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Parallel paleogenomic transects reveal complex genetic history of early European farmers

Ancient DNA studies have established that Neolithic European populations were descended from Anatolian migrants1–8 who received a limited amount of admixture from resident hunter-gatherers3–5,9. Many open questions remain, however, about the spatial and temporal dynamics of population interactions and admixture during the Neolithic period. Using the highest-resolution genome-wide ancient DNA data set assembled to date—a total of 180 samples, 130 newly reported here, from the Neolithic and Chalcolithic of Hungary (6000–2900 BCE, n = 100), Germany (5500–3000 BCE, n = 42), and Spain (5500–2200 BCE, n = 38)—we investigate the population dynamics of Neolithization across Europe. We find that genetic diversity was shaped predominantly by local processes, with varied sources and proportions of hunter-gatherer ancestry among the three regions and through time. Admixture between groups with different ancestry profiles was pervasive and resulted in observable population transformation across almost all cultural transitions. Our results shed new light on the ways that gene flow reshaped European populations throughout the Neolithic period and demonstrate the potential of time-series-based sampling and modeling approaches to elucidate multiple dimensions of historical population interactions.

The population dynamics of the Neolithization process are of great importance for understanding European prehistory [1][2][3][4][5]. The first quantitative model of the Neolithic transition to integrate archaeological and genetic data was the demic diffusion hypothesis [1], which posited that growing population densities among Near Eastern farmers led to a range expansion that spread agriculture to Europe. Ancient DNA analysis has validated major migrations from populations related to Neolithic Anatolians as driving the arrival of farming in Europe [6][7][8][9][10][11][12][13], but the demic diffusion model does not account for the complexities of the interactions between farmers and hunter-gatherers in Europe throughout the Neolithic [2,3,[14][15][16]. For example, ancient DNA has shown that farmers traversed large portions of Europe with limited initial admixture from hunter-gatherers [8,10,12,13,17], and furthermore that farmers and hunter-gatherers lived in close proximity in some locations long after the arrival of agriculture [18,19]. However, genetic data have yet to be used systematically to model the population interactions and transformations during the course of the Neolithic period. Key open questions include whether migrating farmers mixed with hunter-gatherers at each stage of the expansion, and if so how soon after arriving. Additionally, while it has previously been shown that hunter-gatherer ancestry among farmers in several parts of Europe had increased by the Middle Neolithic [10,12,13,20], it is currently unclear whether this was a continuous or discrete process and furthermore whether it involved a continent-wide phenomenon or a variety of parallel, local events.
We compiled a high-resolution data set of 177 Neolithic and Chalcolithic European genomes (pre-dating the arrival of steppe ancestry in the third millennium BCE [10]) from what are now Hungary, Germany, and Spain, of which 127 individuals are newly reported here, 38 with new direct radiocarbon dates ( Figure 1A From what is now Germany, we generated data for 15 new Linearbandkeramik (LBK) EN (∼5500-4850 BCE) and eight new MN (∼4600-3000 BCE) individuals (with new libraries for an additional nine LBK for which we have previously reported data), while from Spain, we sequenced two new EN (∼5500-4500 BCE) and 14 new CA (∼3000-2200 BCE) individuals. After quality control (Methods), we retained 110 samples, which we merged with 50 Neolithic individuals from the literature [9,10,12,22,23]. For population genetic analyses, we focused on a subset of 151 individuals from 15 population groupings for which we had the highest-quality data. We co-analyzed these samples with 25 Neolithic individuals (∼6500-6000 BCE) from northwestern Anatolia [12] to represent the ancestors of the first European farmers (FEF; Supplementary Information section 4) and four primary European hunter-gatherer individuals ("western hunter-gatherers," or WHG): the ∼5700 BCE "KO1" from Hungary [12,22], the ∼5900 BCE "La Braña 1" (LB1) from Spain [12,24], the ∼6100 BCE "Loschbour" from Luxembourg [9], and the ∼12,000 BCE "Villabruna" from northeastern Italy [25].  A principal component analysis (PCA [26]) of our samples showed that, as expected, all of the Neolithic individuals fall along a cline of admixture between FEF and WHG, with varying proportions of hunter-gatherer ancestry (Extended Data Figure 1). All studied population groups are consistent with a common origin in Anatolia (Supplementary Information section 4), and differentiation among the ancestral farmer populations in the three regions is low (Extended Data Figure 1). To investigate genetic structure within the source population(s) of hunter-gatherer ancestry, we combined the four primary WHG individuals with other hunter-gatherers (eastern hunter-gatherers, or "EHG," ∼7000-5000 BCE from Russia [10,12]; "Bichon," ∼10,000 BCE from Switzerland [27]; and "El Mirón," ∼17,000 BCE from Spain [25]) along an approximate east-west transect across Europe.
We computed pairwise outgroup f 3 -statistics, performed multidimensional scaling (MDS) on the resulting matrix, and converted the MDS positions to polar coordinates (Supplementary Information section 5). The correlation between genetic structure and longitude revealed by this analysis ( Figure 1D) suggests that our reference samples can reasonably be used to define a geographic cline among hunter-gatherers within Europe. Computing f 4 -statistics measuring shared drift between our Neolithic samples and the WHG individuals, we observed an increasing trend in hunter-gatherer ancestry over time in each region [10][11][12], although at a slower rate in Hungary than in Germany and Spain, and with limited intra-population structure or heterogeneity ( Figure 2A; Supplementary Information section 6). We find that this hunter-gatherer ancestry is more similar to the eastern WHG individuals (KO1 and Villabruna) farther east and more similar to the western WHG individuals (LB1 and Loschbour) farther west ( Figure 2B). While this pattern does not demonstrate directly where mixture between hunter-gatherers and farmers took place, it suggests that hunter-gatherer ancestry in farmers was to a substantial extent derived from populations from relatively close to where they lived.  To analyze admixed hunter-gatherer ancestry more formally, we modeled Neolithic farmers in an admixture graph framework. We started with a "scaffold" model (Extended Data Figure 3) consisting of Neolithic Anatolians, the four reference WHG individuals, and two outgroups (Mbuti and Kostenki 14 [25,28]). We observed discrete signals of admixture in LB1 and KO1 via f 3 -and f 4 -statistics [29], and both fit best as admixed in To obtain calendar dates of admixture ( Figure 2D [14,[33][34][35]). The greatest differences between Alföld and Transdanubia are observed in the MN, with substantially more hunter-gatherer ancestry in ALPc than LBKT (Figure 2; Extended Data Table 2), and overall, we observe slight trends toward more hunter-gatherer ancestry to the north and east (Extended Data Figure 2), as expected based on the greater archaeological evidence of hunter-gatherer settlement and interactions [33]. By the LN and CA, however, and especially in the Baden period  Table 2). These results are consistent with genetic and archaeological evidence for LBK origins from the early LBKT [35], followed by additional, Central European WHG admixture after about 5500 BCE. Our "Germany MN" grouping shows increased hunter-gatherer ancestry (∼17%, most closely related to Loschbour) and a more recent average date of admixture, reflecting gene flow from hunter-gatherers after the LBK period. We successfully sequenced a total of 16 ). Based on stable isotopes, the first three of these were classified as farmers, while Bla8 had signatures associated with a hunter-gatherer-fisher lifestyle [18]. In accordance with previous results [18], we find that the group of MN farmers experienced admixture with hunter-gatherers, which we now estimate to have been at least 40%. We additionally observe admixture in the individual Bla8, with ∼25% ancestry derived from farmers.
Our results thus provide evidence of asymmetric gene flow between farmers and huntergatherers at Blätterhöhle centered around the relatively late date of 4100 ± 120 BCE (ALDER dates of 10-25 generations).
In Iberia, we again see widespread evidence of local hunter-gatherer admixture, with  We merged libraries from the same individual (for those with more than one) and then combined our new samples with genome-wide data from the literature (ancient individuals as described and as listed in Extended Data Table 1  To test for possible contamination, we used contamMix [48] and ANGSD [49] to estimate rates of apparent heterozygosity in haploid genome regions (mtDNA and the X chromosome in males, respectively). Any samples with > 5% mtDNA mismatching or > 2%

Population assignments for analyses
In most cases, population groupings were used that correspond to archaeological culture assignments based on chronology, geography, and material culture traits. Occasionally, we merged populations that appeared similar genetically in order to increase power: we pooled samples from all phases and groups of the eastern Hungarian MN into a sin-

Acknowledgments
We  Dates of admixture (in generations in the past) as inferred from ALDER through two different methods. On the left are the average individual-level dates used in our main analyses, and on the right are direct estimates for population groups. By default, for group-level estimates, we used all individuals that yielded a date in our standard ALDER procedure, but because of missing data, for some populations we used a subset of individuals (typically those with highest coverage): Starčevo (BAM4a, BAM17b, and LGCS1a; we note that in this case only BAM17b had an ALDER signal individually), ALPc (NE3, NE1, NE4, TISO13a, HELI11a, MEMO2b, and HAJE7a), Tisza (Gorzsa18 and PULE1.24), Baden (GEN22, GEN55, CO1, GEN12a, GEN13a, GEN15a, and GEN17a), LBK (Stuttgart, LBK1992, HAL5, HAL4, HAL2, and HAL19), Extended Data Figure 1. First two principal components from PCA. We merged with populations genotyped on the Affymetrix Human Origins array and computed PCs for a set of 826 present-day western Eurasian individuals (background gray points). We then projected ancient individuals using the "lsqproject" and "shrinkmode" options in smartpca [26]. Shown is a closeup omitting the present-day Bedouin individuals.  Extended Data Figure 3. Scaffold admixture graph used for modeling European Neolithic populations. Dotted lines denote admixture events. Anatolia Neolithic, LB1, and KO1 are modeled as admixed, with Basal Eurasian ancestry, deeper European hunter-gatherer ancestry, and FEF ancestry, respectively. European test populations are fit as a mixture of FEF and ancestry related to one or two of the four WHG individuals (here Villabruna-related as an example). See Supplementary Information section 6 for full details. Extended Data Figure 4. Admixture dates (A) and proportions (B) as a function of time for the Germany (blue) and Iberia (red) time series and simulated data, as in Figure 3. Symbols are as in Figures 1 and 2, here showing population-level averages plus or minus two standard errors. Proportions and standard errors in (B) are normalized by the total hunter-gatherer ancestry in the most recent (rightmost) population in each region. Yellow dashed lines represent continuous admixture simulations: from top to bottom, diminishing 5% per generation, diminishing 3%, diminishing 1%, and uniform. Green solid lines represent pulse-plus-continuous admixture simulations: from top to bottom, all hunter-gatherer ancestry in a pulse at time zero; 3/4 of final hunter-gatherer ancestry in an initial pulse, followed by uniform continuous gene flow; 1/2 in initial pulse and the rest continuous; and 1/4 in initial pulse.