[45].
Results
GeneticdiversityofEurasianwalnutpopulations
All14SSRlociusedinthepresentstudywerehighlypolymorphicinthesampledwalnutpopu-
lations.Atotalof199allelesweredetectedinthe2,008walnuttreesgenotyped,withanaverage
of14.21allelesperlocus.NoneoftheSSRlocishowedevidenceofnullalleles(foracomplete
descriptionoftheSSRloci,seeS5Table).Geneticdiversityparametersvariedgreatlyamong
Eurasianwalnutpopulations(S6Table).Threegeographicareasshowedhighvaluesofallelic
richness(Rs)andunbiasedexpectedheterozygosity(UH ):1)south–centralAsiaincludingsites
E
inTibet(32-DASH),Kashmir–westernHimalaya(34-HUNZAand33-GILGIT,Pakistan),
northernPamirridges(35-SHOULI,Tajikistan),GissarMountain(18-BAKHMAL,Uzbeki-
stan),westernTienShan(19-KARANKULand17-BONSTANLYK,Uzbekistan);2)fourTrans-
Caucasussites(37-LAGOand38-SKRA,Georgia;39-ANATOLIAand40-TRABZONTurkey)
and3)fourBalkanpopulations(41-PAIKO_A,42_PAIKO_B,Greece;45-BRASOV,Romania;
and46-CHISINAU,Moldova)(Fig1).
Asignificantdeclineinallelicrichnesswasfoundwithdecreasinglongitude(r=0.522,
P=0.00015)andincreasinglatitude(r=0.425,P=0.00135).Of199allelesdetected,33were
uniquetoasinglegeographicsite,with23uniquetoAsianandtentoEuropeanwalnutpopu-
lations(S6Table).Overall,F rangedfrom-0.066(74-SANNIO)to0.280(28-GUILI-2),and
IS
wassignificantlygreaterthanzeroin31of91sampledsites,indicatingasurplusof
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Fig1.Geneticdiversityof91walnutpopulationsinEurasia.InverseDistanceWeighted(IDW)interpolationoftheallelicrichnessvalues(Rs)(a)
andunbiasedheterozygosityUH (b)calculatedfor91walnutpopulations(blackdots)inEurasiausing14SSRmarkers(abbreviationsCN=China,
E
UZ=Uzbekistan,KG=Kyrgyzstan,TJ=Tajikistan,PK=Pakistan,IR=Iran,GE=Georgia,TR=Turkey,MD=Moldova,RO=Romania,
HU=Hungary,SK=Slovakia,GR=Greece,IT=Italy,FR=France,ES=Spain).
doi:10.1371/journal.pone.0172541.g001
homozygotesin18(45%)and13(26%)ofAsianandEuropeanwalnutpopulations,respectively
(S6Table).BasedontheTMPmodel,significant(P<0.05)signalsofrecentreductionineffec-
tivepopulationsizeweredetectedinalargeproportionofEuropeansites(73%),butonlyin
four(10%)Asianpopulations(4-SHAIDAN,5-KYZYL,17-BOSTANLYKand36-KARAJ).By
contrast,usingtheMratiotest,weobservedageneticsignatureconsistentwithabottleneckin
allpopulations.TheG–Wvaluesrangedfrom0.28117(36-KARAJ)to0.37939(72-VALCO),
whichissubstantiallylowerthanthecriticalthresholdof0.68(S7Table).
SpatialgeneticstructureofwalnutpopulationsinEurasia
Thegeneticstructureofthe91sampledwalnutpopulationswasevaluatedusingSTRUCTURE
andNeighbor-Joiningtreeclusteringanalysis.STRUCTUREindicatedfourasthemostappro-
priatenumberofpopulationgroups.Thead-hocstatisticΔKofEvannoetal[40]combined
withtheanalysisofLog-likelihooddistributionofdataL(K)asafunctionofK,recognized
K=4asthebestrepresentationoftheunderlyinghierarchicalstructureofthe91walnutpopula-
tionsinEurasia(S2Fig).Cluster1,centeredinwesternandsouth-centralAsia,includedallwal-
nutssampledfromfourTrans-Caucasussites(37-LAGOand38-SKRA,Georgia;39-ANATOLIA
and40-TRABZONTurkey),Alborzridges,Iran(36-KARAJ),Kashmir-westernHimalayas,Paki-
stan(33-GILGITand34-HUNZA)andTibetan-easternHimalaya,China(32-DASH)(Q (cid:21)
1
0.8227)(S8Table).Inaddition,21walnuttrees(58.3%)collectedin19-KARANKUL(eastern
Uzbekistan)andtenof67walnuttrees(14.9%)from28-GONGLIU-2(Xinjiangprovince,China)
wereunambiguouslyassignedtocluster1(Q1(cid:21)0.800)(Fig2).
Cluster2assembledsevenpopulationsfromtheNurataridgelocatedineast-centralUzbek-
istanandfivepopulationsofnorthernandeasternChina,i.e.,foursitesintheeasternTien
Shanmountains,Xinjiangprovince,andonesitefromShandongprovince(Q (cid:21)0.7639).
2
Cluster3comprisedallnineKyrgyzandfourUzbekpopulations(10-Kamchik,11-Yakkatut,
12-Sidjakand13-Charvak)sampledinthewalnutforestsofthewesternTienShanmountains
(Q (cid:21)0.7856).TheremainingthreewalnutpopulationsinthewesternTienShanmountains
3
(14-Nanai,16-Bogustan,17-Bostanlyk)andtwopopulationsfromtheGissarmountains
(15-Djarkurgan)andtheZaaminmountains(18-Bakhmal)ineasternUzbekistanweremainly
admixturesbetweencluster2(0.2013(cid:20)Q (cid:21)0.3740)andcluster3(0.4987(cid:20)Q (cid:21)0.7118).
2 3
Cluster4includedallEuropeanwalnutpopulationssampledinSpain,Italy,France,Slovakia,
Crete(Greece)andHungary(Q (cid:21)0.7648),exceptforfiveeasternmostBalkanpopulations
4
locatedinGreece(Macedonia,41-PAIKO_A,42_PAIKO_B;Peloponnese,43-ARCADIA),
Romania(45-BRASOV),andMoldova(46-CHISINAU)andtheremainingpopulationfrom
Tajikistan(35-SHOULI),whichshowedadmixedprofilesbetweencluster1(0.3101(cid:20)Q (cid:21)
1
0.7420)andcluster4(0.2230(cid:20)Q (cid:21)0.6663)(Fig2,S8Table).
4
SubsequentSTRUCTUREanalysiswithineachofthepreviouslyinferredclustersrevealed
geneticsubstructure,exceptforcluster3(S2FigandS8Table).Theinferredcluster1wascom-
posedoffoursub-clusters(K’=4).Thesefoursub-clusterssplitwalnuttreesof39-ANATOLIA,
40-TRABZON(Turkey),36-KARAJ(Iran)and19-KARANKUL(21samples,easternUzbeki-
stan)(sub-cluster1–1),from38-LAGO,39-SKRA(Georgia)(sub-cluster1–2),28-GONGLIU-2
(tensamples,Xinjiangprovince,China),32-DASH(Tibet,China)(sub-cluster1–3)and33-GIL-
GIT,34-HUNZA(Kashmir,Pakistan)(sub-cluster1–4)(Fig3A).
PLOSONE|DOI:10.1371/journal.pone.0172541 March3,2017 8/24
RethinkingthehistoryofwalnutinEurope
Fig2.Spatialgeneticstructureof91walnutpopulationsinEurasia.Populationstructureinferencefor91walnutpopulationsbyBayesianassignment
usingSTRUCTUREforK=4.SyntheticmapofInverseDistanceWeighted(IDW)interpolationsoftheestimatedmeanpopulationmembershipvalues(Q)
i
(a)andbarplotshowingassignmentprobabilitiesofindividualstoKclusters(b).Abbreviations:CN=China,UZ=Uzbekistan,KG=Kyrgyzstan,
TJ=Tajikistan,PK=Pakistan,IR=Iran,GE=Georgia,TR=Turkey,MD=Moldova,RO=Romania,HU=Hungary,SK=Slovakia,GR=Greece,IT=Italy,
FR=France,ES=Spain.
doi:10.1371/journal.pone.0172541.g002
Twosub-clustersoftheinferredcluster2(K”=2)correspondedtotwodistinctgroupsof
walnutpopulationssampledineast-centralUzbekistan(sub-cluster2–1)andChina(sub-clus-
ter2–2,Fig3B).Finally,twosub-clustersoftheinferredcluster4(K”‘=2)extendedfrom
SpaintonorthwesternHungary(Gyo¨r-Moson-SopronProvince,55-BONY,56-MOSONM)
(sub-cluster4–1)andfromeasternHungaryandSlovakia(CarpathiansMountains)towestern
Crete(44-CHANIA)(sub-cluster4–2)(Fig3C).ANeighbor-JoiningtreebasedonNei’s[39]
geneticdistanceconfirmedthepreviousresultsandshowedthatfiveeasternmostBalkansites
inGreece(MacedoniaandPeloponnese),RomaniaandMoldovamightrepresentthecontact
zonesbetweenEuropean(Cluster4)andsouthwestAsian(Cluster1)germplasmpassing
throughtheAnatolianplateau(sub-cluster1–1)(Fig4).
InferringdemographichistoryofwalnutinEuropeusingABCanalysis
TheABCmethodenabledustotesttendemographicscenarios(S4Table)andtodelineate
postglacialdispersalroutesofwalnutacrossEurope.InthefirststageoftheDIYABCanalysis,
bothdirect(P )andlogisticregression(P )approachesidentifiedscenario5aasthemostlikely
1 2
model(Table1,S3Fig,P =0.3620,95%CIs:0.0000–0.7832,P =0.9474,95%CIs:0.8396–
1 2
1.0000).
FourexcludedscenariosassumedthatpopulationsfromAnatolia(Scenario1a-2a)orthe
Balkans(Scenario3a-4a)servedasauniquesourceforthehuman-mediatedwalnutcoloniza-
tionoftherestofEurope.Scenario5aproposedtwoindependentdispersaleventsofwalnut
fromAnatolia(Pool1)andwesternEurope(Pool4)totheBalkanswithsubsequentgenetic
admixture(Pool2).InthesecondstageoftheDIYABCanalysis,wetestedsixfine-scale
PLOSONE|DOI:10.1371/journal.pone.0172541 March3,2017 9/24
RethinkingthehistoryofwalnutinEurope
Fig3.Spatialgeneticsub-structureoftheinferredclusters1,2and4ofwalnutpopulations.SyntheticmapsofInverseDistanceWeighted(IDW)
interpolationsoftheestimatedmeanpopulationmembershipvalues(Q)andbarplotfor(a)K’,themostprobablenumberofsub-clusters,basedon
i
microsatelliteanalysisof217walnutsamplesofcluster1,(b)forK”,themostprobablenumberofsub-clusters,basedonmicrosatelliteanalysisof280
walnutsamplesofcluster2and(c)forK”‘themostprobablenumberofsub-clusters,basedonmicrosatelliteanalysisof929walnutsamplesofcluster
4.
doi:10.1371/journal.pone.0172541.g003
scenariosalwaysincludingtwo(AnatoliaandwesternEurope,scenario1b-3b)orthreeglacial
refugia(Anatolia,theBalkansandwesternEurope,scenario4b-6b)withadmixtureeventsin
theBalkans(S4Table).Acomparisonofposteriorprobabilitiesofthesixscenariosunambigu-
ouslyindicatedthatscenario6bwasthemostlikelymodel(Table1,S3Fig,P =0.3840,95%
1
PLOSONE|DOI:10.1371/journal.pone.0172541 March3,2017 10/24