The Yamnaya Steppe Herders from Russia

Figure 1: Genetic structure of ancient Europe.

Ancient genomes from Eurasia have revealed three ancestral populations that contributed to contemporary Europeans in varying degrees1. Mesolithic individuals, sampled from Spain all the way to Hungary1, 2, 3, belong to a relatively homogenous group, termed western hunter-gatherers (WHG). The expansion of early farmers (EF) out of the Levant during the Neolithic transition led to major changes in the European gene pool, with almost complete replacement in the south and increased mixing with local WHG further north1, 2, 3, 4, 5. Finally, a later wave originating with the Early Bronze Age Yamnaya from the Pontic steppe, carrying partial ancestry from ancient North Eurasians (ANE) and ancestry from a second, undetermined source, arrived from the east, profoundly changing populations and leaving a cline of admixture in Eastern and Central Europe1, 3, 6. This view, which was initially based on a handful of genomes, was recently confirmed by extensive surveys of Eurasian samples from the Holocene5, 7.

Here, we extend our view of the genetic makeup of early Europeans by both looking further back in time and sampling from the crossroads between the European and Asian continents. We sequenced a Late Upper Palaeolithic (‘Satsurblia’ from Satsurblia cave, 1.4-fold coverage) and a Mesolithic genome (‘Kotias’ from Kotias Klde cave, 15.4-fold) from Western Georgia, at the very eastern boundary of Europe. We term these two individuals Caucasus hunter-gatherers (CHG). To extend our overview of WHG to a time depth similar to the one available for our samples from the Caucasus, we also sequenced a western European Late Upper Palaeolithic genome, ‘Bichon’ (9.5-fold) from Grotte du Bichon, Switzerland. These new genomes, together with already published data, provide us with a much-improved geographic and temporal coverage of genetic diversity across Europe after the Last Glacial Maximum (LGM)8. We show that CHG belong to a new, distinct ancient clade that split from WHG ~45 kya and from Neolithic farmer ancestors ~25 kya. This clade represents the previously undetermined source of ancestry to the Yamnaya, and contributed directly to modern populations from the Caucasus all the way to Central Asia.


Figure 2: The relationship between Caucasus hunter-gatherers (CHG), western hunter-gatherers and early farmers.

The geographical proximity of the Southern Caucasus to the Levant begs the question of whether CHG might be related to early Neolithic farmers with Near Eastern heritage. To address this question formally we reconstructed the relationship among WHG, CHG and EF using available high-quality ancient genomes1, 3. We used outgroup f3-statistics14 to compare the three possible topologies, with the correct relationship being characterized by the largest amount of shared drift between the two groups that form a clade with respect to the outgroup (Fig. 2a; Supplementary Note 4). A scenario in which the population ancestral to both CHG and EF split from WHG receives the highest support, implying that CHG and EF form a clade with respect to WHG. We can reject a scenario in which CHG and WHG form a distinct clade with respect to EF. The known admixture of WHG with EF1, 3, 4, 5 implies that some shared drift is found between WHG and EF with respect to CHG, but this is much smaller than the shared drift between CHG and EF. Thus, WHG split first, with CHG and EF separating only at a later stage.

A partial genome from a 24,000-year-old individual (MA1) from Mal’ta, Siberia6 had been shown to be divergent from other ancient samples and was shown by Lazaridis et al.1, using f4 statistics, to have more shared alleles with nearly all modern Europeans than with an EF genome. This allowed inference of an ANE component in European ancestry, which was subsequently shown to have an influence in later eastern hunter-gatherers and to have spread into Europe via an incursion of Steppe herders beginning ~4,500 years ago5, 7. Several analyses indicate that CHG genomes are not a subset of this ANE lineage. First, MA1 and CHG plot in distinct regions of the PCA and also have very different profiles in the ADMIXTURE analysis (Fig. 1). Second, when we test if CHG shows any evidence of excess allele sharing with MA1 relative to WHG using tests of the form D(Yoruba, CHG; MA1, WHG) no combinations were significantly positive (Supplementary Table 6). Last, we also tested whether the ancestral component inferred in modern Europeans from MA1 was distinct from any that may have been donated from CHG using tests of the form D(Yoruba, MA1; CHG, modern North European population) (Supplementary Table 7). All northern Europeans showed a significant sharing of alleles with MA1 separate to any they shared with CHG.


Figure 3: Distribution of ROH.

Given their geographic origin, it seems likely that CHG and EF are the descendants of early colonists from Africa who stopped south of the Caucasus, in an area stretching south to the Levant and possibly east towards Central and South Asia. WHG, on the other hand, are likely the descendants of a wave that expanded further into Europe. The separation of these populations is one that stretches back before the Holocene, as indicated by local continuity through the Late Palaeolithic/Mesolithic boundary and deep coalescence estimates, which date to around the LGM and earlier. Several analyses show that CHG are distinct from another inferred minor ancestral population, ANE, making them a divergent fourth strand of European ancestry that expands the model of the human colonization of that continent.

The separation between CHG and both EF and WHG ended during the Early Bronze Age when a major ancestral component linked to CHG was carried west by migrating herders from the Eurasian Steppe. The foundation group for this seismic change was the Yamnaya, who we estimate to owe half of their ancestry to CHG-linked sources. These sources may be linked to the Maikop culture, which predated the Yamnaya and was located further south, closer to the Southern Caucasus. Through the Yamanya, the CHG ancestral strand contributed to most modern European populations, especially in the northern part of the continent.


Figure 4: The relationship of Caucasus hunter-gatherers to modern populations.

Finally, we found that CHG ancestry was also carried east to become a major contributor to the Ancestral North Indian component found in the Indian subcontinent. Exactly when the eastwards movement occurred is unknown, but it likely included migration around the same time as their contribution to the western European gene pool and may be linked with the spread of Indo-European languages. However, earlier movements associated with other developments such as that of cereal farming and herding are also plausible.

The discovery of CHG as a fourth ancestral component of the European gene pool underscores the importance of a dense geographical sampling of human palaeogenomes, especially among diverse geographical regions. Its separation from other European ancestral strands ended dramatically with the extensive population, linguistic and technological upheavals of the Early Bronze Age resulting in a wide impact of this ancestral strand on contemporary populations, stretching from the Atlantic to Central and South Asia.

Nature Communications 6, Article number: 8912

Population relationships of samples.

Genome-wide ancient DNA from West Eurasia
We assembled genome-wide data from 230 ancient individuals from West Eurasia dated to between 6500 and 300 bc (Fig. 1a, Extended Data Table 1, Supplementary Data Table 1 and Supplementary Information section 1). To obtain this data set, we combined published data from 67 samples from relevant periods and cultures4–6, with 163 samples for which we report new data, of which 83 have, to our knowledge, never previously been analysed (the remaining 80 samples include 67 whose targeted single nucleotide polymorphism (SNP) coverage we tripled from 390,000 (‘390k capture’) to 1,240,000 (‘1240k capture’)7; and 13 with shotgun data for which we generated new data using our targeted enrichment strategy3,8). The 163 samples for which we report new data are drawn from 270 distinct individuals who we screened for evidence of authentic DNA7. We used in-solution hybridization with synthesized oligonucleotide probes to enrich promising libraries for the targeted SNPs (Methods). The targeted sites include nearly all SNPs on the Affymetrix Human Origins and Illumina 610-Quad arrays, 49,711 SNPs on chromosome X, 32,681 SNPs on chromosome Y, and 47,384 SNPs with evidence of functional importance. We merged libraries from the same individual and filtered out samples with low coverage or evidence of contamination to obtain the final set of individuals. The 1240k capture gives access to genome-wide data from ancient samples with small fractions of human DNA and increases efficiency by targeting sites in the human genome that will actually be analysed. The effectiveness of the approach can be seen by comparing our results to the largest previously published ancient DNA study, which used a shotgun sequencing strategy5

Insight into population transformations
To learn about the genetic affinities of the archaeological cultures for which genome-wide data are reported for the first time here, we studied either 1,055,209 autosomal SNPs when analysing 230 ancient individuals alone, or 592,169 SNPs when co-analysing them with 2,345 present-day individuals genotyped on the Human Origins array4. We removed 13 samples either as outliers in ancestry relative to others of the same archaeologically determined culture, or first-degree relatives (Supplementary Data Table 1). Our sample of 26 Anatolian Neolithic individuals represents the first genome-wide ancient DNA data from the eastern Mediterranean. Our success at analysing such a large number of samples is due to the fact that in the case of 21 of the successful samples, we obtained DNA from the inner ear region of the petrous bone9, which has been shown to increase the amount of DNA obtained by up to two orders of magnitude relative to teeth3. Principal component (PCA) and ADMIXTURE10 analyses show that the Anatolian Neolithic samples do not resemble any present-day near-Eastern populations but are shifted towards Europe, clustering with early European farmers (EEF) from Germany, Hungary and Spain7 (Fig. 1b and Extended Data Fig. 2).

Further evidence that the Anatolian Neolithic and EEF were related comes from the high frequency (47%; n= 15) of Y-chromosome haplogroup G2a typical of ancient EEF samples7 (Supplementary Data Table 1), and the low fixa- tion index (FST; 0.005 – 0.016) between Neolithic Anatolians and EEF (Supplementary Data Table 2). These results support the hypothesis7 of a common ancestral population of EEF before their dispersal along distinct inland/central European and coastal/Mediterranean routes. The EEF are slightly more shifted to Europe in the PCA than are the Anatolian Neolithic (Fig. 1b) and have significantly more admixture from Western hunter-gatherers (WHG), as shown by f4-statistics (|Z| > 6 standard errors from 0) and negative f3-statistics (|Z| > 4)11 (Extended Data Table 2). We estimate that the EEF have 7–11% more WHG admixture than their Anatolian relatives (Extended Data Fig. 2, Supplementary Information section 2).


Genome-wide scan for selection.

The Iberian Chalcolithic individuals from El Mirador cave are genet- ically similar to the Middle Neolithic Iberians who preceded them (Fig. 1b and Extended Data Fig. 2), and have more WHG ancestry than their Early Neolithic predecessors7 (|Z|> 10) (Extended Data Table 2). However, they do not have a significantly different proportion of WHG ancestry (we estimate 23–28%) than the Middle Neolithic Iberians (Extended Data Fig. 2). Chalcolithic Iberians have no evi- dence of steppe ancestry (Fig. 1b and Extended Data Fig. 2), in contrast to central Europeans of the same period5,7. Thus, the steppe-related ancestry that is ubiquitous across present-day Europe4,7 arrived in Iberia later than in central Europe (Supplementary Information section 2). To understand population transformations in the Eurasian steppe, we analysed a time transect of 37 samples from the Samara region spanning ~5600–1500 bc and including the Eastern hunter-gath- erer (EHG), Eneolithic, Yamnaya, Poltavka, Potapovka and Srubnaya cultures. Admixture between populations of Near Eastern ancestry and the EHG7 began as early as the Eneolithic (5200–4000 bc), with some individuals resembling EHG and some resembling Yamnaya (Fig. 1b and Extended Data Fig. 2).


Allele frequencies for five genome-wide significant signals of selection.

The Yamnaya from Samara and Kalmykia, the Afanasievo people from the Altai (3300–3000 bc), and the Poltavka Middle Bronze Age (2900–2200 bc) population that followed the Yamnaya in Samara are all genetically homogeneous, forming a tight ‘Bronze Age steppe’ cluster in PCA (Fig. 1b), sharing predom- inantly R1b Y chromosomes5,7 (Supplementary Data Table 1), and having 48–58% ancestry from an Armenian-like Near Eastern source (Extended Data Table 2) without additional Anatolian Neolithic or EEF ancestry7 (Extended Data Fig. 2). After the Poltavka period, popula- tion change occurred in Samara: the Late Bronze Age Srubnaya have ~17% Anatolian Neolithic or EEF ancestry (Extended Data Fig. 2). Previous work documented that such ancestry appeared east of the Urals beginning at least by the time of the Sintashta culture, and suggested that it reflected an eastward migration from the Corded Ware peoples of central Europe5. However, the fact that the Srubnaya also had such ancestry indicates that the Anatolian Neolithic or EEF ancestry could have come into the steppe from a more eastern source. Further evidence that migrations originating as far west as central Europe may not have had an important impact on the Late Bronze Age steppe comes from the fact that the Srubnaya possess exclusively (n= 6) R1a Y chromosomes (Supplementary Data Table 1), and four of them (and one Poltavka male) belonged to haplogroup R1a-Z93, which is common in central/ south Asians12, very rare in present-day Europeans, and absent in all ancient central Europeans studied to date.

The strongest signal of selection is at the SNP (rs4988235) responsible for lactase persistence in Europe15,16. Our data (Fig. 3) strengthens previous reports that an appreciable frequency of lactase persistence in Europe only dates to the last 4,000 years3,5,17. The allele’s earliest appearance in the dataset is in a central European Bell Beaker sample (individual I0112) dated to between 2450 and 2140 bc. Two other independent signals related to diet are located on chromosome 11 near FADS1 and DHCR7. FADS1 and FADS2 are involved in fatty acid metabolism, and variation at this locus is associated with plasma lipid and fatty acid concentration18. The selected allele of the most significant SNP (rs174546) is associated with decreased triglyceride levels18. This locus has experienced independent selection in non-Eu- ropean populations13,19,20 and is likely to be a critical component of adaptation to different diets. Variants at DHCR7 and NADSYN1 are associated with circulating vitamin D levels21 and the most associ- ated SNP in our analysis, rs7940244, is highly differentiated across closely related northern European populations22,23, suggesting selection related to variation in dietary or environmental sources of vitamin D. Two signals have a potential link to coeliac disease. One occurs at the ergothioneine transporter SLC22A4 that is hypothesized to have experienced a selective sweep to protect against ergothioneine deficiency in agricultural diets24. Common variants at this locus are associated with increased risk for ulcerative colitis, coeliac disease, and irritable bowel disease and may have hitchhiked to high frequency as a result of this sweep24–26. However, the specific variant (rs1050152, L503F) that was thought to be the target did not reach high frequency until relatively recently (Extended Data Fig. 4). The signal at ATXN2/SH2B3—also associated with coeliac disease25—shows a similar pattern (Extended Data Fig. 4). The second strongest signal in our analysis is at the derived allele of rs16891982 in SLC45A2, which contributes to light skin pigmentation and is almost fixed in present-day Europeans but occurred at much lower frequency in ancient populations. In contrast, the derived allele of SLC24A5 that is the other major determinant of light skin pigmentation in modern Europe (and that is not significant in the genome-wide scan for selection) appears fixed in the Anatolian Neolithic, suggesting that its rapid increase in frequency to around 0.9 in Early Neolithic Europe was mostly due to migration (Extended Data Fig. 4). Another pigmentation signal is at GRM5, where SNPs are associated with pigmentation possibly through a regulatory effect on nearby TYR27.



We also find evidence of selection for the derived allele of rs12913832 at HERC2/OCA2, which is at 100% frequency in the European hunter- gatherers we analysed, and is the primary determinant of light eye colour in present-day Europeans28,29. In contrast to the other loci, the range of frequencies in modern populations is within that of ancient populations (Fig. 3). The frequency increases with higher latitude, suggesting a complex pattern of environmental selection. The TLR1–TLR6–TLR10 gene cluster is a known target of selection in Europe, possibly related to resistance to leprosy, tuberculosis or other mycobacteria30–32. There is also a strong signal of selection at the major histocompatibility complex (MHC) on chromosome 6. The strongest signal is at rs2269424 near the genes PPT2 and EGFL8, but there are at least six other apparently independent signals in the MHC (Extended Data Fig. 3); and the entire region is significantly more associated than the genome-wide average (residual inflation of 2.07 in the region on chromosome 6 between 29–34 Mb after genome-wide genomic control correction). This could be the result of multiple sweeps, balancing selection, or increased drift as a result of background selection reducing effective population size in this gene-rich region.

Nature 528, 499–503 (24 December 2015)