Multilocus approaches reveal underestimated species diversity and inter-specific gene flow in pikas (Ochotona) from southwestern China

[Display omitted] •A potential new subgenus represented by the O. syrinx species complex.•Uncover three potential new species.•Mitochondrial introgression was observed from O. cansus to O. curzoniae. The phylogeny of living pikas (Ochotonidae, Ochotona) remains obscure, and pika species diversity in southwestern China has never been well explored. In this study, 96 tissue samples from 11 valid species in three classified subgenera (Pika, Ochotona and Conothoa) from 23 locations were characterized using multilocus sequences of 7031bp. Two mitochondrial (CYT B and COI) and five nuclear gene segments (RAG1, RAG2, TTN, OXAIL and IL1RAPL1) were sequenced. We analysed evolutionary histories using maximum likelihood (RAxML) and Bayesian analyses (BEAST), and we also used molecular species delimitation analyses (BPP) to explore species diversity. Our study supported O. syrinx (O. huangensis) as a distinct clade from all named subgenera. Relationships among subgenera were not fully resolved, which may be due to a rapid diversification in the middle Miocene (∼13.90Ma). Conflicting gene trees implied mitochondrial introgression from O. cansus to O. curzoniae. We uncovered three cryptic species from Shaanxi, Sichuan and Yunnan with strong support, suggesting an underestimation of species diversity in the “sky-island” mountains of southwest China.