The maternal genetic make-up of the Iberian Peninsula between the Neolithic and the Early Bronze Age

1 SCIENTIFIC REPORTS | 7: 15644 | DOI:10.1038/s41598-017-15480-9 www.na u e.com/scien i ic epo s The ma e nal gene ic make-up o he Ibe ian Peninsula be ween he Neoli hic and he Ea ly B onze Age Anna Szécsényi-Nagy 1, Ch is ina Ro h2, Guido B and 3, C is ina Rihue e-He ada 4, C is ina Tejedo -Rod íguez5, Pe a Held2, Íñigo Ga cía-Ma ínez-de-Lag án5, Héc o A cusa Magallón5, S ephanie Zesch6, Co ina Knippe 7, Esz e Bán y8, Susanne F iede ich9, Ha ald Melle 9, P imi i a Bueno Ramí ez10, Rosa Ba oso Be mejo10, Rod igo de Balbín Beh mann10, Ana M. He e o-Co al11, Raúl Flo es Fe nández12, Ca men Alonso Fe nández13, Ja ie Jiménez Eche a ia13, Lau a Rindlisbache 14, Camila Olia 4, Ma ía-Inés F egei o4, Ignacio So iano4, O iol Vicen e 4, Ra ael Micó4, Vicen e Lull4, Jo ge Sole Díaz15, Juan An onio López Padilla15, Consuelo Roca de Togo es Muñoz15, Mau o S. He nández Pé ez16, F ancisco Ja ie Jo e Maes e16, Joaquín Lomba Mau andi17, Azucena A ilés Fe nández17, Ka ina T. Lillios18, Ana Ma ia Sil a 19,20, Miguel Magalhães Ramalho21, Luiz Miguel Oos e beek22, Claudia Cunha23, Anna J. Wa e man24, Jo di Roig Buxó25, And és Ma ínez26, Juana Ponce Ma ínez27, Ma k Hun O iz27, Juan Ca los Mejías-Ga cía 27, Juan Ca los Pece o Espín27, Rosa io C uz-Auñón B iones27, Tiago Tomé28, Edua do Ca mona Balles e o29, João Luís Ca doso30, Ana C is ina A aújo31, Co ina Liesau on Le ow-Vo beck 32, Concepción Blasco Bosqued32, Pa icia Ríos Mendoza32, Ana Pujan e33, José I. Royo-Guillén34, Ma co Au elio Esquemb e Be iá35, Vic o Manuel Dos San os Goncal es36, Rui Pa ei a36, Elena Mo án He nández36, Elena Méndez Izquie do37, Jo ge Vega y Miguel38, Robe o Menduiña Ga cía38, Vic o ia Ma ínez Cal o39, Osca López Jiménez39, Johannes K ause3, Sand a L. Pichle 14, Ra ael Ga ido-Pena32, Michael Kuns 40, Robe o Risch4, Manuel A. Rojo-Gue a41, Wol gang Haak 3,42 & Ku W. Al 43,44 1Labo a o y o A chaeogene ics in he Ins i u e o A chaeology, Resea ch Cen e o he Humani ies, Hunga ian Academy o Sciences, Budapes , Hunga y. 2Ins i u e o O ganismic and Molecula E olu ion, Johannes Gu enbe g Uni e si y, Mainz, Ge many. 3Max Planck Ins i u e o he Science o Human His o y, Jena, Ge many. 4Depa amen o de P ehis o ia, Uni e si a Au ònoma de Ba celona, Ba celona, Spain. 5A cadia-Gene al Founda ion o Valladolid Uni e si y, Valladolid, Spain. 6Ge man Mummy P ojec , Reiss-Engelho n-Museen, Mannheim, Ge many. 7Cu - Engelho n-Zen um A chäome ie gGmbH, Mannheim, Ge many. 8Romano-Ge manic Commission, Ge man A chaeological Ins i u e, F ank u am Main, Ge many. 9S a e O ice o He i age Managemen and A chaeology Saxony-Anhal and S a e Museum o P ehis o y, Halle, Ge many. 10Depa amen o de His o ia y Filoso ía, Uni e sidad de Alcalá de Hena es, Alcalá de Hena es, Spain. 11Depa amen o de P ehis o ia, Uni e sidad Complu ense de Mad id, Mad id, Spain. 12P o essional A chaeologis , Pa la (Mad id), Spain. 13C onos SC A queología y Pa imonio, Bu gos, Spain. 14In eg a i e P ehis o y and A chaeological Science, Uni e si y o Basel, Basel, Swi ze land. 15Museo A queológico de Alican e-MARQ, Alican e, Spain. 16Depa amen o de P ehis o ia, A queología, His o ia An igua, Filología G iega y Filología La ina, Uni e sidad de Alican e, Alican e, Spain. 17Depa amen o de P ehis o ia, A queología, His o ia An igua, His o ia Medie al y Ciencias y Técnicas His o iog á icas, Uni e sidad de Mu cia, Mu cia, Spain. 18Depa men o An h opology, Uni e si y o Iowa, Iowa Ci y, Iowa, Uni ed S a es o Ame ica. 19Labo a o y o P ehis o y, CIAS, Depa men o An h opology, Uni e si y o Coimb a, Coimb a, Po ugal. 20UNIARQ, Uni e si y o Lisbon, Lisboa, Po ugal. 21Labo a ó io Nacional de Ene gia e Geologia, I.P., Museu Geológico, Lisboa, Po ugal. 22Ins i u o Poli écnico de Toma , Ins i u o Te a e Memó ia, Geosciences Cen e o Coimb a Uni e si y, Toma , Po ugal. 23P og ama de Capaci ação Ins i ucional MCTIC/MPEG, Museu Pa aense Emílio Goeldi, Belém, B azil. 24Depa men o Na u al and Applied Sciences, Moun Me cy Uni e si y, Ceda Rapids, Iowa, Uni ed S a es o Ame ica. 25A ago L d, Ba celona, Spain. 26Museo A quelógico de Lo ca, Lo ca, Spain. 27Depa amen o de P ehis o ia y A queología, Uni e sidad de Se illa, Se illa, Spain. 28Uni e sidade Fede al do Pa á, Campus Uni e si á io Recei ed: 8 Feb ua y 2017 Accep ed: 27 Oc obe 2017 Published: xx xx xxxx OPEN www.na u e.com/scien i ic epo s/ 2 SCIENTIFIC REPORTS | 7: 15644 | DOI:10.1038/s41598-017-15480-9 Ag icul u e i s eached he Ibe ian Peninsula a ound 5700 BCE. Howe e , li le is known abou he gene ic s uc u e and changes o p ehis o ic popula ions in di e en geog aphic a eas o Ibe ia. In ou s udy, we ocus on he ma e nal gene ic makeup o he Neoli hic (~ 5500–3000 BCE), Chalcoli hic (~ 3000–2200 BCE) and Ea ly B onze Age (~ 2200–1500 BCE). We epo ancien mi ochond ial DNA esul s o 213 indi iduals (151 HVS-I sequences) om he no heas , cen al, sou heas and sou hwes egions and hus on he la ges a chaeogene ic da ase om he Peninsula o da e. Simila o o he pa s o Eu ope, we obse e a discon inui y be ween hun e -ga he e s and he i s a me s o he Neoli hic. Du ing he subsequen pe iods, we de ec egional con inui y o Ea ly Neoli hic lineages ac oss Ibe ia, howe e he gene ic con ibu ion o hun e -ga he e s is gene ally highe han in o he pa s o Eu ope and a ies egionally. In con as o ancien DNA indings om Cen al Eu ope, we do no obse e a majo u no e in he m DNA eco d o he Ibe ian La e Chalcoli hic and Ea ly B onze Age, sugges ing ha he popula ion his o y o he Ibe ian Peninsula is dis inc in cha ac e . The changeo e om a hun e -ga he e li es yle o a p oduc i e mode o subsis ence i s eme ged a ound 10,000 BCE in he Nea Eas 1–3. This so-called ‘Neoli hic ansi ion’ b ough abou undamen al changes in economy, social s uc u e, demog aphy and human heal h, and laid he ounda ions o ag a ian socie ies and hus o ancien ci iliza ions. O e he cou se o he 7 h and 6 h millennia BCE, ag icul u e sp ead om he Balkans o Cen al Eu ope. Ano he ou e o dissemina ion an along he Medi e anean coas lines o G eece, I aly and he sou h o F ance o he Ibe ian Peninsula and no h o he Pa is Basin and Cen al Eu ope4. Howe e , he p ocess o Neoli hisa ion was non-linea , wi h he a chaeological eco d documen ing in luences o local cul u al a- di ions5. In Ibe ia, he Neoli hic ansi ion, which began a ound 5700 BCE, appea s o ha e been complex, and Mesoli hic and a ming communi ies coexis ed and in e ac ed o as long as wo millennia6. The a e ac ual emains, mainly ce amic, a es o he di e en o igins and modes o Neoli hisa ion on he Ibe ian Peninsula. On one hand s ands a Medi e anean ma i ime coloniza ion by Neoli hic pionee s cha ac e - ized by ce amic wi h clea pa allels o he Ligu ian Imp essa collec ions o I alian o igin7–9. Some Ea ly Neoli hic si es we e also loca ed in he hin e land, sugges ing u he ou es o dissemina ion h ough he Py enees and/o along majo i e s, such as he Eb o10,11,16. On he o he hand, No h A ican in luences and con ac s a e angible in he sou he n Ibe ian Neoli hic12,13. All in all, Ibe ia appea s a mel ing po o in luences and g oups, combining Neoli hic li eways and wi h indigenous mechanisms o adap a ion14. In he Ea ly Neoli hic, we can obse e common ea u es sha ed o e la ge a eas, bu also some egionally es ic ed phenomena15. In he whole e i o y, o example, he e exis ed sophis ica ed sys ems o ag icul u e and li es ock handling, wi h adap able c ops16,17 and seasonal s a egies in lock managemen 18–20. The g oups o he F anco-Ibe ian Ca dial and he Epica dial po e y s yles appea ed a his ime and ecen s udies ha e e ealed mu ual diach onic in luences in he ma e ial cul u e and economies o hese cul u es15,21. An inc easing numbe o bu ials om ha epoch ha e come o ligh in he las yea s22–24. The oldes a e indi idual inhuma ions, some imes g ouped in ceme e ies25. I is also common o ind human emains in ca es, which we e inc easingly used o collec i e bu ials26. F om he la e 5 h millennium BCE onwa d, bu ial monu- men s appea ed, and megali hic ombs became widesp ead27. This phenomenon links Ibe ia wi h o he pa s o Eu ope, indica ing long-dis ance ne wo ks o communica ion. Meanwhile, ce ain pa s o no heas Ibe ia main ained indi idual inhuma ions in pi s, mos ly in small ceme e ies28. Besides megali hic ombs, di ched enclo- su es ex ending o e mo e han 100 hec a es s a ed o domina e he landscapes o sou he n and cen al Ibe ia om 3300 o 3100 BCE, highligh ing ano he widely sp ead Eu opean phenomenon29,30. Du ing he Ibe ian Chalcoli hic pe iod (3000–2200 BCE), o i ied se lemen s wi h s one walls and semi-ci cula bas ions appea ed in he wes e n and sou he n pa s o he Peninsula, while elsewhe e, open se lemen s we e s ill ex an 31. The di e si y in se lemen and bu ial ypes sugges s he exis ence o social s uc u es wi h di e en le els o com- plexi y32. A he same ime, as exchange ne wo ks, ci cula ed p ecious goods such as i o y om A ica and e en Asia o Ibe ia33. F om ~2600 BCE onwa ds, he so-called ‘Bell Beake phenomenon’ became mani es wi h i s cha ac e is- ic po e y, coppe weapons, gold o namen s, and o he p es ige goods, an a chaeological e lec ion o impo - an social and economic changes which sp ead ac oss as egions o wes e n and cen al Eu ope34. Ibe ia’s Bell Beake assemblages a e among he iches and mos di e se in wes e n Eu ope35, bo h in e ms o se lemen s and do Guamá, Belém, B azil. 29Se icio Te i o ial de Cul u a de Valladolid, Valladolid, Spain. 30Depa amen o de Ciencias Sociais e de Ges ao, Uni e sidade Abe a, Lisboa, Po ugal. 31Resea ch Cen e in Biodi e si y and Gene ic Resou ces (CIBIO-InBIO) & Di ec o a e-Gene al o Cul u al He i age (DGPC), Lisboa, Po ugal. 32Depa amen o de P ehis o ia y A queología, Uni e sidad Au ónoma de Mad id, Mad id, Spain. 33A queología Es udios, Mu cia, Spain. 34Technical a chaeologis , Gobie no de A agón, Za agoza, Spain. 35A pa Pa imonio L d, Alican e, Spain. 36Cen o de A queologia da Uni e sidade de Lisboa (Unia q), Lisboa, Po ugal. 37G upo de In es igación HUM-949 TELLUS, P ehis o ia y A queología en el Su de Ibe ia, Uni e sidad de Se illa, Se illa, Spain. 38ARGEA L d., Mad id, Spain. 39GIPSIA L d., Toledo, Spain. 40Ge man A chaeological Ins i u e Mad id, Mad id, Spain. 41Depa amen o de P ehis o ia y A queología, Uni e sidad de Valladolid, Valladolid, Spain. 42School o Biological Sciences, The Uni e si y o Adelaide, Adelaide, Aus ia. 43Cen e o Na u al and Cul u al His o y o Man, Danube P i a e Uni e si y, K ems, Aus ia. 44Depa men o Biomedical Enginee ing and Depa men o En i onmen al Science, Basel Uni e si y, Basel, Swi ze land. Co espondence and eques s o ma e ials should be add essed o A.S.-N. (email: szecsenyi-nagy. [email p o ec ed]) o K.W.A. (email: [email p o ec ed]) www.na u e.com/scien i ic epo s/ 3 SCIENTIFIC REPORTS | 7: 15644 | DOI:10.1038/s41598-017-15480-9 bu ials36. I has hus long been a ocus o a chaeological esea ch, commencing om mig a ionis hypo heses and leading up o cu en social explana ions, whe e he Bell Beake phenomenon is pe cei ed as a package o p es ig- ious objec s exchanged and consumed by eli e g oups and displayed on special occasions37. A ound 2200 BCE, he Chalcoli hic se lemen and une a y p ac ices we e suddenly discon inued, pa icu- la ly in he wes e n and sou he n pa o Ibe ia, whe e mos o he di ched and o i ied se lemen s we e aban- doned and collec i e si es and megali hic ombs we e eplaced by indi idual bu ials31. El A ga g oup began o eme ge in sou heas Ibe ia, wi h la ge and massi ely o i ied u ban cen e s like La Bas ida (Mu cia, n. 28 on Fig.1)38, which managed o con ol a e i o y o o e 35,000 km2 du ing he ollowing 650 yea s. Wi h ega d o i s popula ion his o y, he Ibe ian Peninsula has been he ocus o se e al ecen a chaeogene ic s udies39–44. S udies ocusing no only on m DNA bu also on Y ch omosome ma ke s ha e suppo ed he model o a pionee coloniza ion in he no h-eas e n coas al egions o he Ibe ian Peninsula a he onse o he Ibe ian Neoli hic39,45. On he o he hand, he no he n pa o Spain (Can ab ian inge) a es ed o a a he complex Neoli hic ansi ion42. Recen m DNA and genome-wide analyses ha e pu an emphasis on he gene ic a ini y and sha ed Nea Eas e n ances y be ween he ea ly Ibe ian a me s and he con empo a y Cen al Eu opean Linea bandke amik (LBK) popula ion46–48. The la es m DNA and genomic s udies ha e e ealed inc eased sub- sequen admix u e o hun e -ga he e elemen s du ing he local middle Neoli hic (La Mina; Al o de Reinoso, n. 51–52 on Fig.1)44,47 and he Chalcoli hic (El Po alón; El Mi ado , n. 60–62. on Fig.1) again eminiscen o p ocesses obse ed in Cen al Eu ope41,49,50. Despi e nume ous esea ch p ojec s being ca ied ou o e he pas yea s, he Neoli hic se lemen his o y o Eu ope can s ill only be explained a a b oad scale47,49,51–54. Regional ansec s h ough ime de ailing he de el- opmen s and he cou se o he Neoli hic in cen al Ge many55–57 and in he Ca pa hian Basin58 ha e mos ly been examined by mi ochond ial DNA (m DNA) con ol egion da a. Ou p ojec comple es he la e se ies by ocusing on he a chaeological models and hypo heses ha ha e been pu o wa d o he Ibe ian Peninsula, and whe e diach onic (i.e. ‘ h ough ime’) sampling o ancien DNA allows he de ec ion o demog aphic changes and discon inui ies be ween 5700 and 1500 BCE. Essen ial esea ch ques ions ocus on h ee le els: i) he indi idual si es, ii) he Ibe ian Peninsula as a scene o Neoli hic ansi ion and iii) a compa ison wi h con empo aneous ancien and mode n-day Eu opeans. A key ques ion o ou s udy o he m DNA di e si y on he Ibe ian Peninsula h ough ime was o examine o wha ex en egional and sup a- egional cul u al g oups could be ecognized as gene ically iden i iable en i ies, as shown in o he a eas o Eu ope46,55. A ela ed ques ion was whe he he cul u al b eaks ha can be seen, o ins ance, a he end o he Neoli hic and Chalcoli hic pe iods, we e also accompanied by human popula ion u no e s. Resul s We p ocessed ancien DNA samples o 318 human indi iduals om p ehis o ic Ibe ia and gene a ed ep oducible mi ochond ial hype a iable egion haplo ypes (HVS-I, np 16020–16401) om 151 indi iduals ollowing s ic au hen ica ion c i e ia (see Me hods). M DNA haplog oup classi ica ion o u he 62 samples was based on mul- iplex yping o single nucleo ide polymo phisms (SNPs) (Fig.1, Supplemen a y Tables S1–4). The DNA ampli- ica ion and ep oduc ion success o he HVS-I showed s ong di e ences among samples om di e se egions o he Ibe ian Peninsula: he highes ampli ica ion success a es we e obse ed in no heas Ibe ia (NEI) and highe al i ude egions o cen al Ibe ia (CI) (71–78%), while especially he sou he n pa o he Ibe ian Peninsula (sou heas e n Ibe ian (SEI) g oup and sou hwes Ibe ia (SWI) had e y low ampli ica ion success a es (20–43%). We me ged hese da a wi h p e iously published HVS-I m DNA esul s om 125 p ehis o ic indi iduals (Supplemen a y TablesS5, S15), and sepa a ed he HVS-I da ase o 238, and m DNA haplog oup in o ma ion o 305 p ehis o ic Ibe ian indi iduals in o geog aphically and ch onologically de ined g oups (see Me hods). The esul ing haplog oup composi ions o hese g oups a e p esen ed in Fig.2. Haplog oup based analyses. The Ibe ian p e-Neoli hic m DNA subs a e is s ill poo ly unde s ood. Recen NGS s udies desc ibed hun e -ga he e s belonging o haplog oup U5b in no h Ibe ia43,59, while he sug- ges ed p e- a ming p esence o haplog oups H, N and U442 we e epo ed in s udies ha did no mee ou s ic au hen ica ion c i e ia (see Me hods). The e o e, we used Holocene pe iod hun e -ga he e s om Cen al, Wes and No h Eu ope as p oxy o he Ibe ian p e-Neoli hic m DNA subs a e in ou quan i a i e analyses (Fig.2, Supplemen a y TablesS6, S15). This con inen al hun e -ga he e da ase (HG_HOL, n = 35) con ains p edomi- nan ly U haplog oups (U5b and U5a domina ed, bu also R, U2, U4, U* we e obse ed53,57,58,60–63). In Ibe ia, hap- log oup U5b was ound mos equen ly in he CI Neoli hic da ase (19.4%; n = 62), and showed also ela i e high equency in he CI Chalcoli hic (13.9%; n = 79) and NEI Chalcoli hic-Ea ly B onze Age g oups (14.8%; n = 27). The haplog oup composi ion o he Ibe ian Neoli hic popula ion shows simila i ies o he Ea ly Neoli hic da a om Ana olia (~6500–6000 BCE), he Ca pa hian Basin (~5800–4900 BCE), and Cen al Eu ope (~5500–4000 BCE, ep esen ed by he cen al Ge man Mi elelbe-Saale egion)49,55,58. Haplog oups K, J, T2, HV, V and X a e obse ed in compa able equencies in Ibe ian and Cen al Eu opean g oups. Howe e , he p opo ion o hap- log oup H is highe in he Ibe ian Ea ly Neoli hic (EN) (22.2%; n = 27) han in Cen al Eu ope (15%; n = 160), while he equency o N1a is e y low (3.7% compa ed o 9.4% in Cen al Eu ope). Ano he di e ence wi h ega ds o Cen al Eu ope is he occu ence o haplog oup N* in Neoli hic NEI g oup (Supplemen a y TablesS6, S15). We used he haplog oup equencies o he s udied g oups o p incipal componen analyses (PCA, see Me hods). When compa ed o published ancien DNA da a om he Nea Eas , he wes e n Ana olian Neoli hic popula ion is mos simila o Cen al Eu opean Ea ly-Middle Neoli hic popula ions and o he Neoli hic o no heas Ibe ia (Fig.3, Supplemen a y Fig. S1–2), bu i also shows some a ini ies wi h u he Ibe ian a me s h ough common EN haplog oups (e.g. K, J, T2)49. The p esence o haplog oup N* in he P e-Po e y Neoli hic in Sy ia and in NEI can also be in e p e ed in his ligh 39,64, bu his link needs o be e i ied by whole mi ogenome www.na u e.com/scien i ic epo s/ 4 SCIENTIFIC REPORTS | 7: 15644 | DOI:10.1038/s41598-017-15480-9 Figu e 1. Map o he s udied si es, including he published e e ence da a and iming o a chaeological pe iods on he Ibe ian Peninsula and in Cen al Eu ope. Geog aphic egions, also di e en ia ed in he m DNA analyses, a e indica ed as: NEI: no heas , SEI: sou heas , SWI: sou hwes Ibe ia. Numbe s on he map a e colo ed acco ding o he ch onological pe iods, ep esen ed in he lowe pa o he igu e. Fo he Cen al Eu opean ch onology we used eco ds om he mos impo an compa a i e egion o cen al Ge man Mi elelbe- Saale55. See Supplemen a y TableS5 o u he de ails. Si e codes: 1. Moi a do Sebas ião, 2. Gale ia da Cis e na (Almonda ca e), 3. G u a de Nossa Senho a das Lapas, 4. G u a do Cada al, 5. G u a das Alcobe as, 6. G u a do Poço Velho, 7. G u a dos Ossos, 8. Tholos de Pai Mogo I, 9. Hipogeu de Mon e Canelas I, 10. Hipogeu de Mon e Canelas III, 11. Bolo es, 12. G u a de Malgas a, 13.Valencia de la ConcepciónA ea 9, 14. G u a do Ca alhal de Tu quel, 15. Cob e las C uces, 16. Co a de l’O , 17. Co a de la Sa sa, 18. Co a d’en Pa do, 19. Molinos del Papel, 20. Co a del Ba anc del Migdia, 21. Co a del Can al, 22. Camino de Molino, 23. Fuen e Álamo, 24. Lo ca-Los Tin es, 25. Lo ca-Mad e Me ceda ias, 26. Lo ca-Cas illo de Lo ca, 27. Rincón de Moncada, 28. La Bas ida, 29. Tabayá, 30. Ille a dels Banye s, 319. Co a Bonica, 32. Can Sadu ní, 33. Co a d’A ellane , 34. Els T ocs, 35. San Pau de Camp, 36. Ba anc d’en Ri à, 37. Balma de Sa gan ana, 38. Co a de la Ven osa, 39. Co a de Mon anissel, 40. Miguel Vi es, 41. Can Gambús, 42. Cha es, 43. Valdescusa, 46. Al o de Rodilla, 47. Fuen e Celada, 48. Fuen e Pecina 1, 49. Fuen e Pecina 2, 50. Fuen e Pecina 4, 51. Al o de Reinoso, 52. La Mina, 53. La Ta ayuela, 54. El Juncal, 55. A oyal I, 56. El Hundido, 57. Camino de las Yese as, 58. Humanejos, 59. Valle de las Higue as, 60–61. El Po alón, 62. El Mi ado , 63. Es Fo a de ses A i ges. Backg ound map o Ibe ia (copy igh holde Uni e si a Au ònoma Ba ceolna) was modi ied in Adobe Illus a o CS6 so wa e (www.adobe.com/p oduc s/illus a o ). www.na u e.com/scien i ic epo s/ 5 SCIENTIFIC REPORTS | 7: 15644 | DOI:10.1038/s41598-017-15480-9 analyses. Haplog oup composi ions o all o he Ibe ian g oups a e highly simila o each o he , esul ing in clus- e s on he PCA plo s and Wa d clus e ing ee. Ou la ges Ibe ian da ase s a e om Neoli hic and Chalcoli hic o CI (n = 62 and 79), which show an ele a ed equency o U5b compa ed o he NEI Neoli hic. In e es ingly, we also obse e p obable hun e -ga he e haplog oups U2 and U4, whe eas he cha ac e is ic EN haplog oups N*, N1a a e missing in he CI egion. The sou heas e n Ibe ian da ase (n = 48) con ains eigh La e Neoli hic indi iduals wi h haplog oups K, H and U5b, 28 indi iduals om he Chalcoli hic and 12 om he Ea ly B onze Age (EBA). Simila ly o he CI g oups, SEI shows a mix u e o haplog oups o he NEI g oup, bu some o hem (N*, N1a, U3) a e missing. The numbe o Neoli hic and Chalcoli hic SWI samples (n = 12 and n = 31) is oo small o es ima e he popu- la ion dynamics in he egion eliably (Supplemen a y TableS6). The combined da ase shows a p edominance o haplog oup K (35.7%) and high equency o haplog oups H, J and T (16.7–14.3–14.3%). As Middle Neoli hic (~ 4500–3500 BCE) and La e Neoli hic (~3500–3000 BCE) pe iods o mos si es a e no sepa able a he cu en s a e o a chaeological esea ch, we combine hese da a in ou analyses (MLN). Fishe ’s exac (p = 0.5473) and popula ion con inui y (p = 0.3385–0.3722) es s con i m he connec ion be ween he EN and MLN pe iods o Ibe ia in gene al. Some o he Ibe ian Neoli hic m DNA haplog oups (U2, N*, N1a) a e no obse ed in he successi e Chalcoli hic Ibe ian popula ion (n = 156), whe eas o he s main ain a s eady equency (V, T2, X) h oughou 3500 yea s. An in e es ing excep ion is haplog oup L1b in he La e Chalcoli hic cen al Ibe ia a he si e Camino de las Yese as (n. 57 on Fig.1), nea Mad id. This g oup is mos equen in oday’s Wes -Cen al A ica65, and hin s a a connec ion o he No h-Wes A ican coas s in p ehis o ic imes. In con as o U5b, haplog oup U5a (common in Cen al Eu opean Mesoli hic and La e Neoli hic55,5760) appea s i s in he 3000–2500 BCE pe iod o cen al Ibe ia. O he ypical Cen al Eu opean and Ca pa hian Basin Neoli hic haplog oups such as H5, T1, W and U8, which a e obse ed in la e Cen al Eu opean Neoli hic pe iods, a e missing om he en i e p ehis o ic Ibe ian da ase . These quali a i e di e ences accoun o he sepa a ion o mos o he Ibe ian p ehis o ic g oups om he Cen al Eu opean Neoli hic and Ea ly B onze Age popula ions along he second componen o he PCA, shown on Fig.3. Tes s o popula ion con inui y and Fishe ’s exac es be ween he whole Ibe ian Ea ly and La e Chalcoli hic da ase s esul in non-signi ican p alues (p = 0.5444–0.5578 and 0.9779), which suppo popula ion con inui y be ween he wo pe iods. Un o una ely, a ine ch onological sepa a ion o he Chalcoli hic (CHA) da ase can- no be achie ed o all samples and he e o e we conside only a subse o he CHA da ase (n = 71 o Ea ly and n = 47 o La e CHA) in his analysis (Supplemen a y TableS8). We also es ed whe he his con inui y could be he esul o da a me ging ac oss bigge geog aphic dis ance. Howe e , he gene ic con inui y du ing he Ea ly o La e Chalcoli hic ansi ion also holds a egional scale, as ou popula ion con inui y es canno ejec con inui y be ween he Neoli hic (con aining mainly La e Neoli hic) and CHA g oups o cen al Ibe ia (p = 0.6033–0.6143), and NEI (p = 0.9038–0.7835). This con inuous gene ic makeup is also suppo ed by non-signi ican (p = 0.2264 and 0.4484) di e ences in haplog oup composi ions (Supplemen a y TablesS7–8). Figu e 2. M DNA haplog oup composi ion o he p ehis o ic Ibe ian g oups. Abb e ia ions: Hun e - ga he e s in Eu ope om he Holocene (HG_HOL), no heas Ibe ian Neoli hic (NEI_Neo), no heas Ibe ian Chalcoli hic and Ea ly B onze Age (NEI_CHA_EBA), cen al Ibe ian Neoli hic (CI_Neo), cen al Ibe ian Chalcoli hic (CI_CHA), sou hwes Ibe ian Neoli hic (SWI_Neo), sou hwes Ibe ian Chalcoli hic (SWI_CHA), sou heas Ibe ian Neoli hic (SEI_Neo), Chalcoli hic and B onze Age (SEI_ CHA_EBA). Rela i e haplog oup equencies a e p esen ed in Supplemen a y TableS6. The backg ound map o Ibe ian Peninsula (h ps://en.wikipedia.o g/wiki/File:Relie _Map_o _Spain.png# ile) is licensed unde CC BY-SA 3.0 (h ps:// c ea i ecommons.o g/licenses/by-sa/3.0/) was modi ied in Adobe Illus a o CS6 so wa e (www.adobe.com/ p oduc s/illus a o ). www.na u e.com/scien i ic epo s/ 6 SCIENTIFIC REPORTS | 7: 15644 | DOI:10.1038/s41598-017-15480-9 The Ea ly B onze Age sample se in he Ibe ian p ehis o ic ansec is s ill e y small (a o al o n = 37 indi- iduals om all ou egions s udied). A he m DNA haplog oup le el he EBA does no show new in luences o popula ion changes a he onse o he Ibe ian B onze Age acco ding o esul s om Fishe ’s es and es o popula ion con inui y, when bo h he en i e Peninsula and sou heas e n Ibe ia in pa icula we e conside ed (Supplemen a y TablesS7-8). A mo e de ailed analysis o H subhaplog oups ocuses on 17 SNPs in he coding egion o he human mi og- enome. The la ges p opo ion o he H indi iduals belongs o he subhaplog oup H1 (65.1%), and he second la ges g oup is subhaplog oup H3 (14%), while 18.6% o he H indi iduals canno be assigned o any o he sub- g oups included in he H-PLEX assay (see Me hods, Supplemen a y TablesS3-4)66. H3 is de ec ed in Chalcoli hic indi iduals om cen al, sou heas and sou hwes Ibe ia. H1 is obse ed in each pe iod and egion, bu mo e equen ly in he Chalcoli hic and Ea ly B onze Age han in he Neoli hic. The compa a i e ancien H da a om he Eu opean p ehis o y is oo spa se o in-dep h s a is ical analyses. Howe e i becomes appa en om ou esul s ha he H di e si y in p ehis o ic Ibe ia is di e en om he H di e si y o Cen al Eu ope55,58,67,68, and mo e simila o he Neoli hic popula ions in F ance69,70. No ably, common Cen al Eu opean subhaplog oups H5 and H767,68 ha e no ye been obse ed in Sou hwes Eu ope. O e all, we do no obse e di ec links be ween he Cen al Eu opean La e Neoli hic (ch onologically compa- able o he Chalcoli hic in Ibe ia) and he Ibe ian (en i e and la e) CHA g oups a he haplog oup le el, and hus canno con i m a La e Chalcoli hic expansion owa d Cen al Eu ope as sugges ed by haplog oup H mi ogenome da a68. Haplo ype and sequence based analyses. The m DNA a ia ion in p ehis o ic Ibe ia is u he explo ed by HVS-I sequence and haplo ype analyses o 238 indi iduals om he Peninsula. The haplo ype di e si y o he hun e -ga he e popula ions o Cen al Eu ope (HG_PLEI (Pleis ocene): Hd = 0.879; n = 29, Figu e 3. P incipal componen analysis based on haplog oup equencies o 955 indi iduals om 16 p ehis o ic g oups. The i s wo componen s display 42.8% o he o al a iance. G oups a e colo ed acco ding o hei geog aphical posi ions: b own: Ibe ia, pu ple: F ance, och e: Cen al and Eas -Cen al Eu ope, yellow: Eas e n Eu ope and Nea Eas . Fo abb e ia ions o Ibe ian g oups, see legend o Fig.2. Fu he abb e ia ions: Cen al and No h Eu opean hun e -ga he e s om he Holocene (HG_HOL), Neoli hic Ana olia (ANAT) Yamnaya (YAM), Ea ly Neoli hic Ca pa hian Basin (CB_EN), Ea ly Neoli hic Ge many (GER_EN), Middle Neoli hic Ge many (GER_MN), La e Neoli hic Ge many (GER_LN), Ea ly B onze Age Cen al Eu ope (CEU_ EBA), Neoli hic Gu gy si e in F ance (FRA_GUR), Neoli hic T eilles cul u e in F ance (TRE). Fo u he in o ma ion see Supplemen a y TableS6. www.na u e.com/scien i ic epo s/ 7 SCIENTIFIC REPORTS | 7: 15644 | DOI:10.1038/s41598-017-15480-9 HG_HOL (Holocene): Hd = 0.931; n = 35) p o ides he baseline o his compa a i e se ies. The di e si y is lowe in he Ea ly Neoli hic o Ibe ia (Hd = 0.926; n = 27) bu inc eases in he ollowing Middle-La e Neoli hic (Hd = 0.933; n = 74) and Chalcoli hic pe iods (Hd = 0.944; n = 118). The Ea ly B onze Age shows again a lowe di e si y (Hd = 0.917; n = 16), al hough his could be he esul o he small numbe o in es iga ed indi id- uals (Supplemen a y TableS9). The haplo ype di e si y among he egional g oups is he lowes in he SWI Neoli hic-Chalcoli hic g oup in Ibe ia, and ha along wi h he de ec ed common basal J and K haplo ypes could cause po en ial bias in lineage sha ing esul s p esen ed below. The haplo ype sha ing (SHA) be ween he la ge ch onological g oups de ec s no hun e -ga he e con ibu- ion in he EN g oup o Ibe ia, and a low le el o hun e -ga he e ma e nal lineages in he MLN pe iods (6.8%) (Table1). Du ing he MLN, se e al new hun e -ga he e - ype U5b lineages appea . Be ween he wo successi e Neoli hic pe iods, he haplo ype sha ing amoun s o 50%, and 43.2% o he MLN lineages a e al eady p esen in he EN (as seen om ou ances al SHA). In he Chalcoli hic pe iod, we also obse e a s ong ances al EN con ibu ion (44.9%), wi h only 14.4% o he lineages o igina ing om la e Neoli hic pe iods. The con inui y o lineages is also seen in he smalle EBA da ase , which shows close connec ions o EN, MLN and CHA pe iods, and in which only 18.8% o he haplo ypes a e new (Table1, Supplemen a y TableS10). The Ibe ian ea ly a ming g oups sha e gene ally high numbe o lineages (38.3–82.4%) wi h each o he (Supplemen a y Fig.S3). The Cen al Eu opean Ea ly Neoli hic has he highes numbe o sha ed haplo ypes compa ing o he no heas Ibe ian g oups, bu he sha ing is also high wi h sou heas Ibe ian da ase . In con- as , lineage sha ing wi h Holocene pe iod hun e -ga he e s is he highes in he mos ly Chalcoli hic sou heas Ibe ian g oup (8.6%) and in CI Neoli hic (6.7%). Conce ning empo al succession in NEI egion, he p opo ion o hun e -ga he e lineages inc eases ( om 0 o 4.8%) du ing he Chalcoli hic pe iod (Supplemen a y TableS10). Con inui y be ween Neoli hic and Chalcoli hic g oups is e lec ed by he amoun o sha ed lineages be ween he wo pe iods in CI (61.2%), bu bo h CI da ase also sha e many lineages wi h all o he Ibe ian g oups (Supplemen a y TableS10). Gene ic dis ances (Fs ) a e gene ally low among he Ibe ian p ehis o ic g oups, and none o hem a e signi - ican (Supplemen a y TableS11). We obse e he sho es dis ances om Cen al Eu opean EN-MN in no h- eas Ibe ia. By plo ing he HVS-I based gene ic dis ances a e mul idimensional scaling (MDS, Supplemen a y Fig.S4), la ge-scale ela ionships a e displayed. He e, he NEI popula ions a e wi hin he clus e o he o he Ibe ian g oups, whe eas he Cen al Eu opean and F ench da ase s o m a clus e ha is sligh ly o se . In e es ingly, MDS shows ha some g oups (e.g. Neoli hic NEI) appea less di e en ia ed om he emaining popula ions when compa ed o haplog oup-based PCA. The clus e ing o he g oups on MDS plo was he e o e es ed by he analysis o molecula a iance. The bes -suppo ed g oupings (i.e. wi h he highes a iance among he clus e s and lowes wi hin he clus e s) we e e alua ed by i e a i ely al e na ing he composi ion o he g oup- ings. The Ibe ian g oups di e en ia ed om an EN Cen al Eu opean- Ca pa hian Basin and a MLN-EBA Cen al Eu opean- LN F ench clus e s in his analysis as well, and can be u he di ided in o a Neoli hic-Chalcoli hic CI and a NEI-SEI-SWI clus e (Supplemen a y TableS12). We u he compa ed HVS-I sequences o h ee la ge ch onological g oups (EN, MLN and CHA) o he Ibe ian Peninsula wi h 133 mode n popula ions. Gene ic dis ances o mode n-day popula ions a e gene ally low, and es ic ed o ce ain egion(s) o Eu ope (Supplemen a y Fig.S5, Supplemen a y TableS13). The Ea ly Neoli hic shows ela i ely high a ini y o mode n A ghanis an, Pales ine, I an, and Tu key (Fs = 0.00173– 0.00428), bu also high a ini ies o se e al Eu opean popula ions (e.g. I alian, F ench bu also Belo ussian). The gene ic dis ances o mode n popula ions inc ease wi h he MLN and CHA pe iods, bu show gene al simila i y o mode n-day Eu ope. Recen genomic s udies ha e highligh ed he simila i y o ea ly a me s o mode n Sou h Eu opeans71, especially o mode n day Sa dinians47,49. This pic u e is no e lec ed in ou m DNA da ase , whe e A:SHA n sample De ec ed in he ollowing popula ions: HG_HOL EN MLN CHA EBA m DNA lineages: HG_HOL 35 100 0 6.76 5.08 0 EN 27 0 100 50 53.38 28.13 MLN 74 31.43 66.67 100 52.54 25 CHA 118 34.29 66.67 66.22 100 40.63 EBA 16 0 33.33 31.08 43.22 100 B: ances al SHA De ec ed in he ollowing popula ions: n sample HG_HOL EN MLN CHA EBA m DNA lineages: HG_HOL 35 100 EN 27 0 100 MLN 74 6.76 43.24 50 CHA 118 5.08 44.92 14.41 35.59 EBA 16 0 56.25 18.75 6.25 18.75 Table 1. Resul s o sha ed haplo ype analysis (SHA): pe cen age o sha ed HVS-I haplo ypes among he Ibe ian ch onological g oups (A), and ances al haplo ype analysis wi h he s udied Ibe ian g oups (B). Abb e ia ions: Cen al and No h Eu opean hun e -ga he e s om he Holocene (HG_HOL), Ea ly Neoli hic (EN), Middle and La e Neoli hic (MLN), Chalcoli hic (CHA), Ea ly B onze Age (EBA) in Ibe ia. Fo u he de ails, see Supplemen a y TableS10 and Me hods. www.na u e.com/scien i ic epo s/ 8 SCIENTIFIC REPORTS | 7: 15644 | DOI:10.1038/s41598-017-15480-9 Sa dinians ank only he 67 h closes o he Ibe ian EN g oup ou o 133 mode n popula ions, and 46 h om he MLN g oup (Supplemen a y TableS13). Discussion Du ing he Las Glacial Maximum, he Ibe ian Peninsula, jus as Sou heas Eu ope and he I alian Peninsula, o med a classic Glacial Re uge A ea o Eu opean popula ions, well documen ed by a chaeological e idence72–74. The e o e, i is assumed ha pa s o oday’s Eu opean popula ion a e he descendan s o he esiden s o hese e ugia. This heo y is based on coalescence da e es ima es o some m DNA lineages (H1, H3, V, U5b1), which p eda e he Neoli hic expansion, and also based on he high abundances o hese lineages in Ibe ia oday75–79. Howe e , he F anco-Can ab ian glacial e uge heo y has no ye been con i med by ancien m DNA o genomic analyses, and mo e Ibe ian Pleis ocene and especially Holocene samples need o be in es iga ed a ull mi og- enomic esolu ion. Wi h he end o he Las Ice Age and he beginning o he Holocene ~12,000 BCE, a common Eu opean Mesoli hic popula ion eme ged, which also included a dis inc signal o Nea Eas e n o igin59. The indigenous Ibe ian la e Uppe Paleoli hic and Mesoli hic popula ions we e ep esen ed by whole mi ogenome haplo ypes assigned o haplog oup U5b om No he n Spain43,59. The Ibe ian Peninsula is cha ac e ized by di e se landscapes wi h dis inc economic po en ials. Du ing he ini- ial phase o Neoli hisa ion, i s a me s p obably a i ed in no heas Ibe ia p ima ily along he Medi e anean ou e and om he e sp ead along he coas line and i e s (e.g., he Eb o Valley) in o he hin e land7–9,80. Ou gene ic da a suppo a subs an ial in lux o Neoli hic immig an s o no heas e n Spain, whe e a me line- ages a e mos abundan and di e se, and on o o he egions, like he SEI and CI. We ound he pe sis ence o ‘hun e -ga he e ’ m DNA haplog oups in he Neoli hic o be s onges in Cen al Ibe ia. These gene ic esul s ma ch he a chaeological da a on he mode o Neoli hisa ion desc ibed o hese a eas, so ha he ully es ablished Neoli hic Ibe ian communi ies had dis inc hun e -ga he e componen s16,81,82. The u he he ea ly a me s ad anced in o he inne /cen al and sou he n pa s o he Peninsula, he highe he p opo ion o indigenous hun e -ga he e lineages. Geog aphy appea s o ha e been a decisi e ac o in he ad ance o Neoli hic li eways. P ehis o ic cen al Ibe ia (sou he n and no he n Mese a, Amb ona Valley), howe e , was ne e an isola ed egion su ounded by moun ains, bu a he an impo an hub o inno a ions and impulses coming in o Ibe ia83. On a gene ic le el, Neoli hic cen al Ibe ia showed co esponding mi ogenomic connec ions o no heas and sou heas Ibe ian egions (Supplemen a y TableS7). The Py enees a e also an a ea o special in e es wi h ega d o he ou e o immig a ion o he i s a me s. This s udy includes en samples om he ca e si e o Els T ocs, da ed o he Ea ly and Middle Neoli hic20. Due o he conside able empo al di e ences be ween he occupa ions, he espec i e sample se s can be conside ed as gene ically independen . The 14C da a o he six indi iduals om he ea lies phase clus e closely (Supplemen a y In o ma ion). Genome-wide analyses o i e o hese EN indi iduals e ealed he g oup o be ea ly Neoli hic immig an s47,49,50. Haplog oups N1a, J, T2, K and V appea , while he ypical Ibe ian hun e -gah e e m DNA haplog oup U5b is missing. The e o e, we suspec he p esence o a (s ill) isola ed EN g oup a Els T ocs. The isola ed geog aphic posi ion in he Py enees sugges s immig a ion o his communi y om no h o he Py enees (o e en om Cen al Eu ope ia he Rhone Valley) a he han om he wes along he Medi e anean coas line. This al e na i e ga eway o Spain has also been p oposed based on a chaeobo anical e idence17,84. The i s and ( o ou knowledge) sole Ibe ian appea ance o he m DNA haplog oup N1a in an adul om Els T ocs, which ma ches iden ical HVS-I N1a haplo ype in Ana olia and Cen al Eu ope, migh ha e a i ed on he con inen- al ou e47,49,55,85. The sha ed oo s be ween Sou hwes and Cen al Eu ope we e especially conspicuous in he No heas Ibe ian EN g oup, as he ag eemen in he common m DNA haplog oups such as V, J, K, T2 and X sugges s. The ansi ion om he EN o he MLN is documen ed in ecen ancien genomic s udies, which desc ibed an inc ease o hun e -ga he e elemen s in he a ming popula ions o cen al Ibe ia du ing he MLN pe iod a he si e o La Mina and he Chalcoli hic si es o El Mi ado and El Po alón41,47,49. The Ibe ian Middle Neoli hic genomic da a a e s ill oo sca ce (concen a ed on a single si e La Mina) o d aw gene al conclusions abou admix- u e wi h la e hun e -ga he e s47. La Mina people had 18.9–22.8% hun e -ga he e ances y, double han he EN indi iduals om Els T ocs (6.8–11%)50, and also highe han hei con empo a ies in Cen al Eu ope. These hun e -ga he e p opo ion u he inc eased in he CHA o 27%50. In ou s udy, we could suppo he gene al con inui y o ma e nal lineages be ween MLN-CHA pe iods. The inc ease o hun e -ga he e ma e nal lineages h ough ime is no seen on la ge scale SHA (Table1), bu obse able in egional g oups, such as in NEI, whe e he Fs om HG_HOL dec eases and he amoun o HG_HOL haplo ypes inc eases om he Neoli hic o he CHA-EBA pe iods (0–4.76%). The same end can be seen in ou Fs analyses whe e he CI_CHA (Fs = 0.17406) appea close o he HG_HOL g oup han he CI_Neo g oup (Fs = 0.21267) (Supplemen a y TablesS7-8, S11). Acco ding o he da a cu en ly a ailable, he homogenei y o Chalcoli hic ancien DNA esul s sugges s ha human mobili y and gene ic mixing had gene ally inc eased in Ibe ia by he Chalcoli hic. Al hough we analyzed ou geog aphically sepa a ed CHA g oups in he no heas , cen al as well as in he sou heas and sou hwes o he Ibe ian Peninsula, hey did no exhibi any signi ican di e ences excep o he compa ison o he cen al and he sou heas e n g oup (Fishe ’s es esul s in Supplemen a y TableS7, Fig.2). The de ec ion o he ‘A ican’ Lb1 haplog oup a he La e Chalcoli hic si e Camino de las Yese as (Mad id, cen- al Ibe ia) is ema kable, gi en ha i o y ado nmen s o A ican o igin ha e also been documen ed a his and o he con empo aneous si es86–88. The obse a ion o a wes e n-cen al ‘A ican’ haplog oup alongside A ican a e ac s and aw ma e ials in Coppe Age Ibe ia indica e long dis ance exchange ha a leas occasionally seem o ha e in ol ed mobili y o indi iduals and/o gene low. The Bell Beake phenomenon was a decisi e elemen in he Ibe ian Chalcoli hic, las ing om he La e CHA o he EBA (~2600–1800 BCE)35,36. Ou es s o popula ion con inui y and Fishe ’s es s be ween Ea ly and La e www.na u e.com/scien i ic epo s/ 9 SCIENTIFIC REPORTS | 7: 15644 | DOI:10.1038/s41598-017-15480-9 CHA pe iods suppo ed popula ion con inui y be ween he wo phases. Howe e , ou da a s uc u e did no allow s udying he Ea ly o La e CHA popula ion changes a a egional scale, so ha u he s udies migh e eal local a ia ion wi h ega d o indi iduals wi h Bell Beake / non-Bell Beake cul u al a ilia ions. In e es ingly, we did no ind e idence o di ec gene ic links be ween Chalcoli hic Ibe ia and con empo aneous Cen al Eu ope. Links be ween he wo egions had p e iously been sugges ed based in pa icula on Bell Beake elemen s p esen ac oss a wide geog aphic ange89–91, as well as by he ma e nal gene ic analyses o he El Mi ado si e40. In e es ingly, we also do no ind e idence o in lux in he Eas o Wes di ec ion, as none o he in es iga ed Chalcoli hic indi iduals show ‘s eppe ances y’, which seen in con empo aneous Cen al Eu opean Co ded Wa e and Bell Beake g oups, sugges ing ha eas e n in luxes did no each he Ibe ian Peninsula un il la e pe iods49. An e alua ion o his unexpec ed obse a ion will need u he palaeogene ic s udies. A ound 2200 BCE, he eme gence o he El A ga g oup was e iden ly p eceded by a b eak in Chalcoli hic cul u al adi ions in sou heas Ibe ia. Ye he e a e no appa en new in luences o signals o subs an ial popu- la ion change on he m DNA haplog oup le el a his ime, so ha he obse ed changes may ei he be due o an uphea al o exis ing social s uc u es o an in lux o g oups ha canno be dis inguished om he local pop- ula ion a he p esen le el o gene ic esolu ion, e.g., om Sou heas e n Eu ope, as p e iously p oposed o El A ga . Un a eling hese appa en ly con adic o y da a will ce ainly equi e u he in-dep h analyses bo h on he a chaeological and he a chaeogene ic le el. Conclusion The p esen s udy, based on 213 new and 125 published m DNA da a o p ehis o ic Ibe ian indi iduals sugges s a mo e complex mode o in e ac ion be ween local hun e -ga he e s and incoming ea ly a me s du ing he Ea ly and Middle Neoli hic o he Ibe ian Peninsula, as compa ed o Cen al Eu ope. A cha ac e is ic o Ibe ian popu- la ion dynamics is he p opo ion o au och honous hun e -ga he e haplog oups, which inc eased in ela ion o he dis ance o he Medi e anean coas . In con as , he ea ly a me s in Cen al Eu ope showed compa a i ely li le admix u e o con empo aneous hun e -ga he e g oups. Al eady du ing he i s cen u ies o Neoli hic an- si ion in Ibe ia, we obse e a mix o emale DNA lineages o di e en o igins. Ea lie hun e -ga he e haplog oups we e ound oge he wi h a a ie y o new lineages, which ul ima ely de i e om Nea Eas e n a ming g oups. On he o he hand, some ea ly Neoli hic si es in no heas Ibe ia, especially he ea ly g oup om he ca e si e o Els T ocs in he cen al Py enees, seem o exhibi a ini ies o Cen al Eu opean LBK communi ies. The di e si y o emale linages in he Ibe ian communi ies con inued e en du ing he Chalcoli hic, when popula ions became mo e homogenous, indica ing highe mobili y and admix u e ac oss di e en geog aphic egions. E en hough he sample size a ailable o Ea ly B onze Age popula ions is s ill limi ed, especially wi h ega ds o he El A ga g oup, we obse e no signi ican changes o he mi ochond ial DNA pool un il he end o ou ime ansec (1500 BCE). The expansion o g oups om he eas e n s eppe47,92, which p o oundly impac ed La e Neoli hic and EBA g oups o Cen al and No h Eu ope, canno (ye ) be seen in he con empo aneous popula ion subs a e o he Ibe ian Peninsula a he p esen le el o gene ic esolu ion. This highligh s he dis inc cha ac e o he Neoli hic ansi ion bo h in he Ibe ian Peninsula and elsewhe e and emphasizes he need o u he in-dep h a chaeogene ic s udies o econs uc ing he close ecip ocal ela ionship o gene ic and cul u al p ocesses on he popula ion le el. Ma e ials and Me hods The s udied si es a e dis ibu ed ac oss he Ibe ian Peninsula, wi h an emphasis on he a chaeologically ele an egions on he Medi e anean coas s, in cen al and no he n Spain and in sou he n Po ugal (Fig.1). We a ge ed ep esen a i e si es om he Mesoli hic, Neoli hic, Chalcoli hic and he Ea ly B onze Age o co e 4,000 yea s o p ehis o y on he Ibe ian Peninsula. Gi en ha he Sou he n Eu opean clima e is usually un a o able o DNA p ese a ion, we implemen ed a lexible sampling scheme con ingen on ampli ica ion successes, ex ending sam- pling om si es wi h good ancien DNA p ese a ion and/o including addi ional si es o ob ain su icien da a. Sample speci ic con ex in o ma ion we e supplied by ou colleagues and p ojec pa ne s in Po ugal and Spain o collec ed om p e iously published pape s. All a chaeological si es, ele an adioca bon da es, and indi idu- als inco po a ed in he p esen s udy a e lis ed in Supplemen a y TablesS1-2. Al oge he , 318 indi iduals om 57 a chaeological si es in Spain, Po ugal and Mo occo we e sampled and analyzed o his s udy. Whene e possible we p e e ed samples om ecen exca a ions o e hose ha had been held a museum collec ions o p olonged pe iods o ime. Tee h and bone samples we e aken unde clean condi ions in Ibe ian museums, a he Ins i u e o An h opology a Johannes Gu enbe g Uni e si y in Mainz, o di ec ly on si e in he case o La Mina, A oyal, Els T ocs and in pa a La Bas ida. Mi ochond ial p o iles o 37 indi iduals om ou p ojec (Al o de Reinoso (n = 27), La Mina (n = 5) and Els T ocs (n = 5) we e p e iously published by ou esea ch eam44,47,49. He e we also epo addi ional samples om he si es o La Mina and Els T ocs. The ch onological classi ica ion o he bu ials was based on he a chaeological da a. In o de o a oid possible p oblems due o e minological inconsis encies, we used empo al ( om Mesoli hic o Ea ly B onze Age; based on ela i e ch onology and absolu e da ing) and geog aphic g oupings o assess he popula ion mi ogene ic da a om he Ibe ian Peninsula. We u he dis inguished g oups based on con ex ual a chaeological e idence, e. g. subsis ence s a egies, such as hun e -ga he e s s. ea ly a me s. We a ge ed collec ing wo o h ee ee h om each skele on and sampled bones only when ee h we e no a ailable. Ancien DNA sample p epa a ion. S anda d sample p epa a ion p o ocols we e used du ing he ancien DNA wo k in he Ins i u e o An h opology, Johannes Gu enbe g Uni e si y in Mainz55. Samples we e i s UV i adia ed o 30 min each side and hen, he su ace was emo ed by sho -blas ing wi h aluminium-oxide-ab asi e. Samples we e hen g ound o ine powde using a mixe mill (Re sch). The milling