Anatolia and the Near East have long been recognized as the epicenter of the Neolithic expansion through archaeological evidence. Recent archaeogenetic studies on Neolithic European human remains have shown that the Neolithic expansion in Europe was driven westward and northward by migration from a supposed Near Eastern origin []. However, this expansion and the establishment of numerous culture complexes in the Aegean and Balkans did not occur until 8,500 before present (BP), over 2,000 years after the initial settlements in the Neolithic core area []. We present ancient genome-wide sequence data from 6,700-year-old human remains excavated from a Neolithic context in Kumtepe, located in northwestern Anatolia near the well-known (and younger) site Troy []. Kumtepe is one of the settlements that emerged around 7,000 BP, after the initial expansion wave brought Neolithic practices to Europe. We show that this individual displays genetic similarities to the early European Neolithic gene pool and modern-day Sardinians, as well as a genetic affinity to modern-day populations from the Near East and the Caucasus. Furthermore, modern-day Anatolians carry signatures of several admixture events from different populations that have diluted this early Neolithic farmer component, explaining why modern-day Sardinian populations, instead of modern-day Anatolian populations, are genetically more similar to the people that drove the Neolithic expansion into Europe. Anatolia’s central geographic location appears to have served as a connecting point, allowing a complex contact network with other areas of the Near East and Europe throughout, and after, the Neolithic.

Anatolia: From the Pre-Pottery Neolithic to the End of the Early Bronze Age (10,500–2000 bce).

Archaeological evidence on the westward expansion of farming communities from Eastern Anatolia to the Aegean and the Balkans.

Results and Discussion

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Özdoğan M. Die Neolithische Revolution in Anatolien. 9 Özdoğan M. Anatolia: From the Pre-Pottery Neolithic to the End of the Early Bronze Age (10,500–2000 bce). 13 Gabriel U. Ein Blick zurück – Das fünfte Jahrtausend vor Christus in der Troas. As the earliest Neolithic sites in Anatolia predate the indications of Neolithization in Europe, it was already recognized a century ago that the Neolithic lifestyle must have spread from here to neighboring regions, although the mode of transmission was debated []. In recent years, the topic has generated new momentum through archaeogenetic research, providing evidence for migrations being responsible for the spread of Neolithic life ways []. According to the archaeological record, the Neolithic period in Anatolia spans over 6,000 years—from 11,000 before present (BP) to around 5,000 BP []. After the initial settlements in the Neolithic core area, including the central Anatolian plateau, the development continued within the area but without further expanding the borders [] while maintaining complex interactions with the Levant []. Settlements and pottery finds along the Anatolian west coast from 8,500 BP until 7,500 BP indicate a westward expansion with a large impact on the local demography and the establishment of numerous culture complexes []. The site of Kumtepe, located in northwestern Anatolia and established around 7,000 BP [], is one of the settlements that emerged after this expansion.

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Ancient DNA reveals key stages in the formation of central European mitochondrial genetic diversity. 17 Fernández E.

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et al. Mitochondrial DNA genetic relationships at the ancient Neolithic site of Tell Halula. 18 Green R.E.

Malaspinas A.-S.

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et al. A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing. We generated genome-wide sequence data from two Neolithic individuals excavated at the site of Kumtepe and obtained ∼0.13× genome coverage for Kum6 (6,700 BP) and ∼0.01× for Kum4 (5,500–4,800 BP) ( Tables S1 and S2 ). The sequence data showed evidence of nucleotide misincorporations characteristic of post-mortem degradation [] ( Figure S1 ). Given the low genome coverage of Kum4, it was only possible to use this individual to corroborate the patterns observed in Kum6 (see the Supplemental Experimental Procedures , section S4). We obtained mitochondrial genomes with coverage of 21× for individual Kum6 and 1.5× for Kum4. Kum6 carries the H2a mitochondrial haplogroup ( Supplemental Experimental Procedures , section 3.2; Table S3 ), a haplogroup commonly found in modern-day Eastern Europeans and Caucasians []. Haplogroup H is the most common haplogroup in Europe and the Near East, and it is thought to have originated in the Near East 25,000–30,000 years ago []. It is also frequently observed in early farmers of Europe [] and the Near East []. We estimated mtDNA contamination [] of Kum6 and found low levels of contamination (2.0%, with a 95% confidence interval of 0.0%–5.9%) ( Table S3 ).

25 Alexander D.H.

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Sjögren K.-G.

et al. Genomic diversity and admixture differs for Stone-Age Scandinavian foragers and farmers. 4 Lazaridis I.

Patterson N.

Mittnik A.

Renaud G.

Mallick S.

Kirsanow K.

Sudmant P.H.

Schraiber J.G.

Castellano S.

Lipson M.

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et al. Derived immune and ancestral pigmentation alleles in a 7,000-year-old Mesolithic European. 23 Gamba C.

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et al. Genome flux and stasis in a five millennium transect of European prehistory. 24 Haak W.

Lazaridis I.

Patterson N.

Rohland N.

Mallick S.

Llamas B.

Brandt G.

Nordenfelt S.

Harney E.

Stewardson K.

et al. Massive migration from the steppe was a source for Indo-European languages in Europe. 19 Allentoft M.E.

Sikora M.

Sjögren K.-G.

Rasmussen S.

Rasmussen M.

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Vinner L.

et al. Population genomics of Bronze Age Eurasia. 26 Raghavan M.

Skoglund P.

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Stafford Jr., T.W.

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et al. Upper Palaeolithic Siberian genome reveals dual ancestry of Native Americans. 27 Fu Q.

Li H.

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Johnson P.L.F.

Aximu-Petri A.

Prüfer K.

de Filippo C.

et al. Genome sequence of a 45,000-year-old modern human from western Siberia. 28 Seguin-Orlando A.

Korneliussen T.S.

Sikora M.

Malaspinas A.-S.

Manica A.

Moltke I.

Albrechtsen A.

Ko A.

Margaryan A.

Moiseyev V.

et al. Paleogenomics. Genomic structure in Europeans dating back at least 36,200 years. 3 Skoglund P.

Malmström H.

Omrak A.

Raghavan M.

Valdiosera C.

Günther T.

Hall P.

Tambets K.

Parik J.

Sjögren K.-G.

et al. Genomic diversity and admixture differs for Stone-Age Scandinavian foragers and farmers. 19 Allentoft M.E.

Sikora M.

Sjögren K.-G.

Rasmussen S.

Rasmussen M.

Stenderup J.

Damgaard P.B.

Schroeder H.

Ahlström T.

Vinner L.

et al. Population genomics of Bronze Age Eurasia. 24 Haak W.

Lazaridis I.

Patterson N.

Rohland N.

Mallick S.

Llamas B.

Brandt G.

Nordenfelt S.

Harney E.

Stewardson K.

et al. Massive migration from the steppe was a source for Indo-European languages in Europe. 3 Skoglund P.

Malmström H.

Omrak A.

Raghavan M.

Valdiosera C.

Günther T.

Hall P.

Tambets K.

Parik J.

Sjögren K.-G.

et al. Genomic diversity and admixture differs for Stone-Age Scandinavian foragers and farmers. Figure 3 Ancestry Proportions Inferred from Model-Bases Clustering Show full caption 23 Gamba C.

Jones E.R.

Teasdale M.D.

McLaughlin R.L.

Gonzalez-Fortes G.

Mattiangeli V.

Domboróczki L.

Kővári I.

Pap I.

Anders A.

et al. Genome flux and stasis in a five millennium transect of European prehistory. The map shows population ancestry proportions for modern-day western Eurasian and North African populations determined using ADMIXTURE for K = 9 (note that eastern Eurasian populations were included in the analysis, as well; see Figure S3 for the full set of results). Ancestry proportions for the ancient individuals are shown as bar charts in the lower panel. Ajv58 was grouped with the Mesolithic samples (despite being dated to the Neolithic time period) since it is genetically and culturally more similar to European Mesolithic hunter-gatherers. Ko1 is, genetically, very similar to the hunter-gather gene pool [], but it has been excavated from a farmer context. ADMIXTURE results for other values of K are shown in Figure S3 In order to infer population structure and admixture signatures among Kum6 and other populations, as well as other ancient individuals, we conducted a maximum-likelihood clustering analysis using ADMIXTURE [] ( Figures 3 and S5 ). We included all modern-day Eurasian and North African populations, a set of ancient European farmers and hunter-gatherers with more than 1× coverage [], a Yamnayan individual and individuals with the largest number of SNPs from each Neolithic group from a recent SNP capture study [], six Bronze Age Asian individuals [], and three Paleolithic individuals []. For a model with nine clusters (K = 9; results for higher numbers of clusters are similar, Figure S3 ), three major ancestry components were observed in the ancient individuals. The first one (blue), observed as the main component in all hunter-gatherers, is also found as a minor contribution to all farmers, which is in line with the observed admixture from hunter-gatherers into farmers []. The second (orange) and the third (green) components were observed mostly in farmers to varying degrees (∼5%–68% and ∼0.06%–45% for K = 9, respectively). The orange component is mainly found in present-day Western Europeans, whereas the third component (green) is mostly found in the modern-day Near East and Caucasus, and the highest proportion of this third component among Neolithic individuals was observed in Kum6 (∼45% for K = 9). The notion that this component is West Asian is also supported by its presence in a Bronze Age Armenian sample (51%), which contains less than 2% of the orange component. Interestingly, this “West Asian” component (green) is not related to the potential genetic material brought to Europe by migration during the Bronze Age and recently connected to the Yamnaya culture [], visualized in Figure 3 as light blue, and it is observed in high frequency in modern-day people from southern Asia. The elevated “West Asian” affinity of Kum6 is likely to be the cause of the genetic differentiation observed between Kum6 and all other ancient farmers shown in the PCA plot ( Figure 1 B). A clear decline was observed in the values of the green component over time (average of ∼29% in Early Neolithic, ∼14% in Middle Neolithic, and 2% in Late Neolithic), which is consistent with increased admixture with hunter-gatherer groups []. Our results suggest that the two ancestry components of ancient farmers (orange and green in Figure 3 ) were established at an early stage, probably before the first farmers expanded into Europe, and were maintained in Europe up until the end of Middle Neolithic and that both components are present in various modern-day European populations. Therefore, these observations directly link the early European Neolithic gene pool to western Anatolia.

20 Patterson N.

Moorjani P.

Luo Y.

Mallick S.

Rohland N.

Zhan Y.

Genschoreck T.

Webster T.

Reich D. Ancient admixture in human history. 20 Patterson N.

Moorjani P.

Luo Y.

Mallick S.

Rohland N.

Zhan Y.

Genschoreck T.

Webster T.

Reich D. Ancient admixture in human history. 23 Gamba C.

Jones E.R.

Teasdale M.D.

McLaughlin R.L.

Gonzalez-Fortes G.

Mattiangeli V.

Domboróczki L.

Kővári I.

Pap I.

Anders A.

et al. Genome flux and stasis in a five millennium transect of European prehistory. 29 Hervella M.

Rotea M.

Izagirre N.

Constantinescu M.

Alonso S.

Ioana M.

Lazăr C.

Ridiche F.

Soficaru A.D.

Netea M.G.

de-la-Rua C. Ancient DNA from South-East Europe Reveals Different Events during Early and Middle Neolithic Influencing the European Genetic Heritage. 30 Scheu A.

Powell A.

Bollongino R.

Vigne J.-D.

Tresset A.

Çakırlar C.

Benecke N.

Burger J. The genetic prehistory of domesticated cattle from their origin to the spread across Europe. 7 Özdoğan M. Archaeological evidence on the westward expansion of farming communities from Eastern Anatolia to the Aegean and the Balkans. 9 Özdoğan M. Anatolia: From the Pre-Pottery Neolithic to the End of the Early Bronze Age (10,500–2000 bce). 31 Borić D.

Price T.D. Strontium isotopes document greater human mobility at the start of the Balkan Neolithic. 32 Whitehouse R.

Renfrew C. The Copper Age of peninsular Italy and the Aegean. 33 Barfield L. The Iceman reviewed. Figure 4 Affinities among Ancient Farmers and Admixture Graph Inference Show full caption (A) D statistics testing the consistency of proposed tree topologies of the shape (outgroup, Kum6; early farmer 1, early farmer 2) using genetic data. Negative values suggest strong affinities between Kum6 and early farmer 1 (labels on the left), whereas positive values indicate that Kum6 is closer to early farmer 2 (labels on the right). Significant differences from zero can be interpreted as rejection of the tree topology. Error bars represent ±2 SEs. See also Table S5 (B) TreeMix plot for Kum6 allowing two migration events. The symbols indicate the two different cultural groups: triangles indicate individuals from farming contexts, and circles indicate individuals from hunter-gatherer contexts. We computed D statistics [] to further investigate additional genetic relationships between ancient Europeans with sufficient sequencing coverage (>1×) and Kum6. All proposed tree topologies where the Tyrolean Iceman [] was included as one of the in-groups were rejected (2 < |Z| < 4.6), suggesting gene flow or a more recent shared ancestry between Kum6 and the Tyrolean Iceman ( Figure 4 A). A similar tendency was observed with a Middle Neolithic Hungarian farmer [], (co1), contemporary with the Tyrolean Iceman, despite the limited resolution due to the low coverage of Kum6 and co1. The observed genetic affinity between the Tyrolean Iceman and Kum6 could be interpreted as additional contacts between western Anatolia and Neolithic Europe at a later stage. This scenario is congruent with mitochondrial [] and archaeozoological [] studies, as well as the archaeological indications of multiple waves of contact between the Balkans and Anatolia []. A continuous contact between northwestern Anatolia and southeastern Europe is in not surprising, given the archaeological record, although it has not been detected in the genomic data previously.

19 Allentoft M.E.

Sikora M.

Sjögren K.-G.

Rasmussen S.

Rasmussen M.

Stenderup J.

Damgaard P.B.

Schroeder H.

Ahlström T.

Vinner L.

et al. Population genomics of Bronze Age Eurasia. 24 Haak W.

Lazaridis I.

Patterson N.

Rohland N.

Mallick S.

Llamas B.

Brandt G.

Nordenfelt S.

Harney E.

Stewardson K.

et al. Massive migration from the steppe was a source for Indo-European languages in Europe. 19 Allentoft M.E.

Sikora M.

Sjögren K.-G.

Rasmussen S.

Rasmussen M.

Stenderup J.

Damgaard P.B.

Schroeder H.

Ahlström T.

Vinner L.

et al. Population genomics of Bronze Age Eurasia. 24 Haak W.

Lazaridis I.

Patterson N.

Rohland N.

Mallick S.

Llamas B.

Brandt G.

Nordenfelt S.

Harney E.

Stewardson K.

et al. Massive migration from the steppe was a source for Indo-European languages in Europe. 27 Fu Q.

Li H.

Moorjani P.

Jay F.

Slepchenko S.M.

Bondarev A.A.

Johnson P.L.F.

Aximu-Petri A.

Prüfer K.

de Filippo C.

et al. Genome sequence of a 45,000-year-old modern human from western Siberia. 28 Seguin-Orlando A.

Korneliussen T.S.

Sikora M.

Malaspinas A.-S.

Manica A.

Moltke I.

Albrechtsen A.

Ko A.

Margaryan A.

Moiseyev V.

et al. Paleogenomics. Genomic structure in Europeans dating back at least 36,200 years. 34 Pickrell J.K.

Pritchard J.K. Inference of population splits and mixtures from genome-wide allele frequency data. 3 Skoglund P.

Malmström H.

Omrak A.

Raghavan M.

Valdiosera C.

Günther T.

Hall P.

Tambets K.

Parik J.

Sjögren K.-G.

et al. Genomic diversity and admixture differs for Stone-Age Scandinavian foragers and farmers. 4 Lazaridis I.

Patterson N.

Mittnik A.

Renaud G.

Mallick S.

Kirsanow K.

Sudmant P.H.

Schraiber J.G.

Castellano S.

Lipson M.

et al. Ancient human genomes suggest three ancestral populations for present-day Europeans. Furthermore, the Bronze Age Yamnayan component suggested to be a part of the Corded ware expansion [] is not present in Kum6, and thus is not producing any increased affinity to the ancestors of the Yamnaya culture from north of the Caucasus (D-Denisovan, Yamnaya_RISE; Kum6, early farmer), all Z > −1.7). Contacts to the east, independent of Yamnaya ancestry [] are, however, supported by (1) higher affinity of Kum6 to some Bronze Age Asian cultures when compared to Mesolithic Europeans and (2) higher affinities of Bronze Age Asians to Kum6 compared to early Neolithic Europeans ( Table S5 ). A comparison of Kum6 to an Asian Upper Paleolithic individual (Ust-Ishim []) and a European Upper Paleolithic sample (Kostenki14 []) confirms that Kum6 shows more affinity to early Europeans (Z = 5). Stronger affinities of Kostenki14 to Kum6 than to early Neolithic Europeans ( Table S5 ), however, suggest that Kum6 contains genomic components not found in early Neolithic Europeans. Kum6 is also grouped with farmers in a model-based population-tree analysis [] ( Figure 4 B), and the inferred migration edges point to the same conclusion as the D statistics results, as well as manifest the expected signals from previously published observations [].