Palaeoproteomic evidence identifies archaic hominins associated with the Châtelperronian at the Grotte du Renne


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Palaeoproteomic evidence identifies archaic hominins associated with the Châtelperronian at the Grotte du Renne
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Proceedings of the National Academy of Sciences of the United States of America
Creator:
Welker, Frido
Hajdinjak, Mateja
Talamo, Sahra
Jaouen, Klervia
Dannemann, Michael
David, Francine
Julien, Michèle
Meyer, Matthias
Kelso, Janet
Barnes, Ian
Brace, Selina
Kamminga, Pepijn
Fischer, Roman
Kessler, Benedikt M.
Stewart, John R.
Pääbo, Svante
Collins, Matthew J.
Hublin, Jean-Jacques
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National Academy of Sciences
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Châtelperron Site (Châtelperron, France) ( lcsh )
Renne Cave (France) ( lcsh )
Mass spectrometry ( lcsh )
DNA, Fossil ( lcsh )
Neanderthals ( lcsh )
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serial ( sobekcm )

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In Western Europe, the Middle to Upper Paleolithic transition is associated with the disappearance of Neandertals and the spread of anatomically modern humans (AMHs). Current chronological, behavioral, and biological models of this transitional period hinge on the Châtelperronian technocomplex. At the site of the Grotte du Renne, Arcy-sur-Cure, morphological Neandertal specimens are not directly dated but are contextually associated with the Châtelperronian, which contains bone points and beads. The association between Neandertals and this “transitional” assemblage has been controversial because of the lack either of a direct hominin radiocarbon date or of molecular confirmation of the Neandertal affiliation. Here we provide further evidence for a Neandertal–Châtelperronian association at the Grotte du Renne through biomolecular and chronological analysis. We identified 28 additional hominin specimens through zooarchaeology by mass spectrometry (ZooMS) screening of morphologically uninformative bone specimens from Châtelperronian layers at the Grotte du Renne. Next, we obtain an ancient hominin bone proteome through liquid chromatography-MS/MS analysis and error-tolerant amino acid sequence analysis. Analysis of this palaeoproteome allows us to provide phylogenetic and physiological information on these ancient hominin specimens. We distinguish Late Pleistocene clades within the genus Homo based on ancient protein evidence through the identification of an archaic-derived amino acid sequence for the collagen type X, alpha-1 (COL10α1) protein. We support this by obtaining ancient mtDNA sequences, which indicate a Neandertal ancestry for these specimens. Direct accelerator mass spectometry radiocarbon dating and Bayesian modeling confirm that the hominin specimens date to the Châtelperronian at the Grotte du Renne.
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Volume 113, Issue 40
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6 p.

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Palaeoproteomicevidenceidentifiesarchaichominins associatedwiththeChâtelperronianattheGrotte duRenne FridoWelker a,b,1 ,MatejaHajdinjak c ,SahraTalamo a ,KlerviaJaouen a ,MichaelDannemann c,d ,FrancineDavid e , MichèleJulien e ,MatthiasMeyer c ,JanetKelso c ,IanBarnes f ,SelinaBrace f ,PepijnKamminga g ,RomanFischer h , BenediktM.Kessler h ,JohnR.Stewart i ,SvantePääbo c ,MatthewJ.Collins b ,andJean-JacquesHublin a a DepartmentofHumanEvolution,MaxPlanckInstituteforEvolutionaryAnthropology,04103Leipzig,Germany; b BioArCh,UniversityofYork,YorkYO10 5DD,UnitedKingdom; c DepartmentofEvolutionaryGenetics,MaxPlanckInstituteforEvolutionaryAnthropology,04103Leipzig,Germany; d Medical Faculty,UniversityofLeipzig,04103Leipzig,Germany; e ParisUnitéMixtedeRecherche7041,ArchéologiesetSciencesdel ’ Antiquité,CentreNationaldela RechercheScientifique,92023Nanterre,France; f DepartmentofEarthSciences,NaturalHistoryMuseum,LondonSW75BD,UnitedKingdom; g Naturalis BiodiversityCenter,2300RALeiden,TheNetherlands; h TargetDiscoveryInstitute,NuffieldDepartmentofMedicine,UniversityofOxford,OxfordOX37FZ, UnitedKingdom;and i FacultyofScienceandTechnology,BournemouthUniversity,DorsetBH125BB,UnitedKingdom EditedbyRichardG.Klein,StanfordUniversity,Stanford,CA,andapprovedJuly29,2016(receivedforreviewApril13,2016) InWesternEurope,theMiddletoUpperPaleolithictransitionisassociatedwiththedisappearanceofNeandertalsandthespreadof anatomicallymodernhumans(AMHs).Currentchronological,behavioral,andbiologicalmodelsofthistransitionalperiodhingeonthe Châtelperroniantechnocomplex.AtthesiteoftheGrotteduRenne, Arcy-sur-Cure,morphologicalNea ndertalspecimensarenotdirectly datedbutarecontextuallyassociatedwiththeChâtelperronian, whichcontainsbonepointsandbea ds.TheassociationbetweenNeandertalsandthis “ transitional ” assemblagehasbeencontroversial becauseofthelackeitherofadirecthomininradiocarbondateorof molecularconfirmationoftheN eandertalaffiliation.HereweprovidefurtherevidenceforaNeandertal – Châtelperronianassociation attheGrotteduRennethroughbiomolecularandchronologicalanalysis.Weidentified28additionalho mininspecimensthroughzooarchaeologybymassspectrometry(ZooMS)screeningofmorphologically uninformativebonespecimensfromChâtelperronianlayersatthe GrotteduRenne.Next,weobtainanancienthomininboneproteomethroughliquidchromatography-MS/MSanalysisanderror-tolerantaminoacidsequenceanalysis. Analysisofthispalaeoproteome allowsustoprovidephylogeneti candphysiologicalinformationon theseancienthomininspecimens.W edistinguishLatePleistocene cladeswithinthegenus Homo basedonancientproteinevidence throughtheidentificationofanarchaic-derivedaminoacidsequence forthecollagentypeX,alpha-1( COL10 1 )protein.Wesupportthis byobtainingancientmtDNAsequenc es,whichindicateaNeandertal ancestryforthesespecimens.Dire ctacceleratormassspectometry radiocarbondatingandBayesianmodelingconfirmthatthehominin specimensdatetotheChâtelperronianattheGrotteduRenne. palaeoproteomics | ZooMS | Châtelperronian | Neandertal T ounderstandtheculturalandgeneticinteractionbetweenthe lastNeandertalsandsomeoftheearliestanatomicallymodernhumans(AMHs)inEurope,weneedtoresolvethetaxonomicaffiliationofthehomininsassociatedwiththe “ transitional ” industriescharacterizingthereplacementperiod,suchasthe Châtelperronian(1,2).Thewell-characterizedChâtelperronian lithictechnologyhasrecentlybeenreclassifiedasfullyUpperPaleolithic(3)andisassociatedatseveralsiteswithboneawls,bone pendants,andcolorants(4,5).TheGrotteduRenneatArcy-surCure,France,iscriticaltocompetingbehavioralandchronological modelsfortheChâtelperronian,asatthissitetheChâtelperronian isstratigraphicallyassociat edwithhomininremainsthatare morphologicallyidentifiedasNeandertals(6 – 8).Hypotheses explainingthisassociationrangefrom “ acculturation ” byAMHs (9),toindependentdevelopmentofsuchartifactsbyNeandertals (5),tomovementofpendantsandboneartifactsfromtheoverlyingAurignacianintotheChâtelperronianlayers(10,11),orto movementofthehomininspecimensfromtheunderlying MousterianintotheChâtelperr onianlayers(10,12).Thefirst twohypothesesassumethatthe stratigraphicassociationof thehomininsandtheChâtelper ronianassemblageisgenuine, whereasthelattertwohypothese scounterthattheassociationistheresultoflarge-scale,taphonomicmovementof material.Inallscenarios,themorphologicalidentificationof thesehomininsasNeandertalsisacceptedbutunsupportedby molecularevidence. Totestthechronostratigraphiccoherenceofthesite,Bayesian modelsofradiocarbondatesforthesitehavebeenconstructed (10,13).Theresultsofthesetwomodelscontradicteachotherin theextenttowhicharchaeologicalmaterialmovedbetween theChâtelperronianandnon-Châtelperronianarchaeologicallayers. Furthermore,theyhavebeencriticizedonvariousmethodological aspects(13,14),andthefirst(10)isatoddswithsomearchaeological evidencethatsuggeststhatlarge-sc aledisplacementofmaterialinto Significance ThedisplacementofNeandertalsbyanatomicallymodernhumans (AMHs)50,000 – 40,000yagoinEuropehasconsiderablebiological andbehavioralimplications.T heChâtelperronianattheGrotte duRenne(France)takesacentralroleinmodelsexplainingthe transition,buttheassociationo fhomininfossilsatthissitewith theChâtelperronianisdebated.Hereweidentifyadditional homininspecimensatthesitethroughproteomiczooarchaeology bymassspectrometryscreeningandobtainmolecular(ancient DNA,ancientproteins)andchronometricdatatodemonstrate thattheserepresentNeandertalsthatdatetotheChâtelperronian.Theidentificationofanam inoacidsequencespecifictoa cladewithinthegenus Homo demonstratesthepotentialof palaeoproteomicanalysisinthestudyofhominintaxonomyin theLatePleistoceneandwarrantsfurtherexploration. Authorcontributions:F.W.,J.R.S.,M.J.C.,andJ.-J.H.designedresearch;F.W.,M.H.,S.T.,K.J., M.D.,M.M.,J.K.,andR.F.performedresearch;F.D.,M.J.,I.B.,S.B.,P.K.,R.F.,B.M.K.,andS.P. contributednewreagents/analytictools;F.W.,M.H.,S.T.,K.J.,M.D.,F.D.,M.J.,M.M.,J.K.,I.B., S.B.,P.K.,R.F.,B.M.K.,J.R.S.,S.P.,M.J.C.,andJ.-J.H.analyzeddata;andF.W.,M.J.C.,andJ.-J.H. wrotethepaper. Theauthorsdeclarenoconflictofinterest. ThisarticleisaPNASDirectSubmission. FreelyavailableonlinethroughthePNASopenaccessoption. Datadeposition:ThesequencesreportedinthispaperhavebeendepositedintheProteomeXchange(accessionnos. PXD003190 and PXD003208 )andintheEuropeanNucleotideArchive(accessionno. PRJEB14504 ). 1 Towhomcorrespondenceshouldbeaddressed.Email:frido_welker@eva.mpg.de. Thisarticlecontainssupportinginformationonlineat www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1605834113/-/DCSupplemental . 11162 – 11167 | PNAS | October4,2016 | vol.113 | no.40www.pnas.org/cgi/doi/10.1073/pnas.1605834113

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theChâtelperronianfromeithertheoverlyingorunderlyinglayersis unlikely(14).BothBayesianmodelsareonlyindirecttestsofthe hominin – Châtelperronianassociation,asnodirectradiocarbondates ofthehomininsareavailable. Pendingthediscoveryoffurtherhomininspecimensatother Châtelperroniansites,theChâtelperronianattheGrotteduRenne remainscrucialtoobtainingacoherentbiologicalandchronologicalviewofthetransitionalp eriodinEurope.Ithasbeendemonstratedpreviouslythatpalaeoprot eomicsallowstheidentification ofadditionalhomininspecimensamongunidentifiedPleistocene faunalremains[zooarchaeology bymassspectrometry,orZooMS (15 – 17)],althoughherethevalueofdoingsohasenabledthedirect datingandunambiguousidentificationoftheNeandertalassociationwiththeChâtelperronian.Wesuccessfullyapplythistothe GrotteduRenneChâtelperronian.Weobtainadirecthominin radiocarbondateatthesite,therebydirectlyaddressingthechronostratigraphiccontextofthisspecimeninrelationtothehypothesisregardingmovementofthehomininspecimensfromthe underlyingMousterianintotheChâtelperronianlayersandprovide biomoleculardata(palaeoproteomics,aDNA)onthegeneticancestryoftheGrotteduRenneChâtelperronianhominins.AlthoughproteomicdataonPleistocenehomininbonespecimenshas beenpresentedbefore(17,18),thephylogeneticandphysiological implicationsofsuchdatasetshas,sofar,notbeenfullyexplored. Hereweusethepotentialoferror-tolerantMS/MSdatabase searchesinrelationtothebiologi calquestionsassociatedwiththe Châtelperronian.Suchtechnicaladvanceshavenotbeenappliedto entirepalaeoproteomesfromLat ePleistocenehomininsbefore (19).Inaddition,wedemonstratethattheboneproteomereflects thedevelopmentalstateofanancienthomininindividual. Throughoutourstudy,wedevelopandusetoolsdesignedto minimize,identify,andexcludeproteinandDNAcontamination ( SIAppendix ,Fig.S1 ). Results ZooMSScreening. Wescreened196taxonomicallyunidentifiable ormorphologicallydubiousbonespecimens(commonly < 20mg ofbone)usingZooMSfromtheareasoftheGrotteduRenne thathadpreviouslyyieldedhomininremains(20).Thisrequired ustoconstructacollagentypeI(COL1)sequencedatabaseincludingatleastonespeciesofeachmediumorlarger-sizedgenus inexistenceinWesternEuropeduringtheLatePleistocene(19) ( DatasetS1 ),andfromthisderivedaZooMSpeptidemarker library( DatasetS2 ).ZooMSusesdifferencesintrypticpeptide massesfrom COL1 1 and COL1 2 aminoacidchainsto taxonomicallyidentifyboneandtoothspecimens(15).ThepeptidemarkerlibrarycombinesnewlyobtainedandpublishedCOL1 sequenceswithpublishedZooMSpeptidemarkers(15,21)and enabledustoconfidentlyidentify28bonefragmentswithinthe extant Pan Homo cladetotheexclusionofotherHominidae( SI Appendix ,Figs.S1andS2andTableS1 ).Togetherwithother studies,thisconfirmsthesuita bilityofZooMSasascreening techniquetoidentifyhomininspecimensamongunidentified fragmentarybonespecimens(17,22).WeconfirmedtheseidentificationsforsamplesAR-7,AR-16,andAR-30byanalyzingthe sameextractsusingshotgunproteomicsandspectraassignment againstourCOL1sequencedatabase( SIAppendix ,Fig.S3 ).In eachcase,taxonomicassignmenttothegenus Homo hadthe highestscore( SIAppendix , ZooMSScreening ). Molecularcontaminationisanimportantissuewhenstudying ancientbiomolecules,especiallywhenitconcernsancienthominins.Extractionblankswereincl udedthroughoutallanalysisstages tomonitortheintroductionofpotentialcontamination,although suchcontrolsonlyprovideinsightintocontaminationintroduced duringthelaboratoryanalysis.M ALDI-TOF-MSanalysisofthese blanksshowednopresenceofCOL1peptides( SIAppendix ,Fig. S2 B ).Furthermore,asamarkerofdiageneticalterationofamino acids(23),glutaminedeamidationvaluesbasedonammoniumbicarbonateZooMShomininspectraindicatedthattheanalyzed collagenhasglutaminedeamidationvaluessignificantlydifferent frommodernbonespecimens( t test: P = 2.31E 11)(Fig.1 A ),but similartodeamidationvaluesobtainedforfaunalspecimensanalyzedfromtheGrotteduRenne[ t tests;peptideP1105: P = 0.85; peptideP1706: P = 0.55(24)].Weinterpretthistosupportthe identificationofendogenous, noncontaminatedhomininCOL1. Palaeoproteomic(LC-MS/MS)Analysis. Afteridentifyingadditional homininspecimensattheGrotteduRennebyZooMS,we undertookpalaeoproteomicandgeneticanalysestoestablish whetherthesenewlyidentifiedhomininsrepresentAMHsor Neandertals.Error-tolerantliquidchromatographytandemmass spectrometry(LC-MS/MS)analysisoftheproteincontentofthe ZooMSextractsofAR-7,AR-16,andAR-30andtwoadditional palaeoproteomicextractionsperformedonAR-30resultedinthe identificationof73proteins(Table1and SIAppendix ,TableS2 ). Webaseourassessmentofendogenousandpossiblyexogenous proteinsonfourlinesofevidence.First,weanalyzedourextractionblanksbyLC-MS/MSanalysis.Thisallowedustoidentify severalproteinsintroducedascontaminantsduringtheanalysis (humankeratins,histones,andHBB)(Table1and SIAppendix , COL1 database samples Grotte du Renne 0 25 50 75 100 ModernPermafrostPleistoceneFaunaHominin Glutamine deamidation (%) P1105 P1706 A 0 25 50 75 100 01234 Log observations Deamidation (%) B Mass mass inaccuracy (ppm) 50015002500 -4 0 4 Fig.1. Identificationofdamagedancientproteins.( A )Deamidationvaluesofboneammonium-bicarbonateextractsfordatabasesamplesandArcybone specimensanalyzedusingMALDI-TOF-MS.( B )DeamidationfrequencybasedonspectralcountsobtainedthroughLC-MS/MSanalysis.Clusteranalysisprovides threeclusters(filledtriangles,squares,andcircles).Opencirclesrepresentproteinsthathavetwoorfewerspectralmatchesandwerenotinclud edincluster analysis. COL10 1 isindicatedinpink;0%indicatesnodeamidationand100%indicatescompletedeamidation.( Inset )AR30ppmerrordistributionby peptidemassforassignedspectra,withspectramatchingto COL10 1 inred,opencirclesandthoseto COL10 1 128Ninred,filledcircles. Welkeretal. PNAS | October4,2016 | vol.113 | no.40 | 11163 ANTHROPOLOGY

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TableS2 ),andmatchestotheseproteinsforAR-7,AR-16,and AR-30wereexcludedfromfurtherin-depthanalysis. Second,wesearchedourdataagainstthecompleteUniProt database,whichcontainsadditionalnonhumanandnonvertebrate proteinsfromvarioussourcesthatcouldhavecontaminatedour extracts.Spectralmatcheston onvertebrateproteinscomprise < 1.0%ofthetotalnumberofmatchedspectra,indicatingaminimal presenceofnonvertebratecontamination( SIAppendix ,Fig.S4 ). Thesespectralmatchesweresubsequentlyexcludedfromanalysis. Third,unsupervisedclusteranalysisbasedonglutamineand asparaginedeamidationfrequenciesobservedforallidentified vertebrateproteinsrevealedaclearseparationinthreeclusters (Fig.1 B and SIAppendix ,Fig.S5 ).Thefirstgroupof14proteins displaysalmostnodeamidatedasparagineandglutaminepositions.Manyofthese(keratins,trypsin,bovine CSN2 ),butnotall ( COL4 6 , UBB , DCD ),havepreviouslybeenreportedascontaminants(Fig.1 B ,filledtriangles)(25).Allproteinsidentifiedin ourextractionblankshavedeamidationfrequenciesthatfallinto thisgroup,furthersuggestingthatthese14proteinsarecontaminants.Thesecondandthirdgroupscompriseatotalof35proteins withelevatedlevelsofdeamidatedasparagineandglutamineresidues(Fig.1 B ,filledcirclesandsquares).Theseincludevarious collagensandnoncollagenousproteinspreviouslyreportedin (ancient)boneproteomes,andwei nterpretthattheseproteinsare endogenoustotheanalyzedbonespecimens.Wewereunableto obtainsufficientdeamidationspectralfrequencydatafor24additionalproteins,asinsufficientnumbersofasparagineorglutaminecontainingpeptideswerepresent(Fig.1 B ,opencircles).Someof these24proteinshavepreviouslybeenidentifiedinnonhominin boneproteomesorareinvolvedinboneformationandmaintenance[ POSTN , THBS1 , ACTB , C3 , IGHG1 ,andtheDelta-like protein3( DLL3 )],andarethereforelikelyendogenoustothe bonespecimensaswell. Fourth,afterexclusionofcontaminants,proteincomposition wassimilartoothernonhomininbone palaeoproteomes(25,26). Fortheseproteins,weobservedthepresenceofadditionaldiageneticandinvivoposttranslationalmodifications( SIAppendix ,Fig. S6andTablesS3andS4 ),likewisesuggestingtheretrievalofan endogenoushomininpalaeoproteome.Onthebasisoftheseresults, wesuggestthatmatchestohumanproteinsinpalaeoproteomic analysisshouldbesupportedbyadditionallinesofevidenceto substantiatetheclaimthatthes erepresentproteinsendogenousto theanalyzedtissueanddonotderivefromexogenouscontaminationderivedfromeitherhandlingofthebonespecimenorcontaminationintroducedduringtheanalyticalprocedure. Amongthenoncontaminantproteinsthereareseveralproteins thatare(specifically)expressedby(pre)hypertrophicchondrocytes [ COL10 1 and COL27 1 (27)]andosteoblasts[ DLL3 (28)and COL24 1 (29)]duringboneformation.Thepresenceof COL10 1 isparticularlynoteworthy.Itispreferentiallysecretedby(pre) hypertrophicchondrocytesduringinitialboneossificationin boneformation,includingcranialsutures(30,31),andwould thereforeberemovedfromthebonematrixdependentonthe rateofboneremodelingofagivenmineralizedtissue.Inlinewith this,geneontology(GO)annotationanalysisindicatesenrichment forGObiologicalprocessesrelatedtocartilagedevelopmentand boneossification( SIAppendix ,TableS5 ).Theseobservationsare consistentwithosteologicalandisotopicobservations,whichsuggesttheidentifiedbonespecimensbelongtoabreastfedinfant (see IsotopicAnalysisandRadiocarbonDating ).GOannotation analysisfurtheridentifiedasignificantgroupofbloodmicroparticles(GO:00725262)suchasalbumin( ALB ).Theseproteinshavebeenidentifiedinnonhomininpalaeoproteomesas wellandareconsistentlyincorporatedintothemineralized bonematrix(25,26). Error-tolerantanalysisofMS/MSspectrahastheabilityto identifyaminoacidvariantsnot presentinproteindatabasesor referencegenomes,potentiallyrevealingrelevantphylogenetic information.Weusedanerror-tolerantsearchengine(PEAKS) againstthehumanreferenceprot eomeandcomparedourprotein sequencedataagainstavailable aminoacidsequencevariations knownthroughgenomicresearchformodernhumans(32),a Denisovangenome(33),andthec odingregionsofthreeNeandertals(34).Usingthisapproach,weconfidentlyidentifyfiveproteinsthatcontainatotalofsev enaminoacidpositionswith nonsynonymousSNPswithbothallelesatfrequencies 1.0%in present-dayhumans( SIAppendix ,TableS6 ).Insixcases,weobservedtheancestralHominidaesta teintheproteomedata,whichis alsopresentinDenisovanandNea ndertalproteinsequences(34). TheseincludeonepositionforwhichamajorityofAMHs(93.5%) carryaderivedsubstitution( COL28 1 ;dbSNPrs17177927)and whereourdatacontaintheancest ralposition(aminoacidP).For theseventhcase, COL10 1 ,weobservedanaminoacidstate presentinDenisovans,Neandert als,and0.9%ofmodernhumans haplotypes(46/5,0081000Genomeshaplotypes)( SIAppendix , TablesS6andS9 ),butnotinanyotherHominidae( Pongoabelii , Gorillagorilla ,or Pantroglodytes )( SIAppendix ,TableS7 ). Weidentified COL10 1 inallthreeanalyzedbonespecimens ( SIAppendix ,TableS2 ),butnotinourextractionblank.The deamidationfrequencyobservedfor COL10 1 (Fig.1 B )andthe excretionof COL10 1 by(pre)hypertrophicchondrocytesduring ossification(30)indicateanendogenousoriginoftheidentified COL10 1 peptides.Weidentifyonepeptidefor COL10 1 ( SI Appendix ,TableS8 )thatcontainsanaminoacidpositionindicative ofanarchaicsequence(NeandertalorDenisovan).Thepeptideof interestisrepresentedintwopalaeoproteomicanalysesperformed onAR-30,withthreespectru m-peptidematchesintotal( SIAppendix ,TableS7 ).Correctprecursormassandfragmentionassignmentwerevalidatedmanuallytoexcludefalseassignmentof 13 C-derivedisotopicpeaksasdeamidatedvariantsofthepeptide, whichledtotheexclusionofafourthspectrum( SIAppendix , Palaeoproteomics ).Allthreespectrarepresentsemitrypticpeptides ( SIAppendix ,Fig.S7 ).Asinotherpalaeoproteomes,thepresence ofsuchsemitrypticpeptidesisnotuncommonandislikelythe Table1.Palaeoproteomicsummarystatisticsperanalyzedbonespecimen VariableBlankAR-7AR-16AR-30(ZooMS)AR-30AAR-30B No.ofMS/MSscans73243,07320,56411,29416,92320,634 No.ofmatching MS/MSscans 19(2.6%)7,035(16.3%)4,053(19.7%)2,232(19.8%)3,416(20.2%)3,769(18.3%) No.ofproteingroups74230243940 Endogenous02021112326 Statusunknown01068128 Exogenous7123546 No.ofNCPs — 7771312 No.ofuniqueproteingroups — 42142 NCP,noncollagenousprotein. 11164 | www.pnas.org/cgi/doi/10.1073/pnas.1605834113 Welkeretal.

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resultofproteindiagenesis(23,25,35 – 37).Inaddition,allthree spectracontainahydroxylated prolineonthesameposition ( COL10 1 position135),furtherdemonstratingconsistency amongourpeptide-spectrummatches.Thereplicationofour resultsintwoindependentanalysesandtheinferredpresenceof posttranslationalmodificationsinallthreespectra,oneofwhichis identicallyplaced,furthersupportthenotionthattheseareendogenoustotheanalyzedbonespecimen. Thenucleotidepositionofinter estislocatedatchr6:116442897 (hg19,dbSNPrs142463796),whichcorrespondstoaminoacidposition128in COL10 1 (UniProtQ03692).Forallthreeavailable Neandertalsequences,thispositi oncarriesthenucleotideT(34), whichtranslatesintotheaminoacidN(codon “ Aat, ” 3 to5 ).The positionisheterozygousN/DintheDenisovangenome(33),aDin theUst ’ -Ishim 45,000BPAMHgenome(38),andDin99.1%of modernhumans(32)(codon “ Gat, ” 3 to5 ).Theremaining0.9% ofmodernhumanindividualsanalyzedmatchtheNeandertalsequence.Alltheseindividualsareoutsidesub-SaharanAfrica(Fig.2). Foraminoacidposition COL10 1 128,theaminoacidNrepresents thederivedstate,andtheaminoacidDistheancestralstate( SI Appendix ,TableS7 ). COL10 1IntrogressionintoModernHumans. Whenpresentinmodern humans,thearchaic-likealleleisfoundinpopulationsknownto havearchaicintrogression(39),suchasSoutheastAsia(1 – 6%)and Oceania(33 – 47%),andwithhighfrequencyinPapuaNewGuinea (47%)(Fig.2and SIAppendix ,TableS9 ).Thisarchaic-likealleleis foundonextendedarchaic-likehaplotypesthathaveaminimum lengthof146kb( SIAppendix ,Fig.S8 ).Giventherecombination rateof0.4cM/Mbinthisregion(40)andtheageoftheNeandertal andDenisovansamples(39),wecomputethatthishaplotypelength ismoreconsistentwitharchaicint rogressionthan withincomplete lineagesorting( SIAppendix , ArchaicAncestryofrs142463796in COL10 1 ).Because COL10 1 128Nispresentin < 1%ofpresentdayhumansasaconsequenceofarchaicintrogression,itspresence insampleAR-30suggestsarchaic(Neandertal + Denisovans)ancestryforatleastpartofitsnucleargenome. mtDNAAnalysis. Tosupportthepalaeoproteomicevidence,we extractedmtDNAfromAR-14andAR-30( SIAppendix , Ancient DNA andTableS10 ).ElevatedCtoTsubstitutionfrequenciesat terminalsequenceends(upto12.1%forAR-14and28.1%for AR-30)suggestthatatleastsomeoftherecoveredsequencesfor bothspecimensareofancientorigin(41)( SIAppendix ,Table S11 ).WhenrestrictingtheanalysistothesedeaminatedmtDNA fragments,supportfortheNeandertalbranchinapanelofdiagnosticmtDNApositionsisabove70%( SIAppendix ,Fig.S9 ), butitiswithoutsupportfortheDenisovanbranch.ThisisconfirmedwhenonlydiagnosticpositionsdifferingbetweenNeandertalsandpresent-dayhumansareincluded( SIAppendix ,Table S12 ).TheuniparentalmodeofmaternalinheritanceformtDNA, theabsenceofnotablemtDNAcontaminationfromNeandertal mtDNAintheextractionblanks,andthedominanceofdeaminatedmtDNAsequencesaligningtotheNeandertalmtDNA branchalldemonstratethatAR-14andAR-30aremitochondrial Neandertals.ResidualmodernhumancontaminationinthefractionofdeaminatedsequencesmakesreconstructionofNeandertal mtDNAconsensussequencesimpossibleforbothbonespecimens. WewerethereforeunabletotestwhetherAR-14andAR-30are maternallyrelated.Nevertheless,theaboveanalysesallowusto concludethatbothspecimenscarrymtDNAofthetypeseenin LatePleistoceneNeandertals. IsotopicAnalysisandRadiocarbonDating. Weextractedcollagenfrom specimenAR-14(MAMS-25149)toprovideadirectaccelerator massspectometry(AMS)datefro maGrotteduRennehominin, andtherebyaddressthepossibilitythatthesehomininsderivefrom theunderlyingMiddlePaleolithicMousterian[i.e.,thehypothesis regardingmovementofthehomininspecimensfromtheunderlyingMousterianintotheChâtelperr onianlayers(10)].Inaddition, weextractedcollagenfrom21additionalfaunalbonespecimens, identifiedbyZooMSandfromtheChâtelperronianLayersIXand XattheGrotteduRenne,toprovideanisotopiccontextforthe 15 N and 13 CstableisotopesobtainedforAR-14.Thecollagenquality criteriaarewithinacceptedranges( SIAppendix ,TableS14 ).The stableisotopesindicatethe 15 Nis5.4 ‰ higherforAR-14comparedwithassociatedcarnivores( SIAppendix ,Fig.S11andTables S14andS15 ).Thissuggestsbreastfeedingasamajordietaryprotein source(42)( SIAppendix , StableIsotopeAnalysisandAssociated Fauna ),whichisinagreementwiththepresenceof COL10 1 and thepresenceofanunfusedvertebralhemiarch,andinsupportofthe interpretationthattheseremai nsrepresentabreastfedinfant( SI Appendix ,Fig.S10andTableS13 ). Totestdifferentchronostratigraphicscenarios,weconstructed fourdifferentBayesianmodelstoaddressvariouscriticismsraised againstpreviousmodels:theHublinetal.model,whichincludes allagesandpriorsfollowingref.13;thehominin-modifiedmodel, whichincludesonlyagesfromhominin-modifiedbonespecimens (priorsanddatesfromref.13);theDiscampsetal.model,which excludesradiocarbonagesobtainedfromanareaofthesite consideredreworkedbysome(43)(priorsanddatesfromref.13); andtheHighametal.model,whichincludesallagesandpriors followingref.44(amodelthatalsoincludeshominin-modified bonesonly).TheBayesianCQLcodeforeachofthefourmodels isincludedin SIAppendix , RadiocarbonDatingandBayesian ModelingofAR-14 .AllfourmodelstreattheChâtelperronian LayersIXandXasasinglechronologicalphasebasedonlithic refitsbetweenthesetwolayers(13,45).Themeasuredagefor AR-14,36,840 ± 660 14 CBP,fitswithintheChâtelperronian chronologicalboundariescalculatedinallfourBayesianmodels (Fig.3and SIAppendix ,TableS16 )(10,13).Withaposterior outlierprobabilityof4 – 8%,AR-14isunlikelytoderivefromthe underlyingMousterianortheoverlyingAurignacianattheGrotte duRenne( SIAppendix ,TableS16 ),evenwhenonlyhomininmodifiedbonespecimensareincludedorbonespecimensfrom potentiallyreworkedareasareexcluded(followingref.43). Fig.2. SoutheastAsianfrequencyofthearchaic-likealleleofrs142463796 inmodernhumanpopulations.Thefrequencyofthearchaic-likeallelein modernhumanpopulationsisdisplayedinred[Yorubaallelefrequencyin populationsshowninlightblueforthe1000Genomesdatasetanddarkblue fortheSimonsGenomeDiversityPanel(SGDP)dataset].Thediameteris proportionaltothenumberofindividualsinagivenpopulation,which rangesbetweenoneand16individualsintheSGDPandbetween66and113 individualsinthe1000Genomesphase3data. Welkeretal. PNAS | October4,2016 | vol.113 | no.40 | 11165 ANTHROPOLOGY

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DiscussionandConclusion DespitetheirspatialproximitywithinLayerXandsquaresC7 andC8,noneofthenewlydescribedspecimenscouldbefitted togethertoformlargerfragments. Themorphologicallyinformative specimens,however,seemtorepresentsmallfragmentsofanimmaturecraniumandanunfusedvertebralhemiarchofneonatalage ( SIAppendix ,Fig.S10andTableS13 ).Thisissupportedbyisotopic evidencesuggestingthatthesefragmentsbelongedtoanonweaned infantandbyproteomicevidence intheformofproteinspresentin bonebeforeboneremodelinghasstarted.Allthenewlyidentified specimenswerefoundinclosespatialassociationwithapreviouslydescribedhominintemporalbonefromsquareC7, LayerXb,assignedtoaninfantaround1yold,aswellas10 dentalspecimensfromsquaresC7andC8(6,7).Thesedental specimensoverlapindevelopmentalage,suggestingtheyrepresentoneorpossiblytwoindividualsbetween6and18moold (7).The28newlyidentifiedspec imens,togetherwithalready describedspecimens,maythereforerepresenttheskeletalremainsofasingleinfant. Ourstudyspecificallyaimedtoprovidemolecularsupportthat thehomininremainspresentintheChâtelperronianlayersatthe GrotteduRenneareNeandertals.Moreover,wetestthehypothesisthatthesehomininsderivefromtheunderlyingMousterian(i.e.,thehypothesisregardingmovementofthehominin specimensfromtheunderlyingMous terianintotheC hâtelperronian layers).Thishypothesismustberejectedaccordingtothechronologicaldatapresentedhereandisinaccordancewiththespatial positioningofboththenewlyidentifiedhomininspecimensand thepreviouslyidentifiedspecimens,asthesearelocatedinrows 1 – 6outsidetheslopingareaoftheGrotteduRenneortheareas thatmighthavebeenaffectedbydiggingandlevelingactivities (12,13,46).Wecannotcontributemoresubstantiallyonthehypothesisthatsomeculturalartifacts,inparticularthoseinterpretedasbodyornamentsandboneawls,intheChâtelperronian layersderivefromtheoverlying Aurignacian(10,11).Thishas beencontestedelsewhere(14,47).Thereischronological evidencethatatleastsomeoftheseboneartifactsarerelatively insitu(10),andarchaeologicalargumentsthatlarge-scaledisplacementoflithicartifactsisunlikely(14).SimilarculturalartifactsarealsopresentintheChâtelperronianatQuinçay(4), althoughnohomininremainsarepresentthere.AtQuinçay, therearenolaterUpperPaleolithiclevelsoverlyingtheChâtelperronian,whichispartlysealedbyroofcollapse,andsoartifactintrusionfromoverlyinglevelscannotaccountforthebone artifactassociationwithaChâtelperronianlithicassemblage characterizedtechnologicallyasUpperPaleolithic(3).So,if theseculturalartifactsareconsideredrelativelyinsituateither site,thenthesebelongwithintheChâtelperronianNeandertal culturalrepertoire. OurbiomoleculardataprovideevidencethathomininscontemporaneouswiththeChâtelperronianlayershavearchaicnuclearandNeandertalmitochondrialancestry,supportingprevious morphologicalstudies(6,7).Theyarethereforeamongsomeof thelatestNeandertalsinwesternEurasia,andpossiblecandidates tobeinvolvedingeneflowfromNeandertalsintoAMHs(orvice versa)(48).Futureanalysisofthenucleargenomeoftheseor otherChâtelperronianspecimensmightbeabletoprovidefurtherinsightsintothedirection,extent,andageofgeneflow betweenLatePleistoceneWesternEuropeanNeandertalsand “ incoming ” AMHs(49,50). Ourresultsrevealthattheboneproteomeisadynamictissue, reflectingontogenyduringtheearlystagesofbonedevelopment. Thisrealizationrequiresfurtherinvestigation,asthishasnot beenexploredin-depthpreviouslyinthepalaeoproteomicorthe clinicalliterature(25,26).Wehypothesizethat COL10 1 or othercartilage-associatedproteinscouldberetainedinmineralizedtissuesthathavereducedratesofremodeling(cranial sutures,epiphysealplates),mineralizedtissuesthatdonotremodel(dentine),orboneregionsthatareinintimatecontact withothertissuetypes(trabecularbone,articularsurfaces).The excretionof COL10 1 by(pre)hypertrophicchondrocytesimpliesthisproteinwillbelostfromtheboneproteomeduring bonedevelopmentandmaintenanceandmaynotbepresent routinelyinadultfossilhomininspecimens(51).Thepresenceof suchproteinsinthisstudyisconsistentwiththelevelofontogeneticdevelopmentofthebonespecimenanalyzed,asobserved throughisotopicandmorphologicalanalyses.Asaresultofthese observations,futurequantitative(palaeo)proteomicsmightexplainphenotypicdifferencesobservedbetweenmembersofour genusandotherhominidsbystudyingproteincompositionand invivoproteinmodificationofancienthomininspecimens. Theidentificationof COL10 1 andadditionalproteinspredicted tocarryderivedaminoacidsubstitu tionsspecificforLatePleistocenecladeswithinthegenus Homo (34)inthedatapresentedhere andelsewherefornonhomininpalaeoproteomes(25,26)suggests ancientproteinsareaviableapproachtostudythetaxonomicaffiliationofPleistocenefossilhom inins,inparticularwhenancient DNAispoorlyornotpreserved.So,theanalysisofancientproteins providesasecondbiomolecularmethodcapableofdifferentiating betweenLatePleistocenecladeswithinourgenuswhenadequate error-tolerantsearchalgorithmsareused.Furthermore,thecontextualanalysisoftheseancientp roteinshasthepotentialtoprovideinvivodetailsonancienthomininontogeny,physiology, andphenotype. Methods Wescreened196bonespecimensusingZooMS( SIAppendix ,Fig.S1 )and taxonomicallyidentifiedtheseusingpreviouslypublishedandnewlyobtained ZooMSCOL1peptidemarkermasses(15 ,21).Osteologicalanalysisofbone specimensidentifiedashomininaes uggestthesespecimenspossiblyrepresentanimmatureinfant.LC-MS/MSanalysiswasconductedonthree( SI Appendix ,Fig.S1 )ofthehomininbonespecimens,aspublishedpreviously fornonhomininbonespecimens(19),aswellastwoadditionalanalyses ofonepalaeoproteomicextractgene ratedfollowingamodifiedprotein Fig.3. Bayesianmodelofradiocarbon datesfortheGrotteduRenne. Modelconstructedincludingallrad iocarbondatesreportedinref.13. Archaeologicallayerboundariesareshown,withthedirectdatesonthe GrotteduRenne(AR-14)andtheSaint-Césairehomininshighlightedin red.Posterior/prioroutlierprobabilityforAR-14isshowninsquareparenthesis(moredetailsin SIAppendix , RadiocarbonDatingandBayesian ModelingofAR-14 ). 11166 | www.pnas.org/cgi/doi/10.1073/pnas.1605834113 Welkeretal.

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extractionprotocol(52).Wetookmeasurestoavoidanddetectprotein contaminationthroughoutourpalaeoproteomicworkflow.AncientDNA analysisfollowedprotocolsoutlinedelsewhere(53,54),asdidstableisotopeandradiocarbonanalysisofcollagenextracts(55).Allbiomolecular extractionswereperformedindedica tedfacilitiesattheMaxPlanckInstituteforEvolutionaryAnthropology.Extendedmethodscanbefoundin SIAppendix .COL1databasesamplescanbeaccessedviaProteomeXchange withidentifierPXD003190,andLC-MS/MSdataforAR-7,AR-16,andAR-30via PXD003208.TheDNAsequencesanalyzedinthisstudyweredepositedinthe EuropeanNucleotideArchiveunderaccessionnumberPRJEB14504. ACKNOWLEDGMENTS. 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