Instances of hybridization between mammalian taxa in the wild are rarely documented. To test for introgression between sibling species of horseshoe bat (Rhinolophus yunanensis and R. pearsoni) and two subspecies of the latter (R. p. pearsoni and R. p. chinensis), we sequenced two mtDNA and two ncDNA markers in individuals sampled from multiple localities within their overlapping ranges. The interspecific mtDNA gene tree corresponded to the expected taxonomic divisions, and coalescent-based analyses suggested divergence occurred around 4 MYA. However, these relationships strongly conflicted with those recovered from two independent nuclear gene trees, in which R. yunanensis clustered with R. p. pearsoni to the exclusion of R. p. chinensis. This geographically widespread discordance is best explained by large-scale historical introgression of ncDNA from R. yunanensis to R. pearsoni by male-mediated exchange in mixed species colonies during Pleistocene glacial periods, when ranges may have contracted and overlapped more than at present. Further species tree-gene tree conflicts were detected between R. p. pearsoni and R. p. chinensis, also indicating past and/or current introgression in their overlapping regions. However, here the patterns point to asymmetric mtDNA introgression without ncDNA introgression. Analyses of coalescence times indicate this exchange has occurred subsequent to the divergence of these subspecies from their common ancestor. Our work highlights the importance of using multiple data sets for reconstructing phylogeographic histories and resolving taxonomic relationships.

Rhinolophus (Rhinolophidae) is the second most speciose genus in Chiroptera and has extensively diversified diploid chromosome numbers (from 2n = 28 to 62). In spite of many attempts to explore the karyotypic evolution of this genus, most studies have been based on conventional Giemsa staining rather than G-banding. Here we have made a whole set of chromosome-specific painting probes from flow-sorted chromosomes of Aselliscus stoliczkanus (Hipposideridae). These probes have been utilized to establish the first genome-wide homology maps among six Rhinolophus species with four different diploid chromosome numbers (2n = 36, 44, 58, and 62) and three species from other families: Rousettus leschenaulti (2n = 36, Pteropodidae), Hipposideros larvatus (2n = 32, Hipposideridae), and Myotis altarium (2n = 44, Vespertilionidae) by fluorescence in situ hybridization. To facilitate integration with published maps, human paints were also hybridized to A. stoliczkanus chromosomes. Our painting results substantiate the wide occurrence of whole-chromosome arm conservation in Rhinolophus bats and suggest that Robertsonian translocations of different combinations account for their karyotype differences. Parsimony analysis using chromosomal characters has provided some new insights into the Rhinolophus ancestral karyotype and phylogenetic relationships among these Rhinolophus species so far studied. In addition to Robertsonian translocations, our results suggest that whole-arm (reciprocal) translocations involving multiple non-homologous chromosomes as well could have been involved in the karyotypic evolution within Rhinolophus, in particular those bats with low and medium diploid numbers.

The Vespertilionidae is the largest family in the order Chiroptera and has a worldwide distribution in the temperate and tropical regions. In order to further clarify the karyotype relationships at the lower taxonomic level in Vespertilionidae, genome-wide comparative maps have been constructed between Myotis myotis (MMY, 2n = 44) and six vesper bats from China: Myotis altarium (MAL, 2n = 44), Hypsugo pulveratus (HPU, 2n = 44), Nyctalus velutinus (NVE, 2n = 36), Tylonycteris robustula (TRO, 2n = 32), Tylonycteris sp. (TSP, 2n = 30)and Miniopterus fuliginosus (MFU, 2n = 46) by cross-species chromosome painting with a set of painting probes derived from flow-sorted chromosomes of Myotis myotis. Each Myotis myotis autosomal probe detected a single homologous chromosomal segment in the genomes of these six vesper bats except for MMY chromosome 3/4 paint which hybridized onto two chromosomes in the genome of M. fuliginosus. Our results show that Robertsonian translocation is the main mode of karyotype evolution in Vespertilionidae and that the addition of heterochromatic material also plays an important role in the karyotypic evolution of the genera Tylonycteris and Nyctalus. Two conserved syntenic associations (MMY9 + 23 and 18 + 19) could be the synapomorphic features for the genus Tylonycteris. The integration of our maps with the published maps has enabled us to deduce chromosomal homologies between human and these six vesper bats and provided new insight into the karyotype evolution of the family Vespertilionidae.