Ancient Eurasia K6

[Valorie Carlee]

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We constructed a neighbour-joining (NJ) tree using the whole-genome sequence data set (Fig. 1b and Supplementary Figs 9 and 10) to determine which modern dog population shows the greatest genetic similarity to the ancient samples (Supplementary Note 8). We found that the Early Neolithic HXH and Late Neolithic NGD grouped together as a sister clade to modern European village dogs, while CTC was external to this clade, but still more similar to it than to any other modern population. As shown previously12, East Asian village dogs and breeds are basal to all other dogs.

(a) PCA of village dogs, with breed dogs and ancient dogs projected onto the PC space using SNP array data. (b) PCA of village dogs, breed dogs and ancient dogs using whole-genome SNP data ascertained in the New World wolves.

We note that the position of NGD in our reanalysis does not agree with that reported in Frantz et al.12, where it lies as an outlier in PC2. Such deviation was interpreted by Frantz et al.12 as NGD carrying ancestry from an extinct European population. However, we found that this is due to a technical artifact that occurred because of the inclusion of both the uncalibrated and calibrated version of this ancient genome in the same PCA. Once one of the duplicate data points is removed from the sample set, NGD returns to the modern dog cluster (Supplementary Figs 11 and 12). Fundamental differences in the overall distribution of genetic variance in PC space between the two studies are due to the overall ascertainment of samples. Since our data set contains a substantially more diverse collection of samples, we assert that our PCA results are more reflective of the true range of dog diversity.

We further examined the genetic relatedness between ancient and modern dogs by performing an f3-outgroup analysis21,22 on both the SNP array and whole-genome sequence data sets. We used the golden jackal and Andean fox as outgroups for the SNP array and the whole-genome data sets, respectively. Our results corroborated NJ tree and PCA findings, and showed that all three Neolithic European samples are genetically most similar to modern European dogs (Fig. 3, Supplementary Figs 13 and 14 and Supplementary Note 10).

Our results are consistent with continuity of a European-like genetic ancestry from modern dogs through the entire Neolithic period. However, the slightly displaced position of the ancient samples from the European cluster in the PCAs (particularly for CTC) suggests a complex history. We therefore performed unsupervised clustering analyses with ADMIXTURE (SNP array data; Supplementary Fig. 15) and NGSadmix (whole-genome data; Fig. 4 and Supplementary Fig. 16) (Supplementary Note 9) and found that, unlike contemporary European village dogs, all three ancient genomes possess a significant ancestry component that is present in modern Southeast Asian dogs. This component appears only at very low levels in a minority of modern European village dogs. Furthermore, CTC harbours an additional component that is found predominantly in modern Indian village as well as in Central Asian (Afghan, Mongolian and Nepalese), and Middle Eastern (Saudi Arabian and Qatari) dogs (concordant with its position in the PCA), as well as some wolf admixture.

To disentangle the more complex admixture patterns observed in CTC, we first sought to understand its relationship to HXH given that both samples originate from Germany. Our f3-outgroup analysis revealed that CTC had greater affinity with HXH than with any modern canid or with NGD (Fig. 3b, Supplementary Figs 13b and 14b and Supplementary Note 10). We therefore performed a MixMapper analysis where HXH was set as one of the sources of admixture for CTC, which identified a population ancestral to modern Indian or Saudi Arabian village dogs as the second source of admixture (Supplementary Table 11). Further support for genetic continuity between HXH and CTC was found using ADMIXTUREGRAPH when two alternative demographic models were tested. In the first model (model A), CTC descends from the same population as HXH followed by admixture with an Indian-like population, while in model B both ancient samples descend from independently diverged European lineages (and therefore there was no genetic continuity between the two). Model A provides a much better fit to the data (Fig. 5a and Supplementary Fig. 20), producing only two f4 outliers (no f2 or f3 outliers), one of which was barely significant (Z=3.013). Model B produced 74 outliers (Supplementary Note 11). Even though there is the risk of overfitting the model to the data, the stark difference between the two models points to continuity among German dogs during the Neolithic, along with gene flow into CTC at the end of this era from an outside source carrying the genetic component observed in contemporary Middle Eastern and Central and South Asian dog populations.

The complex pattern of admixture found in CTC is similar to that observed in many modern dog populations in Central Asia (such as Afghanistan) and the Middle East, as shown in our unsupervised clustering analyses (Supplementary Figs 15 and 16). This raises the question of whether CTC and these modern dog populations share a common admixture history and are descended from the same ancestral populations. We performed a MixMapper analysis that included HXH in the scaffold tree and observed that the European-like component of CTC is drawn exclusively from this Early Neolithic German dog population (Supplementary Table 13). To the contrary, modern Afghan dogs generally demonstrate inferred ancestry from modern European village dogs. This suggests that modern Afghan village dogs and CTC are the result of independent admixture events.

The distinct genetic makeup of the European Neolithic dogs compared to modern European dogs indicates that while ancient and contemporary populations share substantial genomic ancestry, some degree of population structure was likely present on the continent. Neolithic dogs would thus represent a now extinct branch that is somewhat diverged from the modern European clade. In addition, our best fit model of modern and ancient canid demography using ADMIXTUREGRAPH involved a topology that would be consistent with a single dog lineage diverging from wolves (Fig. 5a and Supplementary Table 14). Therefore, we attempted to infer the divergence time of HXH and NGD from modern European dogs after the divergence of the Indian lineage that, according to the NJ tree analysis, is the sister clade of the Western Eurasian branch. We note that this is a simplistic bifurcating model of what may have been more complex European geographic structuring and long-term Eurasian dog gene flow.

As a result of the domestication process, specific portions of dog genomes have significantly differentiated from wolves30. To determine the domestication status of the three Neolithic dogs, we assessed haplotype diversity at candidate domestication loci. Using only breed dogs and wolves, a previous study identified 36 candidate domestication loci30 (Supplementary Table 18). However, our analysis of a more diverse sample set that includes village dogs confirms only 18 of these loci as putative domestication targets, the remainder are likely associated with breed formation (Supplementary Table 19 and Supplementary Note 14). HXH appeared homozygous for the dog-like haplotype at all but one of these 18 loci, and thus was often indistinguishable from most modern dogs. The younger NGD appeared dog-like at all but two loci. CTC, however, was heterozygous for the wolf-like haplotype at six loci, compatible with its increased wolf ancestry described above.

However, the admixture events observed in European Neolithic dogs but not in most modern dogs (and even then to a lesser extent) from the same region suggest some degree of population structure on the continent during that period. This is further reflected by HXH and NGD carrying both Southeast Asian ancestry but lacking the ancestry shared between CTC and modern Middle Eastern, Central and South Asian village dogs, even though NGD and CTC are contemporaneous (4,800 and 4,700 years old, respectively). It is likely that under this scenario of population structure, a subpopulation distinct from that of HXH, CTC and NGD eventually became dominant in modern European dogs, which may explain the observed mtDNA turnover from haplogroup C to A, especially if this subpopulation also passed through a strong bottleneck. Additional support for population structure comes from the clustering of all the ancient samples within C1 into a sub-haplogroup distinct from that of modern dogs, while it is also noteworthy that non-C haplogroups, including A, are more apparent in Southeast Europe in the archaeological record12.

CTC shows similar admixture patterns to Central Asian and Middle Eastern modern dog populations. Considering that the age of the samples provides a time frame (between 7,000 and 5,000 years ago) for CTC to obtain its unique ancestry component, and that the cranium was found next to two individuals associated with the Neolithic Corded Ware culture, we speculate that this component was derived from incoming populations of dogs that accompanied steppe people migrating from the East13.

Analyses incorporating admixture in their model show a significant proportion of modern Indian-like ancestry in CTC. However, in addition, there is a potential wolf-like component observed from our NGSadmix and Spacemix analyses, as well as a Southeast Asian component that appears in all three Neolithic dogs. Given such a complex picture of admixture, with four potential sources that must be inferred from a single genome, it is perhaps unsurprising that different methods demonstrate variability in their inferred Indian-like admixture proportions (from 25% in NGSadmix up to 69% in ADMIXTUREGRAPH). We hope that more genomes from Central Europe from this era will help clarify this complicated picture of admixture in the future.

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