SummaryThe 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, 2, 3], who largely replaced the local Mesolithic hunter-gatherers [1, 4, 5, 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.

85between ~12,000-1,400 BCE, from Natufian hunter-gatherers to Bronze Age farmers. 86 We show that the earliest populations of the Near East derived around half their 87 ancestry from a 'Basal Eurasian' lineage that had little if any Neanderthal admixture 88 and that separated from other non-African lineages prior to their separation from each 89 other. The first farmers of the southern Levant (Israel and Jordan) and Zagros 90 Mountains (Iran) were strongly genetically differentiated, and each descended from 91 local hunter-gatherers. By the time of the Bronze Age, these two populations and 92 Anatolian-related farmers had mixed with each other and with the hunter-gatherers of 93 Europe to drastically reduce genetic differentiation. The impact of the Near Eastern 94 farmers extended beyond the Near East: farmers related to those of Anatolia spread 95 westward into Europe; farmers related to those of the Levant spread southward into 96 East Africa; farmers related to those from Iran spread northward into the Eurasian 97 steppe; and people related to both the early farmers of Iran and to the pastoralists of 98 the Eurasian steppe spread eastward into South Asia. 99 Between 10,000-9,000 BCE, humans began practicing agriculture in the Near East 1 . In the 100 ensuing five millennia, plants and animals domesticated in the Near East spread throughout 101 West Eurasia (a vast region that also includes Europe) and beyond. The relative homogeneity 102 of present-day West Eurasians in a world context 2 suggests the possibility of extensive 103 migration and admixture that homogenized geographically and genetically disparate sources 104 of ancestry. The spread of the world's first farmers from the Near East would have been a 105mechanism for such homogenization. To date, however, due to the poor preservation of DNA 106 in warm climates, it has been impossible to study the population structure and history of the 107 first farmers and to trace their contribution to later populations. 108In order to overcome the obstacle of poor DNA preservation, we took advantage of two 109 methodological developments. First, we sampled from the inner ear region of the petrous 110 bone 3,4 that can yield up to ~100 times more endogenous DNA than other skeletal elements 4 . 111Second, we used in-solution hybridization 5 to enrich extracted DNA for about 1.2 million 112 single nucleotide polymorphism (SNP) targets 6,7 , making efficient sequencing practical by 113 filtering out microbial and non-informative human DNA. We merged all sequences extracted 114 from each individual, and randomly sampled a single sequence to represent each SNP, 115 restricting to individuals with at least 9,000 SNPs covered at least once. We obtained 116 genome-wide data passing quality control for 45 individuals on whom we had a median 117 4 coverage of 172,819 SNPs (Methods). We assembled radiocarbon dates for 26 individuals

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These authors contributed equally to this study.Electronic supplementary material is available online at https://dx.