Paleogenomic Evidence for Multi-generational Mixing between Neolithic Farmers and Mesolithic Hunter-Gatherers in the Lower Danube Basin

Abstract

The transition from hunting and gathering to farming involved profound cultural and technological changes. In Western and Central Europe, these changes occurred rapidly and synchronously after the arrival of early farmers of Anatolian origin [1-3], who largely replaced the local Mesolithic hunter-gatherers [1, 4-6]. Further east, in the Baltic region, the transition was gradual, with little or no genetic input from incoming farmers [7]. Here we use ancient DNA to investigate the relationship between hunter-gatherers and farmers in the Lower Danube basin, a geographically intermediate area that is characterized by a rapid Neolithic transition but also by the presence of archaeological evidence that points to cultural exchange, and thus possible admixture, between hunter-gatherers and farmers. We recovered four human paleogenomes (1.1× to 4.1× coverage) from Romania spanning a time transect between 8.8 thousand years ago (kya) and 5.4 kya and supplemented them with two Mesolithic genomes (1.7× and 5.3×) from Spain to provide further context on the genetic background of Mesolithic Europe. Our results show major Western hunter-gatherer (WHG) ancestry in a Romanian Eneolithic sample with a minor, but sizeable, contribution from Anatolian farmers, suggesting multiple admixture events between hunter-gatherers and farmers. Dietary stable-isotope analysis of this sample suggests a mixed terrestrial/aquatic diet. Our results provide support for complex interactions among hunter-gatherers and farmers in the Danube basin, demonstrating that in some regions, demic and cultural diffusion were not mutually exclusive, but merely the ends of a continuum for the process of Neolithization.

Keywords: Eneolithic; Iron Gates; Mesolithic; Neolithic transition; Romania; ancient DNA.

Figure 1

Geographical, Archaeological, and Genetic Information for the Ancient Spanish and Romanian Samples (A) Map showing the location of the archaeological sites: Chan do Lindeiro (Chan_Meso), Canes (Canes1_Meso), Schela Cladovei (SC1_Meso and SC2_Meso), Ostrovul Corbului (OC1_Meso), and Gura Baciului (GB1_Eneo). Gura Baciului is some 250 km north-northeast of the Iron Gates (Schela Cladovei and Ostrovul Corbului), on a small river that eventually connects with the Danube via the River Tisza. Along with the map we include a timeline with the radiocarbon dates of our samples and the time frame of the different prehistoric periods in Romania. (B) Principal-component analysis (PCA). Ancient data (Data S1) were projected onto the first two principal components defined by selected Eurasians from the Human Origins dataset [8, 9]. The Spanish (Chan_Meso and Canes1_Meso) and Romanian (SC1_Meso, SC2_Meso, and OC1_Meso) Mesolithic samples cluster close to European hunter-gatherer samples. The Eneolithic Romanian sample (GB_Eneo) locates in a different region of the plot, between European hunter-gatherer and farmer samples. (C) ADMIXTURE analysis. ADMIXTURE results are shown at K = 17. The Spanish and Romanian hunter-gatherer samples are composed entirely of the “blue” component, which is also found in other European hunter-gatherer samples, with the exception of the oldest Spanish Mesolithic sample, Chan_Meso, which also has a “lilac” component found in South Indians. The Eneolithic individual GB1_Eneo has the “blue” as well as the “orange” component that predominates in early European and Anatolian farmer samples. Error bars in (A) correspond to the radio carbon ages of samples SC1_Meso, OC1_Meso, and GB1_Eneo as reported in Table 1. See also Figures S1–S4 and Table S3.

Figure 2

Outgroup f₃ Statistics Outgroup f₃ statistics of the form f₃(ancient1, ancient2; Mbuti) for (A) SC1_Meso, (B) SC2_Meso, (C) OC1_Meso, and (D) GB1_Eneo. The highest 20 values of the test are given for each sample. The Romanian Mesolithic samples, SC1_Meso, SC2_Meso, and OC1_Meso, share the most drift with each other, followed closely by the WHG individuals. The Romanian Eneolithic sample, GB1_Eneo, also shares the most affinity with WHGs. See also Figure S3 and Data S2 for more values of this test.