Na-DenepopulationsdescendfromthePaleo-EskimomigrationintoAmericaPavelFlegontov1,2,3,*,N.EzgiAltınışık1,PiyaChangmai1,EdwardJ.Vajda4,JohannesKrause5,StephanSchiffels5,*1DepartmentofBiologyandEcology,FacultyofScience,UniversityofOstrava,Ostrava,CzechRepublic2A.A.KharkevichInstituteforInformationTransmissionProblems,RussianAcademyofSciences,Moscow,Russia3InstituteofParasitology,BiologyCentre,CzechAcademyofSciences,ČeskéBudĕjovice,CzechRepublic4DepartmentofModernandClassicalLanguages,WesternWashingtonUniversity,Bellingham,WA,USA5DepartmentofArchaeogenetics,MaxPlanckInstitutefortheScienceofHumanHistory,KahlaischeStr.10,07745Jena,Germany*correspondingauthors:S.S.,emailschiffels@shh.mpg.de,P.F.,emailpavel.flegontov@osu.cz.

AbstractPrehistoryofNativeAmericansoftheNa-Denelanguagefamilyremainscontroversial.GeneticcontinuityofPaleo-Eskimos(SaqqaqandDorsetcultures)andNa-Denewasproposedunderthethree-wavemodelofAmerica’ssettlement;however,recentstudieshaveproducedconflictingresults.Here,weperformedreconstructionanddatingofNa-Denepopulationhistory,usinggenomesequencingdataandacoalescentmethodrelyingonrarealleles(Rarecoal).Wealsoappliedmodel-freeapproachesforanalysisofrarealleleandautosomalhaplotypesharing.AllmethodsdetectedCentralandWestSiberianancestryexclusivelyinafractionofmoderndayNa-Deneindividuals,butnotinotherNativeAmericans.OurresultsareconsistentwithgeneflowfromPaleo-EskimosintotheFirstAmericanancestorsofNa-Dene,andalaterlessextensivebidirectionaladmixturebetweenNa-DeneandNeo-Eskimos.ThedatedgeneflowfromSiberiatoNa-DeneisinagreementwiththeDene-YeniseianlanguagemacrofamilyproposalandwiththesuccessionofarchaeologicalculturesinSiberia.

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TheNa-DenelanguagefamilyincludestheTlingit,Eyak(recentlyextinct),NorthernandSouthernAthabaskanbranches,andoccupiesAlaskaandpartsofCanada,withisolatedgroupsresidingalongtheUSNorthPacificCoastandmuchfurthersouthintheUSA1.Underthethree-wavemodelofAmerica’ssettlement,originallyadvancedin1986basedonasynthesisoflinguistic,dentalandlimitedgeneticdataavailableatthattime2,Na-Dene-speakingethnicgroups,furtherreferredtoasNa-Dene,wereconsidereddescendantsofthesecondmigrationwavefromBeringia.Geneticandarchaeologicaldataaccumulatedsince1986havegenerallysupportedthethree-wavemodel(reviewedbySkoglundandReich3).Thefirstmajormigrationstartedabout16,000yearsbeforepresent(YBP)andrapidlyspreadacrossNorthandSouthAmerica4.WerefertothedescendantsofthismigrationasFirstAmericans.Thesecond,Paleo-Eskimo,migrationassociatedwiththeSaqqaqandDorsetarchaeologicalcultures,whicharepartoftheArcticSmallTooltradition,ASTt,tookplaceabout4,800YBP,longaftertheBeringlandbridgehadbeeninundated5.ThethirdwaveassociatedwiththeThulecultureoccurredatabout1,000YBPandgaverisetomodernYupikinChukotkaandAlaskaandtoInuitthroughouttheAmericanArctic6.Theseethnicgroupsareoftenreferredtoas“Eskimo”,andfollowingRaghavanetal.6,wecallthismigrationNeo-Eskimo.BoththesecondandthethirdwaveswerelargelyrestrictedtotheAmericanArtic.ItwasshownthatgeneticcontinuitycharacterizedthePaleo-Eskimoperiodfromabout4,800to700YBP,andthatNeo-EskimosalsomaintainedgeneticcontinuityfromtheSiberianOldBeringSeaculture,2,200YBP,tomodernYupikandInuit6.Whileitisclearthatthefirstandthethirdwaveshaveleftmoderndescendants,itremainscontroversialwhetherthesecondmigrationwavewascompletelyreplacedbythethirdoneandwhetherithasleftgenetictracesinmodernNa-Dene3,6-8.Archaeologically,itisestablishedthatthelatestPaleo-EskimocultureswerereplacedbytheThuleculture,anditremainsuncertainwhethertheircontactandmaterialexchangewasextensive6,9,10.Accordingtoarecentreassessmentofradiocarbondating,thetemporaloverlapofPaleo-andNeo-Eskimoslasted50tomaximum200yearsintheeasternAmericanArctic6.Usingsinglenucleotidepolymorphism(SNP)arraydataforalargepanelofNativeAmericanpopulations,Reichetal.8haveshownthatChipewyans,aNa-Dene-speakingNorthernAthabaskanethnicgroup,derive16%oftheirancestryfromapopulationrelatedtothe4,000-year-oldSaqqaqancienthumangenomefromWestGreenland11,and84%oftheirancestryisderivedfromFirstAmericansrelatedtoAlgonquins.AhypothesisthatNorthernAthabaskansderivetheirancestryfromadmixtureofFirstAmericansandNeo-Eskimopopulationshasbeenrejectedbyastatisticaltest8.Otherstudieshavechallengedthisconclusion6,7.Basedongenome-widedatafromancientPaleo-andNeo-Eskimos,modernNa-DeneandotherNativeAmericans,theauthorsarguedthat:1/Dakelh,aNorthernAthabaskangroupfromBritishColumbia,hasnoPaleo-Eskimoancestry,butconsiderableadmixturefromNeo-Eskimoswasdetected;2/admixtureofSaqqaqandNeo-EskimoancestorshappenedinBeringiapriortotheirentryintoAmerica;3/Neo-EskimoscompletelyreplacedthePaleo-Eskimopopulationbyabout1300CE6,7.

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Inordertoshedlightontheseconflictingresults,inthisstudyweperformedadetailedreconstructionanddatingoftheNa-Denepopulationhistory,usinganextensivesetofpublicsequencingdata(Fig.1)andarecentlydevelopedcoalescentmethod,Rarecoal12,whichreliesonrareallelesandforwhichwedevelopedanewextensiontoincludeadmixture.Inparallel,wegeneratednewgenotypingdatafromSiberianpopulations,andinferredanddatedadmixtureeventsusingGLOBETROTTER,atoolrelyingonautosomalhaplotypedata13.Wealsoappliedmodel-freeapproachesforanalysisofrarealleleandautosomalhaplotypesharing.

ResultsDatasetcompositionandsampleinformationWecomposedalargesetofsequencingdatacoveringAfrica,Europe,SoutheastAsia,Siberia,andtheAmericas:1,206individualsfrom94populations,includingtheClovis14andSaqqaq11ancientgenomes(Fig.1,Suppl.Table1).Forthepurposeofhaplotypesharinganalysis,wecomposedtwoindependentSNParraydatasetscoveringthesamegeographicregions(Suppl.Table1):1/asetbasedontheHumanOriginsplatform(1,283individualsfrom101populations,includingSaqqaqandClovis);2/asetbasedonvariousIlluminaarrays(645individualsfrom63populations,includingSaqqaq).PropertiesofthedatasetsandtheirversionsusedforvariousanalysesaredescribedinSuppl.Table2.Wealsopresentnewdata,from58Siberianindividuals(Kets,Nganasans,Selkups,andEnets),whichweregenotypedforupto612,164autosomalSNPsontheHumanOriginsarrayplatform(seesampleinformationinSuppl.Table3).Forrareallelesharinganalysesrelyingongenomesequencingdata,wecombinedethnicgroupsintonon-overlappingmeta-populations(Suppl.Table1),sharingacommongeneticbackground:1/Sub-SaharanAfricans;2/ethnicgroupsofEuropeandtheCaucasus,excludingpopulationsmixedwithCentralAsiansorSiberians(seeMethods);3/SoutheastAsians;4/theArcticgroup–peoplesofBeringiaandtheAmericanArcticthatarederivedfromthethird,Neo-Eskimo,waveofAmerica’ssettlementandfrompopulationscloselyrelatedtoitsSiberiansource6;5/Siberians,excludinganypopulationsofthepreviousgroup;6/Na-Deneethnicgroups;7/agroupofnorthernNorthAmericans,otherthanNa-Dene,whicharegeneticallydistinctfrompopulationsfurthertotheSouth3,7,8;8/nativepopulationsofSouth,CentralAmerica,MexicoandsouthernUSA3,7,8.Forhaplotypesharinganalysesrelyingongenotypingdatawithlargerpopulationsizes,amorefine-grainedbreakdownwaspossible(Suppl.Table1).TheArcticgroupwassplitintotheSiberianandAmericanArcticsubgroups;SiberianswithextensiveancientNorthEurasianancestry15,16wereconsideredseparatelyandreferredtoasSiberians+ANE,whileotherSiberiangroupswerecalled‘coreSiberians’.Thebreakdownofethnicgroupsintothesemeta-populationswassupportedbyADMIXTUREanalysisonunlinkedSNPs(Suppl.Fig.1A,B)andbytheprincipalcomponentanalysis(PCA,seeSuppl.Fig.2A,B)andclusteringtrees(Fig.1,Suppl.Fig.3A,B)constructedbyfineSTRUCTURE17basedonautosomalhaplotypesharingpatterns.Meta-populationsmostrelevantforourstudyarethefollowing:Na-Denewith4high-coveragegenomes,32and

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48individualsintheHumanOriginsandIlluminaSNParraydatasets,respectively;northernNorthAmericans(3genomes,34and65genotypedindividuals);otherAmericans(34genomes,151and69genotypedindividuals);Arctic(18genomes,74and56genotypedindividuals)andSiberiangroups(27genomes,221and190genotypedindividuals).

Na-DenestandoutfromtheotherNativeAmericansTomeasuretherelationshipofNativeAmericanpopulationswithSiberianandArcticgroups,welookedforrareSNPallelessharedbetweenthem.Rarevariants,i.e.thoseoccurringataglobalfrequencyoflessthan1%,havebeenshowntohavemorepowertoresolvesubtlerelationshipsthancommonvariants12,18.Wecalculatedallelesharingcounts(ASC)andtheirstandarddeviationsforeachAmericanpopulation(amoderngrouporanancientgenome)andtheSiberianorArcticmeta-populations(Fig.2,seeMethodsfordetails).Totakecareofvariabilityingenomecoverageacrosspopulationsandofdataset-specificSNPcallingbiases,wenormalizedcountsofallelessharedbetweenanAmericangroupandSiberianorArcticmeta-populationsbysimilarcountsofallelessharedwithadistantoutgroup.EuropeansorAfricanswereusedasalternativeoutgroupsinthisstudy.Sinceweexpectedadecayofarecentancestrysignalathigherallelefrequencies12,allstatisticswerecalculatedseparatelyforallelesofvariousfrequencies:occurring2,3,4,…andupto20timesamong2,412haploidchromosomesets,whichcorrespondstofrequenciesfrom0.08%to0.83%.TheSaqqaqancientindividualandNorthernAthabaskans(ChipewyansandDakelh)clearlystandoutfromotherAmericans,accordingtobothSiberianandArcticrelativeallelesharing(Fig.2).ThisresultisexpectedforSaqqaq,sinceitscloserelationshiptotheArcticandSiberiangroupswasshownbyvariousmethods6,7,11,15.Wealsonotethatinouranalysisthe12,600-years-oldClovisancientgenome14doesnotdifferfrommodernSouthandCentralAmericanpopulations.ThesameresultswereobtainedwithAfricansasthenormalizer(Suppl.Fig.4A-B),andwhenonlyprivate(i.e.exclusivelysharedbetweentwometa-populations)alleleswerecounted(Suppl.Fig.4C-F).Asexpected,privatelysharedalleleswerelargelyrestrictedtothelowest-frequencybins:allelecountsfrom2to5,correspondingtofrequenciesfrom0.08%to0.21%.WenotethattheSiberianandArcticsignalsarestrongerintheDakelhgenomes(twoindividuals6,7)ascomparedtotheChipewyangenomes(twoindividuals19),althoughbothpopulationsaresignificantlydifferentfromotherAmericansatlowallelefrequencies.ForDakelh,thesignalisobservedforallelecountsupto10(0.42%frequency,seeFig.2,Suppl.Fig.4A,B),whichsuggestsrelativelyrecentgeneflow12fromSiberianand/orArcticpopulationsintoNorthernAthabaskans.Weinvestigatethesourceofthisgeneflowandattemptitsdatinginthefollowingsections.

Na-DenebelongtothePaleo-EskimowaveofAmerica’ssettlementWhilewehaveshownthatNorthernAthabascanshaveelevatedSiberianandArcticrareallelesharingcomparedtoallotherNativeAmericansinvestigated,thisdoesnotimmediatelysuggestthattheydescendfromthesecond,Paleo-

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Eskimo,settlementwave8vs.thethird,Neo-Eskimo,one6,7.Therefore,wecombinedtheSiberianandArcticallelesharingstatisticsonatwo-dimensionalplotshowingeachAmericanpopulation(Fig.3).Forthatpurpose,wesummedupallelesharingstatisticsforallelecountsfrom2to5–thosedemonstratingmostprominentsignals(Suppl.Fig.4).Forcomparison,wealsogeneratedthesamestatisticswithatake-one-outprocedureforSiberiansandforrepresentativesoftheAmericanArcticgroup(Fig.3).Weobservethateachmeta-populationisscatteredalongalineontheplot,whichreflectssimilarratiosoftheSiberianandArcticallelesharingamongitsmembers(Fig.3A).Thepositionofapopulationalongthelinedependsonthepresenceofotherancestrycomponents.Forexample,Aleuts,havingahighlevelofEuropeanadmixture6,7(seealsoSuppl.Fig.1),liemuchclosertozeroascomparedtotheotherAmericanArcticgroups(Fig.3B).WhileFirstAmericanpopulationsformatightcluster,theDakelhandChipewyanpopulationsareshiftedconsiderablytowardstheSaqqaqindividual.Sinceallelesharingcountsbehavelinearlyunderadmixture,weusedlinearcombinationstocalculateexpectedrelativeallelesharingstatisticsforrecentlyadmixedpopulations:mixturesofFirstAmericanswitheithertheSaqqaqindividualorpopulationsofthethirdmigrationwave.WeusedallmodernFirstAmericans,theGreenlanderInuitandtwoChukotkanYupikthird-wavepopulationsforthissimulation,andassumed70%,75%,…and90%ofFirstAmericanancestryintheadmixedpopulations.NormalizedallelesharingcountsfortheDakelhpopulationmatchthoseforamixedSaqqaq/FirstAmericanpopulationandareclearlydifferentfromthoseofanysimulatedNeo-Eskimo/FirstAmericanmixtures,i.e.areseparatedfromthelatterclusterbymorethanthreestandarderrorintervals(Fig.3B).ThisresultisconsistentwithPaleo-EskimosbeingancestorsofNa-Dene.However,theChipewyanpopulationlieswithintheclusterofthefirstandthirdwavemixtures.UsingAfricansforthenormalization(Suppl.Fig.5A,B,E,F)and/orcountingprivateallelesgivessimilarresults(Suppl.Fig.5C-F).ToinvestigateawiderdiversityofNa-DeneandothernorthernNorthAmericanpopulations,weappliedasimilaranalysisstrategytoadifferenttypeofdata:toautosomalhaplotypesharingstatisticsontheHumanOriginsandIlluminaSNParraydatasets(Suppl.Tables1and2).CumulativelengthsandcountsofsharedautosomalhaplotypeswereproducedwithChromoPainterv.1forpairsofindividuals,intheformofallvs.all“coancestrymatrices”17,thenAmerican-SiberianorAmerican-ArctichaplotypesharingstatisticswerecalculatedforeachAmericanindividualandnormalizedusingadistantoutgroup(seeMethodsfordetails).Two-dimensionalplotsshowingSaqqaq,Na-Dene,andotherrelevantmeta-populationsappearinFig.4andSuppl.Fig.6fortheHumanOriginsdataset,andfortheIlluminadatasetinSuppl.Figs.7and8.MostnorthernNorthAmericanethnicgroupshavepost-ColumbianEuropeanadmixture,highlyvariableamongindividuals7,20.ThesamepatternwasobservedinbothofourdatasetswithADMIXTURE(Suppl.Fig.1),andintheIlluminadatasetanumberofnorthernNorthAmericanandNa-DeneindividualsformedacladewithEuropeans,whileothersclusteredwithSouthAmericans(Suppl.Fig.3B).Moreover,theSiberianandArcticgroupshaveconsiderable

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Europeanadmixture,datedto2,200-2,400YBPintheformer(seeTable1andSuppl.Text1).Thus,weexpectedthepost-ColumbianEuropeanancestryinnorthernNorthAmericansandNa-DenetobiastheSiberianandArctichaplotypesharingstatisticsupwards.Tomitigatethispotentialbias,wepreferredtousehaplotypesharingwithEuropeansasanormalizerandplottedstatisticsforindividuals,sinceweexpectedhighlyvariablelevelsofEuropeanancestryinNorthAmericans.EssentiallysimilarresultswereproducedonbothSNParraydatasets,usingbothEuropeanandAfricannormalizers(Fig.4,Suppl.Figs.6,7,8).HaplotypesharingstatisticsinbothDakelhindividualsandinafractionofChipewyans(3of30)matchthoseofsimulatedFirstAmericanpopulationshavingfrom~20%to~30%ofSaqqaqancestry,andaredifferentfromthoseofanyFirstAmerican/Neo-Eskimomixtures.TenChipewyanindividualsarelessshiftedfromtheclusterofFirstAmericanindividuals,andtheirhaplotypesharingstatisticsmightbeexplainedeitherbyaproportionofSaqqaqancestryofabout10-20%orbyverylowlevelsofChukotkanYupikancestry(<<5%,seeFig.4B).BesidesNorthernAthabaskans,othermajorbranchesoftheNa-DenelanguagefamilywererepresentedintheIlluminaSNParraydataset,namelySouthernAthabaskansfromUSAandTlingitfromwesternCanada(Suppl.Table1).FourDakelh,threeotherNorthernAthabaskans,sixTlingit,oneSouthernAthabaskan,andonlyonenon-Na-Deneindividual(1of7Splatsin)showedasignalofPaleo-Eskimo(Saqqaq)admixture(Suppl.Fig.7B).Notably,oneNorthernAthabaskanindividual(population‘NorthernAthabaskan3’accordingtoRaghavanetal.7)correspondedtoa~30%mixtureofSaqqaqand~70%ofFirstAmericans(Suppl.Fig.7B).AthirdofNorthernAthabaskans(7of20)showedapronouncedsignalofPaleo-Eskimoadmixture,whilemostinvestigatedTlingit(17of23)andSouthernAthabaskans(4of5)didnotshowaclearsignal.However,haplotypesharingstatisticsoffewotherNa-DeneandnorthernNorthAmericanindividualsarecompatiblewithlowlevelsofPaleo-Eskimo(~10%)orInuitancestry(~5%)(Suppl.Fig.7B).OfthefourDakelhwithasignalofPaleo-Eskimoancestry,twoindividualshadcorrespondinggenomesequencingdata6,andtheyhavedemonstratedconsistentresultsthroughoutallanalyses:onsequencingdata(Fig.3,Suppl.Fig.5),onsequencingdatamergedwiththeHumanOriginsSNParray(Fig.4,Suppl.Figs.3A,6),andonIlluminaSNParrays(Suppl.Figs.3B,7,8).Insummary,ourmodel-freeapproachtoanalyzerarealleleandhaplotypesharingrevealsthatafractionofNa-DeneNativeAmericanslikelyhasaconsiderableproportionofPaleo-Eskimoancestry,roughlyfrom10to30%.VirtuallynootherNativeAmericansdemonstratedthesamesignalinouranalysis,despitealargenumberofpopulationsandindividualsinvestigated(37genomesand319genotypedFirstAmericans).

DemographicmodelinganddatingofpopulationmixturesTointerpretourfindingsinamorequantitativeway,webuiltanexplicitdemographicmodelforthepeoplingofNorthAmerica.WeusedRarecoal12toestimatesplittimesandpopulationsizes,aswellasadmixtureevents,inapopulationtreeconnectingEuropeans,SoutheastAsians,Siberians,populations

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oftheNeo-Eskimomigrationwave,NorthernAthabaskans,andNativeSouthAmericans.SamplesizesandadditionaldetailsareprovidedinSuppl.Text1.Rarecoalisasoftwarethatimplementsafastalgorithmtoestimatethejointsitefrequencyspectrumforrareallelesinhundredsofsamples12.Sincetheinitialreport,wehaveimprovedthesoftwareandaddedpulse-likeadmixtureeventsasanewfeature(seeMethods).Themodelwasderivedinaniterativeway:westartedoffwithfittingamodeltothreepopulationsonly(Europeans,SoutheastAsians,andSouthAmericans),andthenaddedonepopulationatatime,re-estimatingallpreviousandnewparameters(seedetailsinSuppl.Text1).Admixtureedgeswereaddedwhenthemodelfitshowedsignificantdeviationsforparticularallelesharingstatistics.Thefinalmodel(Fig.5A,Table1)containssixclades,fourunidirectionaladmixtureedgesandthreebidirectionaledgeswithasymmetricadmixturerates.TheparameterestimatesincludingconfidenceintervalsforthisfinalmodelareshowninTable1.Substantialadmixtureof22.3–23.8%fromSiberians(22genomes)intoNorthernAthabaskanswasrevealedinourmodel,withonly6.5–7%intheoppositedirection(95%confidenceintervalsaregiven).TheSiberian-Athabaskanadmixtureedgewasdatedat6,575–7,030YBP(Table1).AsimplermodelwithouttheAmericanArcticmeta-population(Suppl.Text1)datedtheSiberian-Athabaskanadmixtureat~4,400YBP,whichlikelycorrespondstotheadmixtureofPaleo-EskimosandFirstAmericansthatmustpostdatethePaleo-Eskimoimmigrationaround4,800YBP5.AdmixturewasalsoinferredbetweentheAmericanArcticgroupsandAthabaskans,however,withamuchsmalleradmixtureproportionof6.3–8.5%intoAthabaskansand10.9–12.4%intheoppositedirection,andamuchlaterdateof476–499YBP(Table1).Wecautionthattheassumptionofconstantpopulationsizeswithinbranches,whichisnecessarytokeepthenumberofparametersmanageable,mayleadtooverlynarrowconfidenceintervalsofourestimates.InordertoassesswhethertheSiberianadmixtureinferredinAthabaskansisalsopresentinothernorthernNorthAmericans,wetestedthefinalmodelshownaboveonadatasetwhereAthabaskansarereplacedwithnon-Na-DenespeakingCree(2genomes)andTsimshian(1genome).Onthisdata,westillestimate~10%SiberianadmixtureintonorthernNorthAmericans(comparewith23%fromSiberiansintoAthabaskans).However,thetimeofthisadmixtureevent(~600YBP)isextremelyrecent,andmoreoveraftertheEuropeanadmixtureeventintoSiberians(Fig.5A,Table1).WethinkthatthismayreflectrecentadmixturebetweenAthabaskansandotherNorthernAmericans.Inanycase,thesignalisweakerandtoorecenttoreflectthesamehistoricaladmixtureeventthatisseenintheAthabaskans.Totesttherobustnessofourestimateswesimulatedthefinalsix-populationmodelwiththeAthabaskansunderthecoalescentwithrecombination(seeSuppl.Text1).WethenestimatedparametersfromthesimulateddatausingRarecoalandcheckedwhethertheinferredparametersmatchthesimulatedparameters.TheresultsaresummarizedinSuppl.Fig.1.15.Ascanbeseen,most

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parametersareestimatedveryaccurately,inparticularalltimeestimatesofsplitsandadmixtureevents.SubstantialdeviationbetweenasimulatedandestimatedparameterisseeninthepopulationsizeestimateoftheSiberianbranch,aswellastheancestralbranchofSiberiansandtheAmericanArcticgroups.ThiscouldreflectsubstructureinourSiberianand/orAmericanArcticmeta-populations,whichwasnotpartofthesimulationbutcouldhaveaneffectonmodelestimates.Finally,weattemptedtomapthehigh-coverageSaqqaqandClovisancientgenomesontothemodeledtree.Itishardlypossibletoincorporatesingleindividualsfullyintothemodel,andlowsequencingcoverageofotherPaleo-Eskimogenomesavailable6makesthemmuchlesssuitableforouranalysis.Instead,weevaluatedthelikelihoodofasample’sbranchtomergeontothetree,testingalltimepointsonallbranches,beforetheageofthesample.TheSaqqaqgenome11mostlikelybranchesoffthetreeeitherbeforethesplitofAthabaskansandSouthAmericans,orattheAthabaskanbranchimmediatelyafterthegeneflowfromSiberians(Fig.5B).Thebranchingpointofthe12,600-years-oldClovisgenome14fitsitsexpectedpositionatthebaseoftheAmericanclade(Fig.5C).ThesomewhatsurprisingclusteringoftheSaqqaqgenomeontotheNativeAmericanancestralbranchinourRarecoalanalysismayreflectsubtledifferencesbetweenSaqqaqandtheextantSiberiansandAmericanArcticpopulationsusedforconstructingthemodel(Suppl.Text1).Inallpreviousanalyses6,11,15andinhaplotype-basedanalysesinthisstudy(Fig.1,Suppl.Fig.3B),SaqqaqclusteredwitheithercoreSiberianorSiberianArcticgroups,probablyreflectingthefactthatitbranchedofftheSiberianstempriortotheseparationofthemoderngroups(theChukchi-Saqqaqdivergencewasdatedat4,400–6,400YBP11).ThebranchpointinferredbyRarecoalprobablyreflectsaSiberianancestorofSaqqaq,thatisclosertoNativeAmericanancestorsthantotheancestorsoftheSiberianandAmericanArcticsamplesusedhere.Theotherhigh-likelihoodbranchpointforSaqqaq,ontheAthabaskanbranchaftertheSiberianadmixtureevent,suggeststhattheSiberian-AthabaskangeneflowmodeledherewasmediatedbyPaleo-Eskimos.Inanycase,Paleo-EskimosrepresentthemostlikelyvectorforanyrelativelyrecentgeneflowfromSiberiathatpre-datestheNeo-Eskimomigrationaround1,000YBP,sincenootherancientAmericangrouphasbeenshowntopossessdetectablelevelsof“coreSiberian”ancestry6.Substantiatingthisconclusion,anadmixtureeventbetweenSaqqaqandFirstAmericanswasrevealedinthehistoryofNorthernAthabaskansusingGLOBETROTTER,ahaplotype-basedtoolcapableofinferringanddatinguptotwodistinctadmixtureevents13.GLOBETROTTERoperatesoncoancestrycurves,generatedfromChromoPainterv.213(seeMethodsfordetails).Inouranalysis,two-datecurvesfittheNa-Denedatabetterascomparedtoone-datecurves(Table2,Suppl.Fig.9).GLOBETROTTERalsofindstheclosestproxiesofadmixturepartnersinagivendatasetanddeterminesadmixtureratios.SaqqaqandFirstAmericanswererevealedasmostlikelyadmixturepartners,withSaqqaqcontributioninthe19%–25%range,dependingonadataset(seeTable2),ingoodagreementwiththeRarecoalresultsabove.Usingmeta-populations

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ashaplotypedonorsandfiveNorthernAthabaskansweexpectedtobeadmixedwithPaleo-Eskimos(Fig.4),theSaqqaqadmixturewasdatedat~3,600YBPwitha95%confidenceintervalof488–4,614YBP(Table2).

DiscussionOurresultsareconsistentwithageneflowfromtheSaqqaqPaleo-Eskimos(19–25%admixtureratio)exclusivelyintotheFirstAmericanancestorsofNa-Dene,andamuchlaterandlessextensivebidirectionalgeneflowwasdetectedbetweentheNa-DeneandNeo-Eskimobranches.AsomewhatlowerlevelofSaqqaq-relatedancestryof16%wasreportedusingtheadmixturegraphmethodinChipewyans,aNorthernAthabaskanethnicgroup8.WeemphasizethatonlyafractionofmodernNa-DeneindividualsdisplaysthislevelofSaqqaqancestry,withmostNa-Denebeingadmixedwithothernativegroupsand/orEuropeans.Methodsofrarealleleandautosomalhaplotypeanalysisareespeciallysensitiveforreconstructingrecentpopulationhistorywithinafewthousandyears,andinsomecasesweredemonstratedtooutperformtraditionalmethods12,13,30basedonunlinkedcommongeneticvariants,suchasADMIXTURE,PCA,TreeMix,f3-,f4-,andD-statistics.Inthislight,weconsiderdiscrepanciesbetweenourresultsandthoseofpreviousstudiesinSuppl.Text2.WedatedthePaleo-Eskimoadmixtureatabout3,600YBPusingGLOBETROTTERanalysisbasedonautosomalhaplotypes(Table2).MucholderdatesoftheSiberian-Athabaskangeneflowobtainedinouranalysisbasedonrareallelesharing,about6,500-7,000YBP(Fig.5,Table1),probablycorrespondnottotheadmixtureofPaleo-EskimosandFirstAmericans(thatmustpostdatethePaleo-Eskimoimmigration),buttoatimepointwhenPaleo-EskimoancestorsbranchedofffromtheSiberian-Arcticstem.Thesplitdatesuggestedhereforthisunsampled“ghostpopulation”fitsthearchaeologicalrecordofSiberiaremarkablywell,asdiscussedbelow.Andsplitdatesforothernodesinferredherebroadlyagreewiththedatesproducedwithindependentmethods4,7.ThenewwaveofpopulationfromnortheasternAsiathatarrivedinAlaskaatleast4,800yearsago5displayscleararchaeologicalprecedentsleadingbacktoCentralSiberia.TheriseoftheSyalakhculturethatflourishedacrossmuchofNortheasternSiberiabetween6,500and5,200YBPinvolvedmigrantsfromtheTransbaikalareawhopossiblymixedwithlocalremnantsoftheearlierSumnaginculture(10,500-6,500BP),bringingthebowandarrowandnewtypesofpotterytoNortheasternSiberia21,22.AstheBel’kachiculture(5,200-4,100YBP)developedfromSyalakhalongtheLenaandAldanrivers23,atleastonegroupofthesepeoplemighthavecrossedtheBeringStraitintoAlaskaaround4,800YBP5,givingrisetoPaleo-Eskimos.Thus,theSyalakhculturepeoples,spreadingacrossSiberiaafter6,500YBP,mightrepresentthe“ghostpopulation”thatsplitoffaround6,500-7,000YBPandlatergaverisetomigrantsintoAmerica.

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ThegeographicconnectionbetweenPaleo-EskimosandtherelatedSiberiangroupsprobablybecameseveredassubsequentwavesofhunter-gatherersenteringEasternSiberiafromthewestduringtheLateNeolithic(Ymyakhtakhculture,3,700-2,800YBP)broughtnewculturesandnewlanguagegroups24.ThisphaseofNorthAsianprehistorymostlikelyinvolvedthespreadofYukaghir,Chukchi-KamchatkanandEskimo-Aleutlanguages25,whosepresenceintheextremenortheastofAsiaintervenedgeographicallybetweenPaleo-Eskimos,Na-Dene,andtheirOldWorldcousins.Notably,thedatesoftheSiberian-Arcticsplitobtainedunderourmodel(~4,000-4,200YBP,Table2)alsoagreewiththisscenariothatlinksthespreadoftheYmyakhtakhculture(after3,700YBP)withtheArcticmeta-population,i.e.ancestorsofmodernChukchi-KamchatkanandEskimo-Aleutethnicgroups.ThesuccessofPaleo-EskimosandNa-DeneinoccupyingterritoriespreviouslypopulatedbyFirstAmericans5,28,insomecases(SouthernAthabaskans)movingveryfarfromtheoriginalhomelandinAlaskaandnorthwesternCanada,mightbepartiallyattributedtoarchery,atechnologicaladvancelackingamongthelocalpopulations.Paleo-EskimosquicklyspreadfromAlaskatoGreenlandandLabradorandhavebeencreditedwithintroducingthebowandarrowtopopulationsinEasternCanadaby4,000YPB29,thoughtheDorsetpeople,thelastwaveofPaleo-Eskimos,seemtohavegivenupthistechnologyforhandheldlances10.AnotherimportantobservationconcernsthedistributionofSiberian(Paleo-Eskimo)ancestryamongmodernNorthAmericans.ThemethodsusedinthisstudydetectedCentralandWestSiberianancestryinafractionofNa-Deneindividualsbelongingtoallmajorbranchesofthelanguagefamilyexistingtoday:Tlingit,NorthernAthabaskan(Chipewyans,Dakelh,etc.)andSouthernAthabaskan.Importantly,theCentralandWestSiberianancestryisalmostexclusivetoNa-Dene,andmissinginotherNorthorSouthAmericannativeethnicgroups,includingHaida20,agrouppreviouslyconsideredadivergentmemberoftheNa-Denelanguagefamily26.Thus,thecurrentconsensusviewoftheNa-Denelanguagefamily27andthedistributionofrecentSiberianancestrymatchremarkablywell.Althoughthesmallpopulationsizesdonotallowstatisticallyvalidcomparisons,individualswithnoticeableSaqqaqancestryarelikelymorefrequentamongNorthernAthabaskansascomparedtoTlingitandSouthernAthabaskans,thelatterbeingmixedwithsouthernNativeAmericans(Suppl.Fig.1B).Wespeculatethatamigratingpopulation,startingfromSiberiaaround6,500YBP(theSyalakhculture),enteringtheNewWorldaround4,800YBP,andlatermixingwithFirstAmericans,mighthavecarriedtheDene-Yeniseianlanguages31-36intoNorthAmerica.ThishypotheticallanguagemacrofamilyunitesmultipleNa-DenelanguagesandKet,theonlysurvivingremnantoftheYeniseianfamily,oncewidespreadinSouthandCentralSiberia33,37-40.ForafurtherdescriptionoftheDene-YeniseianhypothesisandareviewoflexicostatisticaldatingestimatesseeSuppl.Text3.AlthoughtheDene-Yeniseianmacrofamilyisnotuniversallyacceptedamonghistoricallinguists41,42(cf.Hamp43),andcorrelationoflinguisticandgenetichistoryisfarfromuniversal,theexistenceoftheexclusiveSiberian-

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Denegeneflowmakesagenealogicalrelationshipofthelanguagefamilies,eitherastheclosestsister-groups35orwithinawiderclade42,anattractiveareaoffutureresearch.Inferredageofthegeneflow,6,500-7,000YBP,possiblycorrespondingtothesplitofNa-DeneandYeniseianprecursorsinSiberia,iscomparabletotheageoftheclassicIndo-Europeanlanguagefamily44,45,suggestingthatinvestigationoftheDene-Yeniseianconnectionlieswithinthereachofcurrentmethodsinhistoricallinguistics.

Methods

SamplecollectionandDNAextractionof58newlyreportedsamplesSalivasamplesoffourSiberianethnicgroups(Enets,Kets,Nganasans,Selkups)werecollectedandDNAextractionswereperformedasdescribedinFlegontovetal.15SamplinglocationsandadditionalinformationisprovidedinSuppl.Table3.

DatasetpreparationInordertoanalyzerareallelesharingpatterns,wecomposedasetofsequencingdatacoveringAfrica,Europe,SoutheastAsia,Siberia,andtheAmericas:1,206individualsfrom94populations(Suppl.Table1).Threesourceswereutilizedtoassemblethegenomedataset:theSimonsGenomeDiversityProject19,Raghavanetal.7,andthe1000GenomesProject18.Weusedvariantcallsgeneratedintherespectivepublications,keptbiallelicautosomalSNPsonlyandappliedafilterbasedonamappabilitymask12.Additionally,weassembledtwoindependentSNPdatasets:seedatasetcompositionsandfiltrationsettingsinSuppl.Tables1and2.Initially,weobtainedphasedautosomalgenotypesforlargeworldwidecollectionsofAffymetrixHumanOriginsorIlluminaSNParraydata(Suppl.Table2),usingShapeItv.2.20withdefaultparametersandwithoutaguidancehaplotypepanel46.Thenweappliedmissingratethresholdsforindividuals(<50%or<51%)andSNPs(<5%)usingPLINKv.1.90b3.3647.Forsomeanalyses,unlinkedSNPswereselectedusinglinkagedisequilibriumfilteringwithPLINK(Suppl.Table2).Tenprincipalcomponents(PC)werecomputedusingPLINKonunlinkedSNPs,andEuclideandistancesdefinedas:

! ", $ = "& − $& ( + "( − $( ( + "* − $* ( + ⋯+ ", − $, (werecalculatedamongindividualswithinpopulations(qnandpnrefertoPCsfrom1to10inapopulation).WeremovedoutliersaccordingtotheEuclideandistances,andpopulationshavingonaverage>5%oftheSiberianancestralcomponentaccordingtoADMIXTURE48analysis(Suppl.Fig.1),e.g.FinnsandRussians,wereexcludedfromtheEuropeanandSoutheastAsianmeta-populations.InthecaseoftheIlluminaSNParraydataset,Na-DenepopulationswereexemptfromPCAoutlierremovalandfromremovalofsupposedrelativesidentifiedbyRaghavanetal.7ItwasdonetopreservemaximaldiversityofNa-DeneandtoensurethatbothDakelhindividualswithsequencingdataavailablewouldbeincluded.Finally,weselectedrelevantmeta-populations,generatingdatasetsof567-1,283individualsfurtheranalyzedwithADMIXTURE48,

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ChromoPainterv.1andfineSTRUCTURE17,ChromoPainterv.2andGLOBETROTTER13(Suppl.Tables1and2).

RareallelesharingstatisticsWedefinetheAlleleSharingCountbetweenpopulationsAandB(ASCABorCA,B)astheaveragenumberofsitesatwhichanindividualfrompopulationAsharesaderivedalleleoffrequencykwithanindividualfrompopulationB:

-.,/ 0 = 143.3/

!.,4!/,4567,84

where3isthenumberofindividualsinthepopulations,dA,istandsforthenumberofderivedallelesatsiteiinpopulationA,andtheterm59,:equals0ifthetotalcountofderivedallelesinthedatasetdoesnotequalk,andis1otherwise.Thesumacrossallsitesiisnormalizedbytheproductofpopulationsizesmultipliedbyfourtogivetheaveragenumberofsharedallelesbetweentworandomlydrawnhaploidchromosomesets.Insteadofcountingderivedalleles,inpracticewecountednon-referencealleles,whichshouldnotmakeadifferenceforlowfrequencies.Totakecareofvariabilityingenomecoverageacrosspopulationsandofdataset-specificSNPcallingbiases,wecalculatednormalizedallelesharingcountsforpopulationsAandB,dividingASCABbyASCAC,wherepopulationCisadistantoutgroup.BecauseweassumethatmutationsoccurasaPoissonprocess,thestandarddeviationofASCABisdefinedas:

∆-.,/ 0 = 1<.,/ 0

-.,/ 0

<.,/ 0 isthenumberofsitesi,atwhichderivedallelesoccurktimesinthedataset.ThestandarddeviationofASCAB/ASCACiscalculatedusingerrorpropagationviapartialderivatives:

∆=>-.,/ =>-.,? 0 = ∆-.,/ 0-.,? 0

(+ -.,/ 0

-.,? 0 ( ∆-.,? 0(

Inpractice,populationAwasanAmericanpopulationorancientgenome,populationBwasrepresentedbySiberianorArcticmeta-populations,andpopulationC–byAfricansorEuropeans(Suppl.Table1).TheresultingstatisticsarereferredtoasrelativeSiberianorArcticallelesharing.SimilarstatisticswerecalculatedforvariousSiberianandArcticpopulationsusingtheleave-one-outprocedure.AllelesharingstatisticswerealsocalculatedforprivateallelesandnormalizedbyregularAfricanorEuropeanASCs.Wecalledasharedalleleprivate,orexclusivelyshared,ifitwaspresentinanAmericanpopulationandSiberiansormembersoftheArcticgroup,butmissinginallothermeta-populations(wedidnotconditiononthepresenceofthisalleleinotherAmericans).

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HaplotypesharingstatisticsSharedhaplotypelength(SHLAB)isdefinedasthetotalgeneticlengthofDNA(incM)thatagivenrecipientindividualAicopiesfromadonorindividualBjunderthemodel13,17.SHLABwascomputedintheallvs.allmannerbyChromoPainterv.117runningwithdefaultparameters.ForeachindividualofarecipientpopulationA(inpracticeanAmericanindividual),SHLABvalueswereaveragedacrossallindividualsofadonorpopulationB(theSiberianorArcticmeta-population),andthennormalizedbythehaplotypesharingstatisticSHLACfortheEuropeanorAfricanoutgroupC.TheresultingstatisticsSHLAB/SHLACarereferredtoasSiberianorArcticrelativehaplotypesharing,andwerevisualizedforseparateindividuals.SimilarstatisticswerecalculatedforSiberianandArcticindividualsusingtheleave-one-outprocedureatthepopulationlevel.

DatingadmixtureeventsusinghaplotypesharingstatisticsWeusedGLOBETROTTER13toinferanddateuptotwoadmixtureeventsinthehistoryofNa-Denepopulations.Todetectsubtlesignalsofadmixturebetweencloselyrelatedpartners,wefollowedthe‘regional’analysisprotocolofHellenthaletal.13UsingChromoPainterv.213,Na-Denechromosomeswere‘painted’asamosaicofhaplotypesderivedfromdonorpopulationsormeta-populations:theSaqqaqancientgenome,SiberianArcticpopulations,AmericanArcticpopulations,northernNorthAmericans,otherAmericans,coreSiberians,SiberianswithANEancestry,SoutheastAsians,Europeans.Na-Deneindividualswereconsideredashaplotyperecipientsonly,whileotherpopulationsormeta-populationswereconsideredasbothdonorsandrecipients.ThatisdifferentfromtheChromoPainterv.1approach,whereallindividualswereconsideredasdonorsandrecipientsofhaplotypesatthesametime,andonlyself-copyingwasforbidden.PaintingsamplesforNa-Dene(thetargetpopulation)and‘copyvectors’forother(meta)populationscalled‘surrogates’servedasaninputofGLOBETROTTER,whichwasrunaccordingtosection6oftheinstructionmanualofMay27,2016.Thefollowingsettingswereused:nostandardizingbya“NULL”individual(null.ind0);fiveiterationsofadmixturedateandproportion/sourceestimation(num.mixing.iterations5);ateachiteration,anysurrogatesthatcontributed≤0.1%tothemixturedescribingthetargetpopulationwereremoved(props.cutoff0.001);thex-axisofcoancestrycurvesspannedtherangefrom0to50cM(curve.range150),withbinsof0.1cM(bin.width0.1).Confidenceintervals(95%)foradmixturedateswerecalculatedbasedon100bootstrapreplicateswithoptionsnull.ind0andnum.admixdates.bootstrap2(fittingtwodateswhenperformingbootstrapping).Generationtimeof29yearswasusedinalldatingcalculations7.TheGLOBETROTTERsoftwareisabletodatenomorethantwoadmixtureevents13,thereforewehadtoreducethecomplexityoforiginalNa-Denepopulationsthatlikelyexperiencedmorethantwomajorwavesofadmixture.Forthatpurpose,onlyasubsetofNa-DeneindividualswasusedfortheGLOBETROTTERanalysis:individualsdemonstratingasignalofPaleo-EskimoadmixtureandalowlevelofEuropeanancestryaccordingtoSiberianandArctichaplotypesharingstatisticswiththeEuropeannormalizer.Inpractice,Na-Dene

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individualslyingintheareaofthetwo-dimensionalplotoccupiedbysimulatedmixturesofthe1stand2nd,butnotthe1stand3rdmigrationwaves(Fig.4),weretreatedasone‘target’population.Thisdefinitionofatargetpopulationwasusedwithmeta-populationsashaplotypedonors.Toincreasetheamountofdatawhenseparatepopulationswereusedforcalculatingcoancestrycurves,weincluded8additionalChipewyanindividualswithevidenceoflow-levelPaleo-orNeo-Eskimoadmixture,i.e.lyingintheareaofthetwo-dimensionalplotwherethetwoclustersofsimulatedmixturesoverlap(Fig.4).

RarecoalanalysisWeusedtheRarecoalprogram(https://github.com/stschiff/rarecoal)tofitdemographicmodelstometa-populations,iterativelyaddingonepopulationatatime(Suppl.Text1).WestartedwithatreeconnectingEuropeans,SoutheastAsians,andNativeAmericansintoasimpletreewithoutadmixture,andused“rarecoalmcmc”toinfermaximumlikelihoodbranchpopulationsizesandsplittimes.Wetheniterativelyaddedadditionalpopulations,andaftereachaddition,were-optimizedthetreeandinspectedthefitsofthemodeltothedata.Whenwesawasignificantdeviationbetweenmodelanddata,weaddedadmixtureedges,informedbytheunder-orover-estimationofaparticularsharingpattern(Suppl.Text1).AfterRarecoal’sinference,werescaledtimeandpopulationsizeparameterstoyearsandrealeffectivepopulationsizeusingamutationrateof1.25´10-8persitepergeneration,andagenerationtimeof29years7.Thefinalmodel,asshowninFigure5,wasthenalsosimulatedusingtheSCRMsimulator49,andweverifiedthatRarecoalwasabletoinferthetrueparametersaftersimulation.Inordertomapthetwoancientgenomes,SaqqaqandClovis,ontothetree,werestrictedtheanalysistovariantsbetweenallelecounts2and4.Weexcludedsingletons,becausetheyarehighlyenrichedforfalsepositivesinancientgenomes,andmainlyusedforpopulationsizeestimation,whichwearelessinterestedininthecaseofancientsamples12.Weused“rarecoalfind”toevaluatethelikelihoodformergingontothetreeatallbranchesandalltimes(afterthedateofthesample).

ADMIXTUREanalysisTheADMIXTUREsoftware48implementsamodel-basedBayesianapproachthatusesblock-relaxationalgorithminordertocomputeamatrixofancestralpopulationfractionsineachindividual(Q)andinferallelefrequenciesforeachancestralpopulation(P).Agivendatasetisusuallymodeledusingvariousnumbersofancestralpopulations(K).WeranADMIXTUREonHumanOrigins-basedandIllumina-baseddatasetsofunlinkedSNPs(Suppl.Table2)using10to25and5to20Kvalues,respectively.Onehundredanalysisiterationsweregeneratedwithdifferentrandomseeds.Thebestrunwaschosenaccordingtothehighestlikelihood.AnoptimalvalueofKwasselectedusing10-foldcross-validation(CV).

fineSTRUCTURE:PCAandclusteringWeusedfineSTRUCTUREv.2.0.7withdefaultparameterstoanalyzetheoutputofChromoPainterv.117.Clusteringtreesofindividualsweregeneratedby

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fineSTRUCTUREbasedoncountsofsharedhaplotypes17.TheclusteringtreesandcoancestrymatriceswerevisualizedusingfineSTRUCTUREGUIv.0.1.017.Finally,PCAwasgeneratedbasedoncountsofsharedhaplotypesandvisualizedusingR.

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AcknowledgementsWearegratefultoallresearchersthatsharedtheirdata:DavidReich,NickPatterson,IainMathieson,SwapanMallick,MaanasaRaghavan,SimonRasmussen,andEskeWillerslev.WealsothankDavidReichforhelpfulcommentsandforcuratingthenewlyreportedHumanOriginsgenotypingdata.P.F.wassupportedbytheInstitutionDevelopmentProgramoftheUniversityofOstravaandbyEUstructuralfundingOperationalProgrammeResearchandDevelopmentforInnovation,projectNo.CZ.1.05/2.1.00/19.0388.AuthorcontributionsP.F.andS.S.havedesignedthestudy,analyzedthedataandwrittenthemanuscript;N.E.A.andP.C.haveanalyzedthedataandpreparedthefiguresandtables;E.J.V.hascontributedthesectionsdealingwithNa-Denelinguisticsandarchaeology;andJ.K.wasinvolvedinsamplegenotypingandinmanuscriptpreparation.

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CompetingFinancialInterestsTheauthorsdeclarenoconflictingfinancialinterests.FigurelegendsFig.1.Genomesequencingdatausedforrarealleleanalysis.Alldataweretakenfrompublishedsources,seeSuppl.Table1.Thedatasetcomposition(numberofpopulations,np,andindividuals,ni,ineachmeta-population)isshowninthetableontheleft.Meta-populationsarecolor-codedinasimilarwaythroughoutallfiguresanddesignatedasfollows:Arctic(abbreviatedasARC);Na-Dene,inthisanalysisrepresentedbytwoNorthernAthabaskangroups,ChipewyanandDakelh(ATHorAth.);EuropeandtheCaucasus(EUR);northernNorthAmericans,excludingNa-Dene,YupikandInuit(NAMorN.N.Am.);SoutheastAsians(SEAorS.E.Asians);Siberians,excludingpopulationsofChukotkaandKamchatka(SIB);nativepopulationsofSouth,CentralAmerica,MexicoandsouthernUSA(SAM).LocationsofSiberian,Arctic,Athabaskan,andNorthAmericanpopulationsareshownonthemapbelow,whichalsoillustratesthreemigrationwavesandtheirapproximatedatesinthousandsofyear(kyr).LocationsoftheSaqqaq(datedatabout4,000YBP)andClovis(12,600YBP)ancientgenomesareshownwithasterisks.OntopaclusteringtreeconstructedwithfineSTRUCTUREonaversionofHumanOriginsSNParraydataset(655individualsand58populations,seeSuppl.Table2)illustratesrelationshipsofthemeta-populations.ForadetailedversionofthesametreeseeSuppl.Fig.3A.Fig.2.RelativerareallelesharingcountsandtheirstandarddeviationscalculatedforeachAmericanpopulationorancientgenomeandtheSiberian(A)orArctic(B)meta-populations.Allstatisticswerecalculatedseparatelyforallelesofvariousfrequency:occurring2,3,4,…andupto20timesinthesetof2,412chromosomes.Totakecareofvariabilityingenomecoverageacrosspopulationsandofdataset-specificSNPcallingbiases,wenormalizedthecountsofallelessharedbyagivenAmericanpopulationandtheArcticorSiberianmeta-populationsbysimilarcountsofallelessharedwithdistantoutgroups–Europeans(thisfigure)orAfricans(Suppl.Fig.4A,B).SaqqaqandNorthernAthabaskans(ChipewyansandDakelh)standoutfromnorthernNorthAmericans(N.N.Am.)andotherFirstAmericanpopulations.Fig.3.Two-dimensionalplotsofSiberianandArcticallelesharingcountsnormalizedusingtheEuropeanmeta-population.(A)Aplotshowingstatisticsforallpopulationsandstandarddeviations.Meta-populationsarecolor-codedaccordingtothelegend.(B)AnenlargedareaoftheplotshowingsimulatedmixturesofanymodernFirstAmericanpopulationandtheSaqqaqancientindividual(from10%to60%),andsimilarmixtureswithanythird-wavepopulation(from10%to30%ofGreenlanderInuitorChukotkanYupikancestry).Somepopulationnamesareindicatedontheplot.Fig.4.Two-dimensionalplotsofSiberianandArctichaplotypesharingstatisticsnormalizedusingtheEuropeanmeta-population.(A)Aplotshowingstatisticsforindividualsofallrelevantmeta-populations,color-codedaccordingtothe

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legend.(B)AnenlargedareaoftheplotshowingstatisticsforAmericanindividualsandsimulatedmixturesofanymodernFirstAmericanpopulationandtheSaqqaqancientindividual(from10%to30%),andsimilarmixtureswiththeChukotkanYupik(Eskimo)population(from5%to20%ofYupikancestry).Averagevaluesofthestatisticsinpopulationswereusedtocalculatethesimulatedstatistics.Fig.5.Adatedsix-populationdemographicmodelwithasymmetricmigrationconstructedusingRarecoal.ForacompletelistofparameterestimationsseeTable2.Meta-populationsareabbreviatedasfollows:AmericanArctic(Am.Arc.),Athabaskan(Ath.),European(Eur.),SouthAmerican(S.Am.),SoutheastAsian(S.E.A.),Siberian(Sib.).(A)Forthreeedgesmostimportantforourstudy(European-Siberian,Siberian-Athabaskan,Athabaskan-AmericanArctic),separateestimationsofgeneflowinbothdirectionswereperformed.Toreducetheoverallnumberofparameters,theseadmixtureeventswereenforcedtooccuratthesametime.Forthesamepurpose,EuropeanadmixtureinAmericans(intheAmericanArctic,AthabaskanandSouthAmericangroups)wasmodeledasunidirectionalwiththeageof500years,andtheseedgesareomittedforclarity(seetheirparametersinTable2).Effectivepopulationsizes(in1000)areshowninred.MostlikelybranchingpointsfortheSaqqaq(B)andClovis(C)ancientgenomeswerealsoestimatedusingRarecoal.

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TablesTable1.Finalparameterestimatesincludingposteriorprobabilitydistributionquantilesforthesix-populationdemographicmodelwithasymmetricmigration.Meta-populationsareabbreviatedasfollows:AmericanArctic(AARC),Athabaskan(ATH),European(EUR),northernNorthAmerican(NAM),SouthAmerican(SAM),SoutheastAsian(SEA),Siberian(SIB).SeeSuppl.Text1forfurtherdetailsonpopulationsusedintheanalysis.Theright-mostcolumnshowsparametersforamodelwheretheAthabaskangroupwasreplacedbynorthernNorthAmericans.Parametersmostimportantforourdiscussionareshadedingrey. Parameter Maximum

Likelihoodestimate(ATH)

2.5%posteriorquantile

50%posteriorquantile(Median)

97.5%posteriorquantile

MaximumLikelihoodestimate(NAM)

effectivepopulationsizes

EUR 25,101 25,015 25,094 25,182 25,714SEA 44,242 43,943 44,347 44,720 44,620SIB 13,568 13,115 13,445 13,710 10,303AARC 1,173 1,149 1,177 1,193 780ATH 1,851 1,803 1,847 1,890 5,280SAM 6,552 6,485 6,589 6,717 5,664EUR-SEA… 9,315 9,272 9,316 9,359 9,341SEA-SIB… 9,012 8,961 9,031 9,106 8,753SIB…-SAM… 147 146 148 149 141SAM-ATH 1,762 1,750 1,765 1,783 1,610SIB-AARC 27,469 27,202 27,621 28,003 28,612

splittimes EUR-SEA… 36,095y 35,980y 36,131y 36,279y 35,588ySEA-SIB… 20,402y 20,373y 20,410y 20,450y 20,374ySIB…-SAM… 20,290y 20,217y 20,253y 20,289y 20,271ySAM-ATH 9,744y 9,591y 9,714y 9,829y 11,792ySIB-AARC 4,126y 4,011y 4,116y 4,195y 2,580y

admixtureproportionsanddates

EUR®SIB 16.1% 15.9% 16.1% 16.3% 16.6%SIB®EUR 8.0% 7.9% 8.0% 8.2% 6.7%dateEUR«SIB* 2,327y 2,198y 2,299y 2,402y 1,753yEUR®AARC** 25.0% 24.8% 25.0% 25.2% 18.8%EUR®SAM** 2.7% 2.6% 2.7% 2.7% 3.1%EUR®ATH** 0.7% 0.4% 0.7% 1.0% 29.2%SIB®ATH 22.9% 22.3% 23.0% 23.8% 9.6%ATH®SIB 6.8% 6.5% 6.8% 7.0% 1.6%dateSIB«ATH* 6,940y 6,575y 6,812y 7,030y 595yAARC®ATH 7.6% 6.3% 7.5% 8.5% 4.8%ATH®AARC 11.5% 10.9% 11.6% 12.4% 17.4%dateAARC«ATH* 490y 476y 492y 499y 5ySAM®AARC 7.6% 7.2% 7.4% 7.7% 21.9%dateSAM®AARC 488y 473y 481y 495y 2,570y

*Toreducetheoverallnumberofparameters,admixtureeventsinbothdirectionswereenforcedtooccuratthesametime.**Forthesamepurpose,EuropeanadmixtureinAmericans(AARC,ATH,NAM,andSAMgroups)wasmodeledasunidirectionalwiththeageof500years.

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Table2.DatingadmixtureeventsinthehistoryofNa-Denepopulationsusinghaplotypesharingdata,generatedwithChromoPainterv.2andanalyzedwiththeGLOBETROTTERapproach13.Coancestrycurvesapproximatingtwodistinctadmixturedateshavealwaysdemonstratedabetterfittothedata(Suppl.Fig.9),andfitstatisticsforthesecurvesareshowninthistable,aswellasinferredmixturepartners,mixtureproportions,datesandtheir95%confidenceintervals. dataset HumanOrigins HumanOrigins haplotypedonors 9meta-populationsa) 54populations targetpopulation 2Dakelh,

3Chipewyansb)2Dakelh,

11Chipewyansc) p-valueforanyadmixtureevent 0.015 0coancestrycurvesfortwoadmixturedates

goodness-of-fit,max.valueacrossallcurves 0.483 0.738additionalgoodness-of-fitexplainedbyaddingaseconddate,max.valueacrossallcurves

0.105 0.288

surrogatepairsreflectingthePaleo-Eskimoadmixtureeventd)

SARCvs.SAM

SIB+ANEvs.SAM

Itelmen(SARC)vs.Pima(SAM)

Ket(SIB+ANE)vs.Surui(SAM)

goodness-of-fit 0.155 0.274 0.151 0.123additionalgoodness-of-fit 0.099 0.056 0.137 0.035

probabilityat1cMdistance 0.989 0.991 0.977 0.971admixtureevent1

inferreddate,YBP 425 14495%confidenceinterval,YBP 29–522 68–184

source1 26%SAM 35%Cree(NAM)source2 74%SAM 65%Kaqchikel(SAM)

admixtureevent2

inferreddate,YBP 3,587 2,21095%confidenceinterval,YBP 488–4,614 697–3,135

source1 25%Saqqaq 19%Saqqaqsource2 75%SAM 81%Cree(NAM)

a)Thefollowingmeta-populationswereused:1/SiberianArctic(abbreviatedasSARC);2/AmericanArctic(AARC);3/EuropeandtheCaucasus(EUR);4/northernNorthAmericans,excludingNa-Dene,YupikandInuit(NAM);5/SoutheastAsians(SEA);6/nativepopulationsofSouth,CentralAmerica,MexicoandsouthernUSA(SAM);7/theSaqqaqancientgenome;8/SiberianswithextensiveancientNorthEurasianancestry(SIB+ANE);9/coreSiberians(cSIB).b)TomakeadmixturehistoryofthetargetpopulationlesscomplexandamenabletoGLOBETROTTERanalysis,onlyNa-DeneindividualswithpriorevidenceofPaleo-EskimoadmixtureandwithnoevidenceofsignificantEuropeanadmixture(Fig.4)wereused,seeMethodsfordetails.c)AdditionalChipewyanindividuals,withevidenceofPaleo-Eskimoand/orNeo-Eskimoadmixturewereincluded(Fig.4).d)Twocurveswiththehighestpositiveslopeintherangeofgeneticdistancesfrom1to3cMwereconsidered,i.e.thosereflectingtheoldestdetectableadmixtureevent.

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FiguresFig.1.

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Fig.2.

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Fig.3.

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Fig.4.

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Fig.5.

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