Significance One of the most enduring and widely debated questions in prehistoric archaeology concerns the origins of Europe’s earliest farmers: Were they the descendants of local hunter-gatherers, or did they migrate from southwestern Asia, where farming began? We recover genome-wide DNA sequences from early farmers on both the European and Asian sides of the Aegean to reveal an unbroken chain of ancestry leading from central and southwestern Europe back to Greece and northwestern Anatolia. Our study provides the coup de grâce to the notion that farming spread into and across Europe via the dissemination of ideas but without, or with only a limited, migration of people.

Abstract Farming and sedentism first appeared in southwestern Asia during the early Holocene and later spread to neighboring regions, including Europe, along multiple dispersal routes. Conspicuous uncertainties remain about the relative roles of migration, cultural diffusion, and admixture with local foragers in the early Neolithization of Europe. Here we present paleogenomic data for five Neolithic individuals from northern Greece and northwestern Turkey spanning the time and region of the earliest spread of farming into Europe. We use a novel approach to recalibrate raw reads and call genotypes from ancient DNA and observe striking genetic similarity both among Aegean early farmers and with those from across Europe. Our study demonstrates a direct genetic link between Mediterranean and Central European early farmers and those of Greece and Anatolia, extending the European Neolithic migratory chain all the way back to southwestern Asia.

It is well established that farming was introduced to Europe from Anatolia, but the extent to which its spread was mediated by demic expansion of Anatolian farmers, or by the transmission of farming technologies and lifeways to indigenous hunter-gatherers without a major concomitant migration of people, has been the subject of considerable debate. Paleogenetic studies (1⇓⇓–4) of late hunter-gatherers (HG) and early farmers indicate a dominant role for migration in the transition to farming in central and northern Europe, with evidence of only limited hunter-gatherer admixture into early Neolithic populations, but increasing toward the late Neolithic. However, the exact origin of central and western Europe’s early farmers in the Balkans, Greece, or Anatolia remains an open question.

Recent radiocarbon dating indicates that by 6,600–6,500 calibrated (cal) BCE sedentary farming communities were established in northwestern Anatolia at sites such as Barcın, Menteşe, and Aktopraklık C and in coastal western Anatolia at sites such as Çukuriçi and Ulucak, but did not expand north or west of the Aegean for another several hundred years (5). All these sites show material culture affinities with the central and southwestern Anatolian Neolithic (6).

Early Greek Neolithic sites, such as the Franchthi Cave in the Peloponnese, Knossos in Crete, and Mauropigi, Paliambela, and Revenia in northern Greece date to a similar period (7⇓–9). The distribution of obsidian from the Cycladic islands, as well as similarities in material culture, suggest extensive interactions since the Mesolithic and a coeval Neolithic on both sides of the Aegean (8). Although it has been argued that in situ Aegean Mesolithic hunter-gatherers played a major role in the “Neolithization” of Greece (7), the presence of domesticated forms of plants and animals indicates nonlocal Neolithic dispersals into the area.

We present five ancient genomes from both, the European and Asian sides of the northern Aegean (Fig. 1); despite their origin from nontemperate regions, three of them were sequenced to relatively high coverage (∼2–7×), enabling diploid calls using a novel SNP calling method that accurately accounts for postmortem damage (SI Appendix, SI5. Genotype Calling for Ancient DNA). Two of the higher-coverage genomes are from Barcın, south of the Marmara Sea in Turkey, one of the earliest Neolithic sites in northwestern Anatolia (individuals Bar8 and Bar31). On the European side of the Aegean, one genome is from the early Neolithic site of Revenia (Rev5), and the remaining two are from the late and final Neolithic sites of Paliambela (Pal7) and Kleitos (Klei10), dating to ∼2,000 y later (Table 1). Estimates of mitochondrial contamination were low (0.006–1.772% for shotgun data) (Table 1; SI Appendix, SI4. Analysis of Uniparental Markers and X Chromosome Contamination Estimates.). We found unprecedented deamination rates of up to 56% in petrous bone samples, indicating a prehistoric origin for our sequence data from nontemperate environments (SI Appendix, Table S5).

Fig. 1. North Aegean archaeological sites investigated in Turkey and Greece.

Table 1. Neolithic and Mesolithic samples analyzed

Uniparental Genetic Systems The mtDNA haplogroups of all five Neolithic individuals are typical of those found in central European Neolithic farmers and modern Europeans, but not in European Mesolithic hunter-gatherers (1). Likewise, the Y-chromosomes of the two male individuals belong to haplogroup G2a2, which has been observed in European Neolithic farmers (3, 10); in Ötzi, the Tyrolean Iceman (11); and in modern western and southwestern Eurasian populations, but not in any pre-Neolithic European hunter-gatherers (12). The mitochondrial haplogroups of two additional less well-preserved Greek Mesolithic individuals (Theo1, Theo5; SI Appendix, Table S6) belong to lineages observed in Neolithic farmers from across Europe; consistent with Aegean Neolithic populations, unlike central European Neolithic populations, being the direct descendants of the preceding Mesolithic peoples who inhabited broadly the same region. However, we caution against over-interpretation of the Aegean Mesolithic mtDNA data; additional genome-level data will be required to identify the Mesolithic source population(s) of the early Aegean farmers.

Functional Variation Sequences in and around genes underlying the phenotypes hypothesized to have undergone positive selection in Europeans indicate that the Neolithic Aegeans were unlikely to have been lactase persistent but carried derived SLC24A5 rs1426654 and SLC45A2 rs16891982 alleles associated with reduced skin pigmentation. Because our Aegean samples predate the period when the rs4988235 T-allele associated with lactase persistence in Eurasia reached an appreciable frequency in Europe, around 4 kya (12⇓–14), and because this allele remains at relatively low frequencies (<0.15) in modern Greek, Turkish, and Sardinian populations (15), this observation is unsurprising. However, despite their relatively low latitude, four of the Aegean individuals are homozygous for the derived rs1426654 T-allele in the SLC24A5 gene, and four carry at least one copy of the derived rs16891982 G-allele in the SLC45A2 gene. This suggests that these reduced-pigmentation–associated alleles were at appreciable frequency in Neolithic Aegeans and that skin depigmentation was not solely a high-latitude phenomenon (SI Appendix, SI12. Functional Markers). The derived rs12913832 G-allele in the HERC2 domain of the OCA2 gene was heterozygous in one individual (Klei10), but all other Aegeans for whom the allelic state at this locus could be determined were homozygous for the ancestral allele, indicating a lack of iris depigmentation in these individuals. Examination of several SNPs in the TCF7L2 gene region indicates that the two Neolithic Anatolian individuals, Bar8 and Bar31, are likely to have carried at least one copy of a haplotype conferring reduced susceptibility to type 2 diabetes (T2D); the Klei10 and Rev5 individuals also carry a tag allele associated with this haplotype. Consistent with these observations, it has been previously estimated that this T2D-protective haplotype, which shows evidence for selection in Europeans, East Asians, and West Africans, originated ∼11,900 y ago in Europe (16). A number of loci associated with inflammatory disease displayed the derived alleles, including rs2188962 C > T in the SLC22A5/IRF1 region, associated with Crohn’s disease; rs3184504 C > T in the SH2B3/ATXN2 region, associated with rheumatoid arthritis, celiac disease, and type 1 diabetes; and rs6822844 G > T in the IL2/IL21 region, associated with rheumatoid arthritis, celiac disease, and ulcerative colitis. Interestingly, we observe derived states for six of eight loci in a protein–protein interaction network inferred to have undergone concerted positive selection 2.6–1.2 kya in Europeans (17), suggesting that any recent selection on these loci acted on standing variation present at already appreciable frequency (SI Appendix, SI12. Functional Markers).

Hunter-Gatherer Admixture Given that the Aegean is the likely origin of European Neolithic farmers, we used Bar8 and Bar31 as putative sources to assess the extent of hunter-gatherer admixture in European farmers through the Neolithic. f4 statistics of the form f4 (Neolithic farmer, Anatolian, HG, ‡Khomani) indicated small but significant amounts of hunter-gatherer admixture into both Spanish and Hungarian early farmer genomes, and interestingly, the Early Neolithic Greek genome. Our mixture modeling analysis also inferred a small genetic contribution from the Loschbour hunter-gatherer genome (3–9%) to each of the Early Neolithic Hungarian and German genomes, but evidence of a smaller contribution to any Aegean genomes (0–6%). These results suggest that mixing between migrating farmers and local hunter-gatherers occurred sporadically at low levels throughout the continent even in the earliest stages of the Neolithic. However, consistent with previous findings (3), both f4 statistics and ADMIXTURE analysis indicate a substantial increase in hunter-gatherer ancestry transitioning into the Middle Neolithic across Europe, whereas Late Neolithic farmers also demonstrate a considerable input of ancestry from steppe populations (SI Appendix, SI8. Proportions of Ancestral Clusters in Neolithic Populations of Europe and Fig. S32).

Relation to Modern Populations Most of the modern Anatolian and Aegean populations do not appear to be the direct descendants of Neolithic peoples from the same region. Indeed, our mixture model comparison of the Aegean genomes to >200 modern groups (2) indicates low affinity between the two Anatolian Neolithic genomes and six of eight modern Turkish samples; the other two were sampled near the Aegean Sea at a location close to the site of the Neolithic genomes. Furthermore, when we form each Anatolian Neolithic genome as a mixture of all modern groups, we infer no contributions from groups in southeastern Anatolia and the Levant, where the earliest Neolithic sites are found (SI Appendix, Figs. S22 and S30 and Table S30; Dataset S3). Similarly, comparison of allele sharing between ancient and modern genomes to those expected under population continuity indicates Neolithic-to-modern discontinuity in Greece and western Anatolia, unless ancestral populations were unrealistically small (SI Appendix, SI9. Population Continuity). Instead, our mixing analysis shows that each Aegean Neolithic genome closely corresponds to modern Mediterraneans (>68% contributions from southern Europe) and in particular to Sardinians (>25%), as also seen in the PCA and outgroup f3 statistics with few substantial contributions from elsewhere. Modern groups matching the Neolithics—mostly from the Mediterranean and North Africa—strikingly match more to Bar8 from northwestern Anatolia than to the LBK genome from Stuttgart in Germany, indicating that the LBK genome experienced processes such as drift and admixture that were independent from the Mediterranean expansion route, consistent with the dual expansion model.

Concluding Remarks Over the past 7 years, ancient DNA studies have transformed our understanding of the European Neolithic transition (1⇓⇓–4, 12, 13), demonstrating a crucial role for migration in central and southwestern Europe. Our results further advance this transformative understanding by extending the unbroken trail of ancestry and migration all of the way back to southwestern Asia. The high levels of shared drift between Aegean and all available Early Neolithic genomes in Europe, together with the inferred unique drift between Neolithic Aegeans and Early Neolithic genomes from Northern Spain to the exclusion of Early Neolithic genomes from central Europe, indicate that Aegean Neolithic populations can be considered the root for all early European farmers and that at least two independent colonization routes were followed. A key remaining question is whether this unbroken trail of ancestry and migration extends all the way back to southeastern Anatolia and the Fertile Crescent, where the earliest Neolithic sites in the world are found. Regardless of whether the Aegean early farmers ultimately descended from western or central Anatolian, or even Levantine hunter-gatherers, the differences between the ancient genomes presented here and those from the Caucasus (20) indicate that there was considerable structuring of forager populations in southwestern Asia before the transition to farming. The dissimilarity and lack of continuity of the Early Neolithic Aegean genomes to most modern Turkish and Levantine populations, in contrast to those of early central and southwestern European farmers and modern Mediterraneans, is best explained by subsequent gene flow into Anatolia from still unknown sources.

Acknowledgments We thank Songül Alpaslan for help with sampling in Barcın and Eleni Stravopodi for help with sampling in Theopetra. Z.H. and R.M. are supported by a Marie Curie Initial Training Network (BEAN/Bridging the European and Anatolian Neolithic, GA 289966) awarded to M.C., S.J.S., D.G.B., M.G.T., and J. Burger. C.P., J. Burger and S.K. received funding from DFG (BU 1403/6-1). C.P. and J. Burger received funding from the Alexander von Humboldt Foundation. C.S. and M.S. were supported by the European Union (EU) SYNTHESYS/Synthesis of Systematic Resources GA 226506-CP-CSA-INFRA, DFG: (BO 4119/1) and Volkswagenstiftung (FKZ: 87161). L.M.C. is funded by the Irish Research Council (GOIPG/2013/1219). A.S. was supported by the EU CodeX Project 295729. K. Kotsakis, S.T., D.U.-K., P.H., and C.P. were cofinanced by the EU Social Fund and Greek national funds research funding program THALES. C.P., M.U., K. Kotsakis, S.T., and D.U.-K. were cofinanced by the EU Social Fund and the Greek national funds research funding program ARISTEIA II. M.C. was supported by Swiss NSF Grant 31003A_156853. A.K. and D.W. were supported by Swiss NSF Grant 31003A_149920. S.L. is supported by the BBSRC (Grant BB/L009382/1). L.v.D. is supported by CoMPLEX via EPSRC (Grant EP/F500351/1). G.H. is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (Grant 098386/Z/12/Z) and by the National Institute for Health Research University College London Hospitals Biomedical Research Centre. M.G.T. and Y.D. are supported by a Wellcome Trust Senior Research Fellowship Grant 100719/Z/12/Z (to M.G.T.). J. Burger is grateful for support by the University of Mainz and the HPC cluster MOGON (funded by DFG; INST 247/602-1 FUGG). F.G. was supported by Grant 380-62-005 of the Netherlands Organization for Scientific Research.

Footnotes Author contributions: Z.H., S.K., G.H., K. Kirsanow, K.R.V., D.W., M.G.T., C.P., and J. Burger designed research; Z.H., S.K., L.v.D., S.L., L.W., M.U., D.W., and C.P. performed research; K. Kotsakis, P.H., S.T., N.K.-A., D.U.-K., C.Z., F.A., Ç.Ç., B.H., F.G., and C.P. provided archaeological background information; K. Kotsakis, S.T., N.K.-A., D.U.-K., C.Z., F.A., and F.G. provided samples; K. Kotsakis, S.T., D.U.-K., Ç.Ç., B.H., F.G., S.J.S., and C.P. provided text; M.S., A.S., D.W., and M.G.T. contributed new reagents/analytic tools; Z.H., S.K., G.H., C.S., Y.D., D.D.-d.-M., L.v.D., S.L., A.K., V.L., K. Kirsanow, L.M.C., R.M., J. Blöcher, C.L., K.R.V., D.W., and J. Burger analyzed data; and Z.H., S.K., G.H., D.D.-d.-M., L.v.D., S.L., K. Kirsanow, S.G., D.M.B., Ç.Ç., B.H., S.J.S., D.G.B., M.C., K.R.V., M.G.T., C.P., and J. Burger wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: Mitochondrial genome sequences have been deposited in the GenBank database (KU171094–KU171100). Genomic data are available at the European Nucleotide Archive under the accession no. PRJEB11848 in BAM format.

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