Abstract
Horseshoe bats (genus Rhinolophus, family Rhinolophidae) represent an important group within chiropteran phylogeny due to their distinctive traits, including constant high-frequency echolocation, rapid karyotype evolution, and unique immune system. Advances in evolutionary biology, supported by high-quality reference genomes and comprehensive whole-genome data, have significantly enhanced our understanding of species origins, speciation mechanisms, adaptive evolutionary processes, and phenotypic diversity. However, genomic research and understanding of the evolutionary patterns of Rhinolophus are severely constrained by limited data, with only a single published genome of R. ferrumequinum currently available. In this study, we constructed a high-quality chromosome-level reference genome for the intermediate horseshoe bat ( R. affinis). Comparative genomic analyses revealed potential genetic characteristics associated with virus tolerance in Rhinolophidae. Notably, we observed expansions in several immune-related gene families and identified various genes functionally associated with the SARS-CoV-2 signaling pathway, DNA repair, and apoptosis, which displayed signs of rapid evolution. In addition, we observed an expansion of the major histocompatibility complex class II (MHC-II) region and a higher copy number of the HLA- DQB2 gene in horseshoe bats compared to other chiropteran species. Based on whole-genome resequencing and population genomic analyses, we identified multiple candidate loci (e.g., GLI3) associated with variations in echolocation call frequency across R. affinis subspecies. This research not only expands our understanding of the genetic characteristics of the Rhinolophus genus but also establishes a valuable foundation for future research.