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The extremely rich palaeontological record of the horse family, also known as equids, has provided many examples of macroevolutionary change over the last ~55 Mya. This family is also one of the most documented at the palaeogenomic level, with hundreds of ancient genomes sequenced. While these data have advanced understanding of the domestication history of horses and donkeys, the palaeogenomic record of other equids remains limited. In this study, we have generated genome‐wide data for 25 ancient equid specimens spanning over 44 Ky and spread across Anatolia, the Caucasus, Central Asia and Mongolia. Our dataset includes the genomes from two extinct species, the European wild ass, Equus hydruntinus, and the sussemione Equus ovodovi. We document, for the first time, the presence of sussemiones in Mongolia and their survival around ~3.9 Kya, a finding that should be considered when discussing the timing of the first arrival of the domestic horse in the region. We also identify strong spatial differentiation within the historical ecological range of Asian wild asses, Equus hemionus, and incomplete reproductive isolation in several groups yet considered as different species. Finally, we find common selection signatures at ANTXR2 gene in European, Asian and African wild asses. This locus, which encodes a receptor for bacterial toxins, shows no selection signal in E. ovodovi, but a 5.4‐kb deletion within intron 7. Whether such genetic modifications played any role in the sussemione extinction remains unknown. [ABSTRACT FROM AUTHOR]

DNA hybridization‐capture techniques allow researchers to focus their sequencing efforts on preselected genomic regions. This feature is especially useful when analysing ancient DNA (aDNA) extracts, which are often dominated by exogenous environmental sources. Here, we assessed, for the first time, the performance of hyRAD as an inexpensive and design‐free alternative to commercial capture protocols to obtain authentic aDNA data from osseous remains. HyRAD relies on double enzymatic restriction of fresh DNA extracts to produce RNA probes that cover only a fraction of the genome and can serve as baits for capturing homologous fragments from aDNA libraries. We found that this approach could retrieve sequence data from horse remains coming from a range of preservation environments, including beyond radiocarbon range, yielding up to 146.5‐fold on‐target enrichment for aDNA extracts showing extremely low endogenous content (<1%). Performance was, however, more limited for those samples already characterized by good DNA preservation (>20%–30%), while the fraction of endogenous reads mapping on‐ and off‐target was relatively insensitive to the original endogenous DNA content. Procedures based on two instead of a single round of capture increased on‐target coverage up to 3.6‐fold. Additionally, we used methylation‐sensitive restriction enzymes to produce probes targeting hypomethylated regions, which improved data quality by reducing post‐mortem DNA damage and mapping within multicopy regions. Finally, we developed a fully automated hyRAD protocol utilizing inexpensive robotic platforms to facilitate capture processing. Overall, our work establishes hyRAD as a cost‐effective strategy to recover a set of shared orthologous variants across multiple ancient samples. [ABSTRACT FROM AUTHOR]