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Both molecular data and archaeological evidence strongly support an African origin for the domestic donkey. Recent genetic studies further suggest that there were two distinct maternal lineages involved in its initial domestication. However, the exact introduction time and the dispersal process of domestic donkeys into ancient China are still unresolved. To address these questions, we retrieved three near-complete mitochondrial genomes from donkey specimens excavated from Gaoling County, Shaanxi Province, and Linxia Basin, Gansu Province, China, dated at 2,349-2,301, 469-311, and 2,160-2,004 cal. BP, respectively. Maximum-likelihood and Bayesian phylogenetic analyses reveal that the two older samples fall into the two different main lineages (i.e., clade Ⅰ and clade Ⅱ) of the domestic donkey, suggesting that the two donkey maternal lineages had been introduced into Midwestern China at least at the opening of Silk Road (approximately the first century BC). Bayesian analysis shows that the split of the two donkey maternal lineages is dated at 0.323 Ma (95% CI: 0.583–0.191 Ma) using root-tip dating calibrations based on near-complete mitogenomes, supporting the hypothesis that modern domestic donkeys go back to at least two independent domestication events. Moreover, Bayesian skyline plot analyses indicate an apparent female population increase between 5,000 and 2,500 years ago for clade I followed by a stable population size to the present day. In contrast, clade II keeps a relatively stable population size over the past 5,000 years. Overall, our study provides new insights into the early domestication history of Chinese domestic donkeys.

The extant diversity of horses (family Equidae) represents a small fraction of that occurring over their evolutionary history. One such lost lineage is the subgenus Sussemionus, which is thought to have become extinct during the Middle Pleistocene. However, recent molecular studies and morphological analysis have revealed that one of their representatives, E. ovodovi, did exist in Siberia during the Late Pleistocene. Fossil materials of E. ovodovi have thus far only been found in Russia. In this study, we extracted DNA from three equid fossil specimens excavated from northeastern China dated at 12,770-12,596, 29,525-28,887 and 40,201-38,848 cal. yBP, respectively, and retrieved three near-complete mitochondrial genomes from the specimens. Phylogenetic analyses cluster the Chinese haplotypes together with previously published Russian E. ovodovi, strongly supporting the assignment of these samples to this taxon. The molecular identification of E. ovodovi in northeastern China extends the known geographical range of this fossil species by several thousand kilometers to the east. The estimated coalescence time of all E. ovodovi haplotypes is approximately 199 Kya, with the Chinese haplotypes coalescing approximately 130 Kya. With a radiocarbon age of 12,770-12,596 cal. yBP, the youngest sample in this study represents the first E. ovodovi sample dating to the terminal Pleistocene, moving the extinction date of this species forwards considerably compared to previously documented fossils. Overall, comparison of our three mitochondrial genomes with the two published ones suggests a genetic diversity similar to several extant species of the genus Equus.