Pandas, Giant

We tested for admixture between cave bears and their nearest extant relatives, polar bears and brown bears, with the D statistic (ABBA, BABA test)^{2 (link),21 (link)}. All D statistics calculated during this study are reported in the Supplementary information (Supplementary Data 1–4). To avoid bias resulting from ancient DNA cytosine deamination (C- > T error) damage, we restricted our analysis to transversion sites. To test for significance, we applied the weighted block jackknife^{2 (link)}. Because of the low contiguity of the giant panda genome, we used 1 Mb non-overlapping blocks, rather than the 5 Mb non-overlapping blocks used in previous studies^{2 (link),10 (link),12} , which would exclude most of the panda scaffolds (N50 = 1,281,781)^{19 (link)}. Despite their smaller size, 1 Mb non-overlapping bins are adequate for testing cave bear introgression into brown bears because they are substantially longer than the longest estimated length of introgressed blocks of cave bear ancestry which is 175 kb (Supplementary Table 5). We consider results more than three standard errors different from zero (Z > 3) as strong evidence of admixture, and more than two standard errors different from zero (Z > 2) as providing moderate evidence of admixture.
We investigated four alternative outgroups for admixture tests. These were, in order of increasing phylogenetic distance from the focal clade^{13 (link)}: American black bear, Asiatic black bear, spectacled bear and giant panda. All outgroups resulted in a significant signal of admixture between all cave bears and all modern brown bears, following the divergence of polar bears and brown bears (Supplementary Fig. 6). In contrast, tests involving the Late Pleistocene brown bear similarly supported admixture between brown bears and cave bears when the less divergent outgroups were used, but the two most divergent outgroups resulted in a reversal of the admixture signal (Supplementary Fig. 6). We attribute this effect to accumulated errors in the ancient pseudohaploid sequences as a result of both sequencing error and spurious read mapping, both of which tend to occur at higher rates in ancient compared to modern DNA datasets. Specifically, at sites where the outgroup has a private allele, accumulated errors in an ancient sample occupying the P2 position would convert a proportion of these BBBA sites to BABA sites, causing that individual to appear unadmixed relative to P1. This effect is amplified with more divergent outgroups since they will posses more private alleles relative to the ingroup. For the American black bear outgroup, we also observed consistently elevated D values relative to those generated using other outgroups, suggesting differential allele sharing between American black bear and P1 and P2 ingroup lineages (polar and brown bear, in this case). We calculated D statistics to assess this imbalance using the giant panda as outgroup, and found a significant excess of derived alleles shared between American black bear and polar bears, relative to brown bears, in almost all comparisons (Supplementary Fig. 7). This may reflect admixture between American black bear and polar bear, admixture with a ghost lineage, ancestral population structure, or the transfer of archaic alleles into brown bears by a cave bear vector, but we did not investigate these alternative explanations further. All other outgroups did not show any clear or consistent imbalance in allele sharing with either ingroup lineage. Overall, we selected Asiatic black bear as the most appropriate outgroup, being sufficiently closely related to the ingroup to avoid artefacts associated with ancient datasets (Supplementary Fig. 6), and showing no consistent pattern of differential allele sharing with either polar bears or brown bears (Supplementary Fig. 7). Asiatic black bear was thus used as outgroup for all subsequent admixture tests.
To quantify the amount of admixture, we used the f̂statistic^{21 (link)}, which is the excess of shared derived alleles between the admixed individual and candidate introgressor standardized by the maximum excess of shared derived alleles expected in an entirely (100%) introgressed individual. All f̂values calculated during this study are reported in the Supplementary Data 5 and 6. The f̂expected value is best calculated by using individuals that we hypothesize best approximate the diversity within the introgressing populations. We considered the European cave bears to best represent the diversity within a potentially introgressing cave bear lineage. For brown bear introgressors, we selected Eurasian brown bears as best representing diversity in a potential brown bear introgressor. As with the D statistic, we determined significance based on weighted block jackknife with 1 Mb blocks.