Karyotype evolution and phylogenetic conflict in Eurasian wild asses illuminated by a chromosome-level genome assembly of the Mongolian wild ass (Equus hemionus hemionus).

The evolutionary history and taxonomy of the Eurasian wild ass (Equus hemionus) remain contentious due to limited genomic resources and conflicting phylogenetic signals. Here, we present the first chromosome-level genome assembly of the Mongolian wild ass (E. h. hemionus). The assembly reveals a diploid number of 2n = 52 and identifies a lineage-specific chromosomal fusion homologous to horse chromosomes 8q and 15, which is uniquely shared with E. kiang. By integrating autosomal, mitochondrial, and Y-chromosomal data with paleogenomes, we resolved a well-supported phylogeny identifying four major clades: European, Indian Subcontinent, East Asian, and Middle Eastern lineages. Our analyses uncovered widespread cytonuclear discordance, largely attributable to introgression events and incomplete lineage sorting. Divergence times coincided with major Pleistocene glacial cycles, a temporal pattern consistent with peripatric speciation during these intervals, with the Hindu Kush region identified as a probable Pleistocene refugium and diversification center. These results provide robust phylogenetic evidence that challenges the current IUCN taxonomic framework, which regards these evolutionarily distinct lineages as regional subpopulations due to previously alleged lack of monophyletic support. Our findings provide a critical genomic foundation for conserving evolutionarily significant units within these threatened species.