Mitochondrial genomes of Middle Pleistocene horses from the open-air site complex of Schöningen.

Deep-time palaeogenomics offers rare insights into macroevolutionary events for both extant and extinct species. Aside from a Middle Pleistocene genome from North American permafrost (780-560 ka) and a number of Late Pleistocene specimens, most ancient horse DNA studies have focused on tracing the origins of domestication and subsequent periods. Here we present mitochondrial genomes from two Equus mosbachensis specimens from Schöningen, Germany, a Middle Pleistocene archaeological site complex with direct and repeated evidence of hominin-horse interactions on the shore of a palaeolake. Using petrous bone sampling, targeted enrichment and damage-aware and polarization-free mitochondrial DNA reconstruction methods, we extend the range of genome recovery in open-air sites to ~300,000 years ago. Phylogenetic analyses position these mitochondrial DNAs in two distinct, deeply divergent lineages, basal to both previously sequenced ancient Eurasian specimens and all modern-day horses. The Schöningen horse mitochondrial DNA data reveal a previously unrecognized diversification event within the clade, ultimately giving rise to modern-day horses, that is molecularly dated to ~570 ka and provides genetic support for the morphological species assignment. By extending the recoverable limits of ancient DNA from Middle Pleistocene open-air sites, our molecular findings bridge a temporal and geographic gap, providing insights on early evolutionary events within the genus Equus.