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The genus Myotis is one of the most diverse and widely distributed mammals, providing a good model for studies of speciation and diversification across large geographic scales. However, the classification within this genus has long been chaotic. Taxonomic revisions based on multiple data sources are essential and urgent. In this study, morphometrics and genetic markers with different modes of inheritance were used to clarify the taxonomy of Myotis distributed in China. Based on 173 mitochondrial Cytb sequences and five morphological characteristics, 114 specimens collected nationwide over the past 20 years were assigned to 11 Myotis species. All Chinese samples classified into M. davidii and M. longipes were revised to M. alticraniatus and M. laniger. Then, two nuclear fragments (Rag2 and Chd1) and Cytb sequences from representative individuals of Chinese Myotis were used for multilocus phylogeny reconstruction and genetic divergence evaluation. The phylogenetic relationships were clearly demonstrated in the species tree: M. alticraniatus and M. laniger; M. fimbriatus, M. pilosus, M. macrodactylus, and M. petax; and M. pequinius, M. chinensis, and M. blythii formed three strongly supported monophyletic clades. Mitochondrial divergence was almost 10 times that of nuclear divergence, with interspecific K2P distances ranging from 8% to 20% for Cytb and 0.3% to 2.3% for concatenated nuclear genes. Low levels of genetic divergence were observed between M. alticraniatus and M. laniger, as well as M. fimbriatus and M. pilosus. These results provide new insights into the taxonomy and phylogeny of Myotis bats in China and are important for the future research and conservation of Chinese Myotis. [ABSTRACT FROM AUTHOR]

• Multiple evidences were used to delimit species of Rhinolophus macrotis complex. • A new taxonomic treatment is proposed for the R. osgoodi. • Evolutionary processes were reconstructed following species delimitation. • Hybridization and introgression have contributed to the reticulate evolution. Species delimitation and evolutionary reconstruction remain challenging for non-model species that have experienced reticulate evolution and exhibit conflicting patterns of differentiation among multiple lines of evidence, such as mitochondrial and nuclear data and phenotypes. Here, we applied an integrative taxonomic approach to a case study of Rhinolophus macrotis complex, whose taxonomic status remains controversial, to provide insight into the systematics and evolutionary history of these species. By integrating traditional genetic markers with different modes of inheritance, genome-wide SNPs as well as phenotypic characteristics, we clarified the presence of three closely related species, R. episcopus , R. siamensis , and R. osgoodi , within this complex, and proposed a new taxonomic treatment for R. osgoodi. Our results suggested that hybridization and introgression are the main causes of low mtDNA divergence in these species. Combined with the demographic inference, we deduced that glacial–interglacial cycles drove geographic isolation and secondary contacts of these species, then promoted hybridization and lineage fusion among them, finally resulting in a reticulate evolutionary pattern. Overall, our study highlights the importance of combining multiple types of data to delimit species, especially those with conserved morphology, and to reveal the sophisticated processes of speciation. [ABSTRACT FROM AUTHOR]