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.nature.com/scientificreports The maternal genetic make-up of the Iberian Peninsula between the Neolithic and the Early Bronze Age Anna Szécsényi-Nagy 1, Christina Roth2, Guido Brandt3, Cristina Rihuete-Herrada 4, Cristina Tejedor-Rodríguez5, Petra Held2, Íñigo García-Martínez-de-Lagrán5, Héctor Arcusa Magallón5, Stephanie Zesch6, Corina Knipper7, Eszter Bánffy8, Susanne Friederich9, Harald Meller9, Primitiva Bueno Ramírez10, Rosa Barroso Bermejo10, Rodrigo de Balbín Behrmann10, Ana M. Herrero-Corral11, Raúl Flores Fernández12, Carmen Alonso Fernández13, Javier Jiménez Echevarria13, Laura Rindlisbacher14, Camila Oliart4, María-Inés Fregeiro4, Ignacio Soriano4, Oriol Vicente 4, Rafael Micó4, Vicente Lull4, Jorge Soler Díaz15, Juan Antonio López Padilla15, Consuelo Roca de Togores Muñoz15, Mauro S. Hernández Pérez16, Francisco Javier Jover Maestre16, Joaquín Lomba Maurandi17, Azucena Avilés Fernández17, Katina T. Lillios18, Ana Maria Silva 19,20, Miguel Magalhães Ramalho21, Luiz Miguel Oosterbeek22, Claudia Cunha23, Anna J. Waterman24, Jordi Roig Buxó25, Andrés Martínez26, Juana Ponce Martínez27, Mark Hunt Ortiz27, Juan Carlos Mejías-García 27, Juan Carlos Pecero Espín27, Rosario Cruz-Auñón Briones27, Tiago Tomé28, Eduardo Carmona Ballestero29, João Luís Cardoso30, Ana Cristina Araújo31, Corina Liesau von Lettow-Vorbeck 32, Concepción Blasco Bosqued32, Patricia Ríos Mendoza32, Ana Pujante33, José I. Royo-Guillén34, Marco Aurelio Esquembre Beviá35, Victor Manuel Dos Santos Goncalves36, Rui Parreira36, Elena Morán Hernández36, Elena Méndez Izquierdo37, Jorge Vega y Miguel38, Roberto Menduiña García38, Victoria Martínez Calvo39, Oscar López Jiménez39, Johannes Krause3, Sandra L. Pichler 14, Rafael Garrido-Pena32, Michael Kunst40, Roberto Risch4, Manuel A. Rojo-Guerra41, Wolfgang Haak 3,42 & Kurt W. Alt43,44 1Laboratory of Archaeogenetics in the Institute of Archaeology, Research Centre for the Humanities, Hungarian Academy of Sciences, Budapest, Hungary. 2Institute of Organismic and Molecular Evolution, Johannes Gutenberg University, Mainz, Germany. 3Max Planck Institute for the Science of Human History, Jena, Germany. 4Departamento de Prehistoria, Universitat Autònoma de Barcelona, Barcelona, Spain. 5Arcadia-General Foundation of Valladolid University, Valladolid, Spain. 6German Mummy Project, Reiss-Engelhorn-Museen, Mannheim, Germany. 7Curt- Engelhorn-Zentrum Archäometrie gGmbH, Mannheim, Germany. 8Romano-Germanic Commission, German Archaeological Institute, Frankfurt am Main, Germany. 9State Office for Heritage Management and Archaeology Saxony-Anhalt and State Museum of Prehistory, Halle, Germany. 10Departamento de Historia y Filosofía, Universidad de Alcalá de Henares, Alcalá de Henares, Spain. 11Departamento de Prehistoria, Universidad Complutense de Madrid, Madrid, Spain. 12Professional Archaeologist, Parla (Madrid), Spain. 13Cronos SC Arqueología y Patrimonio, Burgos, Spain. 14Integrative Prehistory and Archaeological Science, University of Basel, Basel, Switzerland. 15Museo Arqueológico de Alicante-MARQ, Alicante, Spain. 16Departamento de Prehistoria, Arqueología, Historia Antigua, Filología Griega y Filología Latina, Universidad de Alicante, Alicante, Spain. 17Departamento de Prehistoria, Arqueología, Historia Antigua, Historia Medieval y Ciencias y Técnicas Historiográficas, Universidad de Murcia, Murcia, Spain. 18Department of Anthropology, University of Iowa, Iowa City, Iowa, United States of America. 19Laboratory of Prehistory, CIAS, Department of Anthropology, University of Coimbra, Coimbra, Portugal. 20UNIARQ, University of Lisbon, Lisboa, Portugal. 21Laboratório Nacional de Energia e Geologia, I.P., Museu Geológico, Lisboa, Portugal. 22Instituto Politécnico de Tomar, Instituto Terra e Memória, Geosciences Centre of Coimbra University, Tomar, Portugal. 23Programa de Capacitação Institucional MCTIC/MPEG, Museu Paraense Emílio Goeldi, Belém, Brazil. 24Department of Natural and Applied Sciences, Mount Mercy University, Cedar Rapids, Iowa, United States of America. 25Arrago Ltd, Barcelona, Spain. 26Museo Arquelógico de Lorca, Lorca, Spain. 27Departamento de Prehistoria y Arqueología, Universidad de Sevilla, Sevilla, Spain. 28Universidade Federal do Pará, Campus Universitário Received: 8 February 2017 Accepted: 27 October 2017 Published: xx xx xxxx OPEN www.nature.com/scientificreports/ 2 SCIENTIFIC REPORTS | 7: 15644 | DOI:10.1038/s41598-017-15480-9 Agriculture first reached the Iberian Peninsula around 5700 BCE. However, little is known about the genetic structure and changes of prehistoric populations in different geographic areas of Iberia. In our study, we focus on the maternal genetic makeup of the Neolithic (~ 5500–3000 BCE), Chalcolithic (~ 3000–2200 BCE) and Early Bronze Age (~ 2200–1500 BCE). We report ancient mitochondrial DNA results of 213 individuals (151 HVS-I sequences) from the northeast, central, southeast and southwest regions and thus on the largest archaeogenetic dataset from the Peninsula to date. Similar to other parts of Europe, we observe a discontinuity between hunter-gatherers and the first farmers of the Neolithic. During the subsequent periods, we detect regional continuity of Early Neolithic lineages across Iberia, however the genetic contribution of hunter-gatherers is generally higher than in other parts of Europe and varies regionally. In contrast to ancient DNA findings from Central Europe, we do not observe a major turnover in the mtDNA record of the Iberian Late Chalcolithic and Early Bronze Age, suggesting that the population history of the Iberian Peninsula is distinct in character. The changeover from a hunter-gatherer lifestyle to a productive mode of subsistence first emerged around 10,000 BCE in the Near East1–3. This so-called ‘Neolithic transition’ brought about fundamental changes in economy, social structure, demography and human health, and laid the foundations for agrarian societies and thus for ancient civilizations. Over the course of the 7th and 6th millennia BCE, agriculture spread from the Balkans to Central Europe. Another route of dissemination ran along the Mediterranean coastlines of Greece, Italy and the south of France to the Iberian Peninsula and north to the Paris Basin and Central Europe4. However, the process of Neolithisation was non-linear, with the archaeological record documenting influences of local cultural traditions5. In Iberia, the Neolithic transition, which began around 5700 BCE, appears to have been complex, and Mesolithic and farming communities coexisted and interacted for as long as two millennia6. The artefactual remains, mainly ceramic, attest to the different origins and modes of Neolithisation on the Iberian Peninsula. On one hand stands a Mediterranean maritime colonization by Neolithic pioneers characterized by ceramic with clear parallels to the Ligurian Impressa collections of Italian origin7–9. Some Early Neolithic sites were also located in the hinterland, suggesting further routes of dissemination through the Pyrenees and/or along major rivers, such as the Ebro10,11,16. On the other hand, North African influences and contacts are tangible in the southern Iberian Neolithic12,13. All in all, Iberia appears a melting pot of influences and groups, combining Neolithic lifeways and with indigenous mechanisms of adaptation14. In the Early Neolithic, we can observe common features shared over large areas, but also some regionally restricted phenomena15. In the whole territory, for example, there existed sophisticated systems of agriculture and livestock handling, with adaptable crops16,17 and seasonal strategies in flock management18–20. The groups of the Franco-Iberian Cardial and the Epicardial pottery styles appeared at this time and recent studies have revealed mutual diachronic influences in the material culture and economies of these cultures15,21. An increasing number of burials from that epoch have come to light in the last years22–24. The oldest are individual inhumations, sometimes grouped in cemeteries25. It is also common to find human remains in caves, which were increasingly used for collective burials26. From the late 5th millennium BCE onward, burial monuments appeared, and megalithic tombs became widespread27. This phenomenon links Iberia with other parts of Europe, indicating long-distance networks of communication. Meanwhile, certain parts of northeast Iberia maintained individual inhumations in pits, mostly in small cemeteries28. Besides megalithic tombs, ditched enclosures extending over more than 100 hectares started to dominate the landscapes of southern and central Iberia from 3300 to 3100 BCE, highlighting another widely spread European phenomenon29,30. During the Iberian Chalcolithic period (3000–2200 BCE), fortified settlements with stone walls and semi-circular bastions appeared in the western and southern parts of the Peninsula, while elsewhere, open settlements were still extant31. The diversity in settlement and burial types suggests the existence of social structures with different levels of complexity32. At the same time, as exchange networks, circulated precious goods such as ivory from Africa and even Asia to Iberia33. From ~2600 BCE onwards, the so-called ‘Bell Beaker phenomenon’ became manifest with its characteristic pottery, copper weapons, gold ornaments, and other prestige goods, an archaeological reflection of important social and economic changes which spread across vast regions of western and central Europe34. Iberia’s Bell Beaker assemblages are among the richest and most diverse in western Europe35, both in terms of settlements and do Guamá, Belém, Brazil. 29Servicio Territorial de Cultura de Valladolid, Valladolid, Spain. 30Departamento de Ciencias Sociais e de Gestao, Universidade Aberta, Lisboa, Portugal. 31Research Centre in Biodiversity and Genetic Resources (CIBIO-InBIO) & Directorate-General for Cultural Heritage (DGPC), Lisboa, Portugal. 32Departamento de Prehistoria y Arqueología, Universidad Autónoma de Madrid, Madrid, Spain. 33Arqueología Estudios, Murcia, Spain. 34Technical archaeologist, Gobierno de Aragón, Zaragoza, Spain. 35Arpa Patrimonio Ltd, Alicante, Spain. 36Centro de Arqueologia da Universidade de Lisboa (Uniarq), Lisboa, Portugal. 37Grupo de Investigación HUM-949 TELLUS, Prehistoria y Arqueología en el Sur de Iberia, Universidad de Sevilla, Sevilla, Spain. 38ARGEA Ltd., Madrid, Spain. 39GIPSIA Ltd., Toledo, Spain. 40German Archaeological Institute Madrid, Madrid, Spain. 41Departamento de Prehistoria y Arqueología, Universidad de Valladolid, Valladolid, Spain. 42School of Biological Sciences, The University of Adelaide, Adelaide, Austria. 43Center for Natural and Cultural History of Man, Danube Private University, Krems, Austria. 44Department of Biomedical Engineering and Department of Environmental Science, Basel University, Basel, Switzerland. Correspondence and requests for materials should be addressed to A.S.-N. (email: szecsenyi-nagy. [email protected]) or K.W.A. (email: [email protected]) www.nature.com/scientificreports/ 3 SCIENTIFIC REPORTS | 7: 15644 | DOI:10.1038/s41598-017-15480-9 burials36. It has thus long been a focus of archaeological research, commencing from migrationist hypotheses and leading up to current social explanations, where the Bell Beaker phenomenon is perceived as a package of prestigious objects exchanged and consumed by elite groups and displayed on special occasions37. Around 2200 BCE, the Chalcolithic settlement and funerary practices were suddenly discontinued, particularly in the western and southern part of Iberia, where most of the ditched and fortified settlements were abandoned and collective sites and megalithic tombs were replaced by individual burials31. El Argar group began to emerge in southeast Iberia, with large and massively fortified urban centers like La Bastida (Murcia, n. 28 on Fig.1)38, which managed to control a territory of over 35,000 km2 during the following 650 years. With regard to its population history, the Iberian Peninsula has been the focus of several recent archaeogenetic studies39–44. Studies focusing not only on mtDNA but also on Y chromosome markers have supported the model of a pioneer colonization in the north-eastern coastal regions of the Iberian Peninsula at the onset of the Iberian Neolithic39,45. On the other hand, the northern part of Spain (Cantabrian fringe) attested to a rather complex Neolithic transition42. Recent mtDNA and genome-wide analyses have put an emphasis on the genetic affinity and shared Near Eastern ancestry between the early Iberian farmers and the contemporary Central European Linearbandkeramik (LBK) population46–48. The latest mtDNA and genomic studies have revealed increased subsequent admixture of hunter-gatherer elements during the local middle Neolithic (La Mina; Alto de Reinoso, n. 51–52 on Fig.1)44,47 and the Chalcolithic (El Portalón; El Mirador, n. 60–62. on Fig.1) again reminiscent of processes observed in Central Europe41,49,50. Despite numerous research projects being carried out over the past years, the Neolithic settlement history of Europe can still only be explained at a broad scale47,49,51–54. Regional transects through time detailing the developments and the course of the Neolithic in central Germany55–57 and in the Carpathian Basin58 have mostly been examined by mitochondrial DNA (mtDNA) control region data. Our project completes the latter series by focusing on the archaeological models and hypotheses that have been put forward for the Iberian Peninsula, and where diachronic (i.e. ‘through time’) sampling of ancient DNA allows the detection of demographic changes and discontinuities between 5700 and 1500 BCE. Essential research questions focus on three levels: i) the individual sites, ii) the Iberian Peninsula as a scene of Neolithic transition and iii) a comparison with contemporaneous ancient and modern-day Europeans. A key question of our study of the mtDNA diversity on the Iberian Peninsula through time was to examine to what extent regional and supra-regional cultural groups could be recognized as genetically identifiable entities, as shown in other areas of Europe46,55. A related question was whether the cultural breaks that can be seen, for instance, at the end of the Neolithic and Chalcolithic periods, were also accompanied by human population turnovers. Results We processed ancient DNA samples of 318 human individuals from prehistoric Iberia and generated reproducible mitochondrial hypervariable region haplotypes (HVS-I, np 16020–16401) from 151 individuals following strict authentication criteria (see Methods). MtDNA haplogroup classification of further 62 samples was based on multiplex typing of single nucleotide polymorphisms (SNPs) (Fig.1, Supplementary Tables S1–4). The DNA amplification and reproduction success of the HVS-I showed strong differences among samples from diverse regions of the Iberian Peninsula: the highest amplification success rates were observed in northeast Iberia (NEI) and higher altitude regions of central Iberia (CI) (71–78%), while especially the southern part of the Iberian Peninsula (southeastern Iberian (SEI) group and southwest Iberia (SWI) had very low amplification success rates (20–43%). We merged these data with previously published HVS-I mtDNA results from 125 prehistoric individuals (Supplementary TablesS5, S15), and separated the HVS-I dataset of 238, and mtDNA haplogroup information of 305 prehistoric Iberian individuals into geographically and chronologically defined groups (see Methods). The resulting haplogroup compositions of these groups are presented in Fig.2. Haplogroup based analyses. The Iberian pre-Neolithic mtDNA substrate is still poorly understood. Recent NGS studies described hunter-gatherers belonging to haplogroup U5b in north Iberia43,59, while the suggested pre-farming presence of haplogroups H, N and U442 were reported in studies that did not meet our strict authentication criteria (see Methods). Therefore, we used Holocene period hunter-gatherers from Central, West and North Europe as proxy for the Iberian pre-Neolithic mtDNA substrate in our quantitative analyses (Fig.2, Supplementary TablesS6, S15). This continental hunter-gatherer dataset (HG_HOL, n = 35) contains predominantly U haplogroups (U5b and U5a dominated, but also R, U2, U4, U* were observed53,57,58,60–63). In Iberia, haplogroup U5b was found most frequently in the CI Neolithic dataset (19.4%; n = 62), and showed also relative high frequency in the CI Chalcolithic (13.9%; n = 79) and NEI Chalcolithic-Early Bronze Age groups (14.8%; n = 27). The haplogroup composition of the Iberian Neolithic population shows similarities to the Early Neolithic data from Anatolia (~6500–6000 BCE), the Carpathian Basin (~5800–4900 BCE), and Central Europe (~5500–4000 BCE, represented by the central German Mittelelbe-Saale region)49,55,58. Haplogroups K, J, T2, HV, V and X are observed in comparable frequencies in Iberian and Central European groups. However, the proportion of haplogroup H is higher in the Iberian Early Neolithic (EN) (22.2%; n = 27) than in Central Europe (15%; n = 160), while the frequency of N1a is very low (3.7% compared to 9.4% in Central Europe). Another difference with regards to Central Europe is the occurrence of haplogroup N* in Neolithic NEI group (Supplementary TablesS6, S15). We used the haplogroup frequencies of the studied groups for principal component analyses (PCA, see Methods). When compared to published ancient DNA data from the Near East, the western Anatolian Neolithic population is most similar to Central European Early-Middle Neolithic populations and to the Neolithic of northeast Iberia (Fig.3, Supplementary Fig. S1–2), but it also shows some affinities with further Iberian farmers through common EN haplogroups (e.g. K, J, T2)49. The presence of haplogroup N* in the Pre-Pottery Neolithic in Syria and in NEI can also be interpreted in this light39,64, but this link needs to be verified by whole mitogenome www.nature.com/scientificreports/ 4 SCIENTIFIC REPORTS | 7: 15644 | DOI:10.1038/s41598-017-15480-9 Figure 1. Map of the studied sites, including the published reference data and timing of archaeological periods on the Iberian Peninsula and in Central Europe. Geographic regions, also differentiated in the mtDNA analyses, are indicated as: NEI: northeast, SEI: southeast, SWI: southwest Iberia. Numbers on the map are colored according to the chronological periods, represented in the lower part of the figure. For the Central European chronology we used records from the most important comparative region of central German Mittelelbe- Saale55. See Supplementary TableS5 for furtherdetails. Site codes: 1. Moita do Sebastião, 2. Galeria da Cisterna (Almonda cave), 3. Gruta de Nossa Senhora das Lapas, 4. Gruta do Cadaval, 5. Gruta das Alcobertas, 6. Gruta do Poço Velho, 7. Gruta dos Ossos, 8. Tholos de Pai Mogo I, 9. Hipogeu de Monte Canelas I, 10. Hipogeu de Monte Canelas III, 11. Bolores, 12. Gruta de Malgasta, 13.Valencia de la ConcepciónArea 9, 14. Gruta do Carvalhal de Turquel, 15. Cobre las Cruces, 16. Cova de l’Or, 17. Cova de la Sarsa, 18. Cova d’en Pardo, 19. Molinos del Papel, 20. Cova del Barranc del Migdia, 21. Cova del Cantal, 22. Camino de Molino, 23. Fuente Álamo, 24. Lorca-Los Tintes, 25. Lorca-Madre Mercedarias, 26. Lorca-Castillo de Lorca, 27. Rincón de Moncada, 28. La Bastida, 29. Tabayá, 30. Illeta dels Banyets, 319. Cova Bonica, 32. Can Sadurní, 33. Cova d’Avellaner, 34. Els Trocs, 35. Sant Pau de Camp, 36. Barranc d’en Rifà, 37. Balma de Sargantana, 38. Cova de la Ventosa, 39. Cova de Montanissel, 40. Miguel Vives, 41. Can Gambús, 42. Chaves, 43. Valdescusa, 46. Alto de Rodilla, 47. Fuente Celada, 48. Fuente Pecina 1, 49. Fuente Pecina 2, 50. Fuente Pecina 4, 51. Alto de Reinoso, 52. La Mina, 53. La Tarayuela, 54. El Juncal, 55. Arroyal I, 56. El Hundido, 57. Camino de las Yeseras, 58. Humanejos, 59. Valle de las Higueras, 60–61. El Portalón, 62. El Mirador, 63. Es Forat de ses Aritges. Background map of Iberia (copyright holder Universitat Autònoma Barceolna) was modified in Adobe Illustrator CS6 software (www.adobe.com/products/illustrator). www.nature.com/scientificreports/ 5 SCIENTIFIC REPORTS | 7: 15644 | DOI:10.1038/s41598-017-15480-9 analyses. Haplogroup compositions of all other Iberian groups are highly similar to each other, resulting in clusters on the PCA plots and Ward clustering tree. Our largest Iberian datasets are from Neolithic and Chalcolithic of CI (n = 62 and 79), which show an elevated frequency of U5b compared to the NEI Neolithic. Interestingly, we also observe probable hunter-gatherer haplogroups U2 and U4, whereas the characteristic EN haplogroups N*, N1a are missing in the CI region. The southeastern Iberian dataset (n = 48) contains eight Late Neolithic individuals with haplogroups K, H and U5b, 28 individuals from the Chalcolithic and 12 from the Early Bronze Age (EBA). Similarly to the CI groups, SEI shows a mixture of haplogroups of the NEI group, but some of them (N*, N1a, U3) are missing. The number of Neolithic and Chalcolithic SWI samples (n = 12 and n = 31) is too small to estimate the population dynamics in the region reliably (Supplementary TableS6). The combined dataset shows a predominance of haplogroup K (35.7%) and high frequency of haplogroups H, J and T (16.7–14.3–14.3%). As Middle Neolithic (~ 4500–3500 BCE) and Late Neolithic (~3500–3000 BCE) periods of most sites are not separable at the current state of archaeological research, we combine these data in our analyses (MLN). Fisher’s exact (p = 0.5473) and population continuity (p = 0.3385–0.3722) tests confirm the connection between the EN and MLN periods of Iberia in general. Some of the Iberian Neolithic mtDNA haplogroups (U2, N*, N1a) are not observed in the successive Chalcolithic Iberian population (n = 156), whereas others maintain a steady frequency (V, T2, X) throughout 3500 years. An interesting exception is haplogroup L1b in the Late Chalcolithic central Iberia at the site Camino de las Yeseras (n. 57 on Fig.1), near Madrid. This group is most frequent in today’s West-Central Africa65, and hints at a connection to the North-West African coasts in prehistoric times. In contrast to U5b, haplogroup U5a (common in Central European Mesolithic and Late Neolithic55,5760) appears first in the 3000–2500 BCE period of central Iberia. Other typical Central European and Carpathian Basin Neolithic haplogroups such as H5, T1, W and U8, which are observed in later Central European Neolithic periods, are missing from the entire prehistoric Iberian dataset. These qualitative differences account for the separation of most of the Iberian prehistoric groups from the Central European Neolithic and Early Bronze Age populations along the second component of the PCA, shown on Fig.3. Tests of population continuity and Fisher’s exact test between the whole Iberian Early and Late Chalcolithic datasets result in non-significant p values (p = 0.5444–0.5578 and 0.9779), which support population continuity between the two periods. Unfortunately, a finer chronological separation of the Chalcolithic (CHA) dataset cannot be achieved for all samples and therefore we consider only a subset of the CHA dataset (n = 71 for Early and n = 47 for Late CHA) in this analysis (Supplementary TableS8). We also tested whether this continuity could be the result of data merging across bigger geographic distance. However, the genetic continuity during the Early to Late Chalcolithic transition also holds at regional scale, as our population continuity test cannot reject continuity between the Neolithic (containing mainly Late Neolithic) and CHA groups of central Iberia (p = 0.6033–0.6143), and NEI (p = 0.9038–0.7835). This continuous genetic makeup is also supported by non-significant (p = 0.2264 and 0.4484) differences in haplogroup compositions (Supplementary TablesS7–8). Figure 2. MtDNA haplogroup composition of the prehistoric Iberian groups. Abbreviations: Huntergatherers in Europe from the Holocene (HG_HOL), northeast Iberian Neolithic (NEI_Neo), northeast Iberian Chalcolithic and Early Bronze Age (NEI_CHA_EBA), central Iberian Neolithic (CI_Neo), central Iberian Chalcolithic (CI_CHA), southwest Iberian Neolithic (SWI_Neo), southwest Iberian Chalcolithic (SWI_CHA), southeast Iberian Neolithic (SEI_Neo), Chalcolithic and Bronze Age (SEI_ CHA_EBA). Relative haplogroup frequencies are presented in Supplementary TableS6. The background map of Iberian Peninsula (https://en.wikipedia.org/wiki/File:Relief_Map_of_Spain.png#file) is licensed under CC BY-SA 3.0 (https:// creativecommons.org/licenses/by-sa/3.0/) was modified in Adobe Illustrator CS6 software (www.adobe.com/ products/illustrator). www.nature.com/scientificreports/ 6 SCIENTIFIC REPORTS | 7: 15644 | DOI:10.1038/s41598-017-15480-9 The Early Bronze Age sample set in the Iberian prehistoric transect is still very small (a total of n = 37 individuals from all four regions studied). At the mtDNA haplogroup level the EBA does not show new influences or population changes at the onset of the Iberian Bronze Age according to results from Fisher’s test and test of population continuity, when both the entire Peninsula and southeastern Iberia in particular were considered (Supplementary TablesS7-8). A more detailed analysis of H subhaplogroups focuses on 17 SNPs in the coding region of the human mitogenome. The largest proportion of the H individuals belongs to the subhaplogroup H1 (65.1%), and the second largest group is subhaplogroup H3 (14%), while 18.6% of the H individuals cannot be assigned to any of the subgroups included in the H-PLEX assay (see Methods, Supplementary TablesS3-4)66. H3 is detected in Chalcolithic individuals from central, southeast and southwest Iberia. H1 is observed in each period and region, but more frequently in the Chalcolithic and Early Bronze Age than in the Neolithic. The comparative ancient H data from the European prehistory is too sparse for in-depth statistical analyses. However it becomes apparent from our results that the H diversity in prehistoric Iberia is different from the H diversity of Central Europe55,58,67,68, and more similar to the Neolithic populations in France69,70. Notably, common Central European subhaplogroups H5 and H767,68 have not yet been observed in Southwest Europe. Overall, we do not observe direct links between the Central European Late Neolithic (chronologically comparable to the Chalcolithic in Iberia) and the Iberian (entire and late) CHA groups at the haplogroup level, and thus cannot confirm a Late Chalcolithic expansion toward Central Europe as suggested by haplogroup H mitogenome data68. Haplotype and sequence based analyses. The mtDNA variation in prehistoric Iberia is further explored by HVS-I sequence and haplotype analyses of 238 individuals from the Peninsula. The haplotype diversity of the hunter-gatherer populations of Central Europe (HG_PLEI (Pleistocene): Hd = 0.879; n = 29, Figure 3. Principal component analysis based on haplogroup frequencies of 955 individuals from 16 prehistoric groups. The first two components display 42.8% of the total variance. Groups are colored according to their geographical positions: brown: Iberia, purple: France, ochre: Central and East-Central Europe, yellow: Eastern Europe and Near East. For abbreviations of Iberian groups, see legend of Fig.2. Further abbreviations: Central and North European hunter-gatherers from the Holocene (HG_HOL), Neolithic Anatolia (ANAT) Yamnaya (YAM), Early Neolithic Carpathian Basin (CB_EN), Early Neolithic Germany (GER_EN), Middle Neolithic Germany (GER_MN), Late Neolithic Germany (GER_LN), Early Bronze Age Central Europe (CEU_ EBA), Neolithic Gurgy site in France (FRA_GUR), Neolithic Treilles culture in France (TRE). For further information see Supplementary TableS6. www.nature.com/scientificreports/ 7 SCIENTIFIC REPORTS | 7: 15644 | DOI:10.1038/s41598-017-15480-9 HG_HOL (Holocene): Hd = 0.931; n = 35) provides the baseline for this comparative series. The diversity is lower in the Early Neolithic of Iberia (Hd = 0.926; n = 27) but increases in the following Middle-Late Neolithic (Hd = 0.933; n = 74) and Chalcolithic periods (Hd = 0.944; n = 118). The Early Bronze Age shows again a lower diversity (Hd = 0.917; n = 16), although this could be the result of the small number of investigated individuals (Supplementary TableS9). The haplotype diversity among the regional groups is the lowest in the SWI Neolithic-Chalcolithic group in Iberia, and that along with the detected common basal J and K haplotypes could cause potential bias in lineage sharing results presented below. The haplotype sharing (SHA) between the larger chronological groups detects no hunter-gatherer contribution in the EN group of Iberia, and a low level of hunter-gatherer maternal lineages in the MLN periods (6.8%) (Table1). During the MLN, several new hunter-gatherer-type U5b lineages appear. Between the two successive Neolithic periods, the haplotype sharing amounts to 50%, and 43.2% of the MLN lineages are already present in the EN (as seen from our ancestral SHA). In the Chalcolithic period, we also observe a strong ancestral EN contribution (44.9%), with only 14.4% of the lineages originating from later Neolithic periods. The continuity of lineages is also seen in the smaller EBA dataset, which shows close connections to EN, MLN and CHA periods, and in which only 18.8% of the haplotypes are new (Table1, Supplementary TableS10). The Iberian early farming groups share generally high number of lineages (38.3–82.4%) with each other (Supplementary Fig.S3). The Central European Early Neolithic has the highest number of shared haplotypes comparing to the northeast Iberian groups, but the sharing is also high with southeast Iberian dataset. In contrast, lineage sharing with Holocene period hunter-gatherers is the highest in the mostly Chalcolithic southeast Iberian group (8.6%) and in CI Neolithic (6.7%). Concerning temporal succession in NEI region, the proportion of hunter-gatherer lineages increases (from 0 to 4.8%) during the Chalcolithic period (Supplementary TableS10). Continuity between Neolithic and Chalcolithic groups is reflected by the amount of shared lineages between the two periods in CI (61.2%), but both CI dataset also share many lineages with all other Iberian groups (Supplementary TableS10). Genetic distances (Fst) are generally low among the Iberian prehistoric groups, and none of them are significant (Supplementary TableS11). We observe the shortest distances from Central European EN-MN in northeast Iberia. By plotting the HVS-I based genetic distances after multidimensional scaling (MDS, Supplementary Fig.S4), large-scale relationships are displayed. Here, the NEI populations are within the cluster of the other Iberian groups, whereas the Central European and French datasets form a cluster that is slightly offset. Interestingly, MDS shows that some groups (e.g. Neolithic NEI) appear less differentiated from the remaining populations when compared to haplogroup-based PCA. The clustering of the groups on MDS plot was therefore tested by the analysis of molecular variance. The best-supported groupings (i.e. with the highest variance among the clusters and lowest within the clusters) were evaluated by iteratively alternating the composition of the groupings. The Iberian groups differentiated from an EN Central European- Carpathian Basin and a MLN-EBA Central European- LN French clusters in this analysis as well, and can be further divided into a Neolithic-Chalcolithic CI and a NEI-SEI-SWI cluster (Supplementary TableS12). We further compared HVS-I sequences of three larger chronological groups (EN, MLN and CHA) of the Iberian Peninsula with 133 modern populations. Genetic distances to modern-day populations are generally low, and restricted to certain region(s) of Europe (Supplementary Fig.S5, Supplementary TableS13). The Early Neolithic shows relatively high affinity to modern Afghanistan, Palestine, Iran, and Turkey (Fst = 0.00173– 0.00428), but also high affinities to several European populations (e.g. Italian, French but also Belorussian). The genetic distances to modern populations increase with the MLN and CHA periods, but show general similarity to modern-day Europe. Recent genomic studies have highlighted the similarity of early farmers to modern South Europeans71, especially to modern day Sardinians47,49. This picture is not reflected in our mtDNA dataset, where A:SHA n sample Detected in the following populations: HG_HOL EN MLN CHA EBA mtDNA 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: ancestral SHA Detected in the following populations: n sample HG_HOL EN MLN CHA EBA mtDNA 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. Results of shared haplotype analysis (SHA): percentage of shared HVS-I haplotypes among the Iberian chronological groups (A), and ancestral haplotype analysis with the studied Iberian groups (B). Abbreviations: Central and North European hunter-gatherers from the Holocene (HG_HOL), Early Neolithic (EN), Middle and Late Neolithic (MLN), Chalcolithic (CHA), Early Bronze Age (EBA) in Iberia. For further details, see Supplementary TableS10 and Methods. www.nature.com/scientificreports/ 8 SCIENTIFIC REPORTS | 7: 15644 | DOI:10.1038/s41598-017-15480-9 Sardinians rank only the 67th closest to the Iberian EN group out of 133 modern populations, and 46th from the MLN group (Supplementary TableS13). Discussion During the Last Glacial Maximum, the Iberian Peninsula, just as Southeast Europe and the Italian Peninsula, formed a classic Glacial Refuge Area for European populations, well documented by archaeological evidence72–74. Therefore, it is assumed that parts of today’s European population are the descendants of the residents of these refugia. This theory is based on coalescence date estimates for some mtDNA lineages (H1, H3, V, U5b1), which predate the Neolithic expansion, and also based on the high abundances of these lineages in Iberia today75–79. However, the Franco-Cantabrian glacial refuge theory has not yet been confirmed by ancient mtDNA or genomic analyses, and more Iberian Pleistocene and especially Holocene samples need to be investigated at full mitogenomic resolution. With the end of the Last Ice Age and the beginning of the Holocene ~12,000 BCE, a common European Mesolithic population emerged, which also included a distinct signal of Near Eastern origin59. The indigenous Iberian late Upper Paleolithic and Mesolithic populations were represented by whole mitogenome haplotypes assigned to haplogroup U5b from Northern Spain43,59. The Iberian Peninsula is characterized by diverse landscapes with distinct economic potentials. During the initial phase of Neolithisation, first farmers probably arrived in northeast Iberia primarily along the Mediterranean route and from there spread along the coastline and rivers (e.g., the Ebro Valley) into the hinterland7–9,80. Our genetic data support a substantial influx of Neolithic immigrants to northeastern Spain, where farmer lineages are most abundant and diverse, and on to other regions, like the SEI and CI. We found the persistence of ‘hunter-gatherer’ mtDNA haplogroups in the Neolithic to be strongest in Central Iberia. These genetic results match the archaeological data on the mode of Neolithisation described for these areas, so that the fully established Neolithic Iberian communities had distinct hunter-gatherer components16,81,82. The further the early farmers advanced into the inner/central and southern parts of the Peninsula, the higher the proportion of indigenous hunter-gatherer lineages. Geography appears to have been a decisive factor in the advance of Neolithic lifeways. Prehistoric central Iberia (southern and northern Meseta, Ambrona Valley), however, was never an isolated region surrounded by mountains, but rather an important hub for innovations and impulses coming into Iberia83. On a genetic level, Neolithic central Iberia showed corresponding mitogenomic connections to northeast and southeast Iberian regions (Supplementary TableS7). The Pyrenees are also an area of special interest with regard to the route of immigration of the first farmers. This study includes ten samples from the cave site of Els Trocs, dated to the Early and Middle Neolithic20. Due to the considerable temporal differences between the occupations, the respective sample sets can be considered as genetically independent. The 14C data of the six individuals from the earliest phase cluster closely (Supplementary Information). Genome-wide analyses of five of these EN individuals revealed the group to be early Neolithic immigrants47,49,50. Haplogroups N1a, J, T2, K and V appear, while the typical Iberian hunter-gahterer mtDNA haplogroup U5b is missing. Therefore, we suspect the presence of a (still) isolated EN group at Els Trocs. The isolated geographic position in the Pyrenees suggests immigration of this community from north of the Pyrenees (or even from Central Europe via the Rhone Valley) rather than from the west along the Mediterranean coastline. This alternative gateway to Spain has also been proposed based on archaeobotanical evidence17,84. The first and (to our knowledge) sole Iberian appearance of the mtDNA haplogroup N1a in an adult from Els Trocs, which matches identical HVS-I N1a haplotype in Anatolia and Central Europe, might have arrived on the continental route47,49,55,85. The shared roots between Southwest and Central Europe were especially conspicuous in the Northeast Iberian EN group, as the agreement in the common mtDNA haplogroups such as V, J, K, T2 and X suggests. The transition from the EN to the MLN is documented in recent ancient genomic studies, which described an increase of hunter-gatherer elements in the farming populations of central Iberia during the MLN period at the site of La Mina and the Chalcolithic sites of El Mirador and El Portalón41,47,49. The Iberian Middle Neolithic genomic data are still too scarce (concentrated on a single site La Mina) to draw general conclusions about admixture with late hunter-gatherers47. La Mina people had 18.9–22.8% hunter-gatherer ancestry, double than the EN individuals from Els Trocs (6.8–11%)50, and also higher than their contemporaries in Central Europe. These hunter-gatherer proportion further increased in the CHA to 27%50. In our study, we could support the general continuity of maternal lineages between MLN-CHA periods. The increase of hunter-gatherer maternal lineages through time is not seen on large scale SHA (Table1), but observable in regional groups, such as in NEI, where the Fst from HG_HOL decreases and the amount of HG_HOL haplotypes increases from the Neolithic to the CHA-EBA periods (0–4.76%). The same trend can be seen in our Fst analyses where the CI_CHA (Fst = 0.17406) appear closer to the HG_HOL group than the CI_Neo group (Fst = 0.21267) (Supplementary TablesS7-8, S11). According to the data currently available, the homogeneity of Chalcolithic ancient DNA results suggests that human mobility and genetic mixing had generally increased in Iberia by the Chalcolithic. Although we analyzed four geographically separated CHA groups in the northeast, central as well as in the southeast and southwest of the Iberian Peninsula, they did not exhibit any significant differences except for the comparison of the central and the southeastern group (Fisher’s test results in Supplementary TableS7, Fig.2). The detection of the ‘African’ Lb1 haplogroup at the Late Chalcolithic site Camino de las Yeseras (Madrid, central Iberia) is remarkable, given that ivory adornments of African origin have also been documented at this and other contemporaneous sites86–88. The observation of a western-central ‘African’ haplogroup alongside African artefacts and raw materials in Copper Age Iberia indicate long distance exchange that at least occasionally seem to have involved mobility of individuals and/or gene flow. The Bell Beaker phenomenon was a decisive element in the Iberian Chalcolithic, lasting from the Late CHA to the EBA (~2600–1800 BCE)35,36. Our tests of population continuity and Fisher’s tests between Early and Late www.nature.com/scientificreports/ 9 SCIENTIFIC REPORTS | 7: 15644 | DOI:10.1038/s41598-017-15480-9 CHA periods supported population continuity between the two phases. However, our data structure did not allow studying the Early to Late CHA population changes at a regional scale, so that further studies might reveal local variation with regard to individuals with Bell Beaker / non-Bell Beaker cultural affiliations. Interestingly, we did not find evidence for direct genetic links between Chalcolithic Iberia and contemporaneous Central Europe. Links between the two regions had previously been suggested based in particular on Bell Beaker elements present across a wide geographic range89–91, as well as by the maternal genetic analyses of the El Mirador site40. Interestingly, we also do not find evidence for influx in the East to West direction, as none of the investigated Chalcolithic individuals show ‘steppe ancestry’, which seen in contemporaneous Central European Corded Ware and Bell Beaker groups, suggesting that eastern influxes did not reach the Iberian Peninsula until later periods49. An evaluation of this unexpected observation will need further palaeogenetic studies. Around 2200 BCE, the emergence of the El Argar group was evidently preceded by a break in Chalcolithic cultural traditions in southeast Iberia. Yet there are no apparent new influences or signals of substantial population change on the mtDNA haplogroup level at this time, so that the observed changes may either be due to an upheaval of existing social structures or an influx of groups that cannot be distinguished from the local population at the present level of genetic resolution, e.g., from Southeastern Europe, as previously proposed for El Argar. Unraveling these apparently contradictory data will certainly require further in-depth analyses both on the archaeological and the archaeogenetic level. Conclusion The present study, based on 213 new and 125 published mtDNA data of prehistoric Iberian individuals suggests a more complex mode of interaction between local hunter-gatherers and incoming early farmers during the Early and Middle Neolithic of the Iberian Peninsula, as compared to Central Europe. A characteristic of Iberian population dynamics is the proportion of autochthonous hunter-gatherer haplogroups, which increased in relation to the distance to the Mediterranean coast. In contrast, the early farmers in Central Europe showed comparatively little admixture of contemporaneous hunter-gatherer groups. Already during the first centuries of Neolithic transition in Iberia, we observe a mix of female DNA lineages of different origins. Earlier hunter-gatherer haplogroups were found together with a variety of new lineages, which ultimately derive from Near Eastern farming groups. On the other hand, some early Neolithic sites in northeast Iberia, especially the early group from the cave site of Els Trocs in the central Pyrenees, seem to exhibit affinities to Central European LBK communities. The diversity of female linages in the Iberian communities continued even during the Chalcolithic, when populations became more homogenous, indicating higher mobility and admixture across different geographic regions. Even though the sample size available for Early Bronze Age populations is still limited, especially with regards to the El Argar group, we observe no significant changes to the mitochondrial DNA pool until the end of our time transect (1500 BCE). The expansion of groups from the eastern steppe47,92, which profoundly impacted Late Neolithic and EBA groups of Central and North Europe, cannot (yet) be seen in the contemporaneous population substrate of the Iberian Peninsula at the present level of genetic resolution. This highlights the distinct character of the Neolithic transition both in the Iberian Peninsula and elsewhere and emphasizes the need for further in-depth archaeogenetic studies for reconstructing the close reciprocal relationship of genetic and cultural processes on the population level. Materials and Methods The studied sites are distributed across the Iberian Peninsula, with an emphasis on the archaeologically relevant regions on the Mediterranean coasts, in central and northern Spain and in southern Portugal (Fig.1). We targeted representative sites from the Mesolithic, Neolithic, Chalcolithic and the Early Bronze Age to cover 4,000 years of prehistory on the Iberian Peninsula. Given that the Southern European climate is usually unfavorable for DNA preservation, we implemented a flexible sampling scheme contingent on amplification successes, extending sampling from sites with good ancient DNA preservation and/or including additional sites to obtain sufficient data. Sample specific context information were supplied by our colleagues and project partners in Portugal and Spain or collected from previously published papers. All archaeological sites, relevant radiocarbon dates, and individuals incorporated in the present study are listed in Supplementary TablesS1-2. Altogether, 318 individuals from 57 archaeological sites in Spain, Portugal and Morocco were sampled and analyzed for this study. Whenever possible we preferred samples from recent excavations over those that had been held at museum collections for prolonged periods of time. Teeth and bone samples were taken under clean conditions in Iberian museums, at the Institute of Anthropology at Johannes Gutenberg University in Mainz, or directly on site in the case of La Mina, Arroyal, Els Trocs and in part at La Bastida. Mitochondrial profiles of 37 individuals from our project (Alto de Reinoso (n = 27), La Mina (n = 5) and Els Trocs (n = 5) were previously published by our research team44,47,49. Here we also report additional samples from the sites of La Mina and Els Trocs. The chronological classification of the burials was based on the archaeological data. In order to avoid possible problems due to terminological inconsistencies, we used temporal (from Mesolithic to Early Bronze Age; based on relative chronology and absolute dating) and geographic groupings to assess the population mitogenetic data from the Iberian Peninsula. We further distinguished groups based on contextual archaeological evidence, e. g. subsistence strategies, such as hunter-gatherers vs. early farmers. We targeted collecting two to three teeth from each skeleton and sampled bones only when teeth were not available. Ancient DNA sample preparation. Standard sample preparation protocols were used during the ancient DNA work in the Institute of Anthropology, Johannes Gutenberg University in Mainz55. Samples were first UV irradiated for 30 min each side and then, the surface was removed by shot-blasting with aluminium-oxide-abrasive. Samples were then ground to fine powder using a mixer mill (Retsch). The milling