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

Deep-time paleogenomics 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 (mtDNA) 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 paleolake. Using petrous bone sampling, targeted enrichment and novel damage-aware and polarization-free mtDNA reconstruction methods, we extend the range of genome recovery in open-air sites to ~300,000 years ago. Phylogenetic analyses position these mtDNAs in two distinct, deeply divergent lineages, basal to both previously sequenced ancient Eurasian specimens and all modern-day horses. The Schöningen horse mtDNA data reveals 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 new insights on early evolutionary events within the genus Equus.