Species identification of zokors in the east of the Qinghai-Xizang Plateau based on both morphology and DNA barcoding

Species classification and identification constitute the cornerstone of biological research. Integrating DNA barcoding with classical taxonomic approaches can enhance the reliability of species identification. In this study, the traditional morphology and mitochondrial cytochrome b gene (Cyt b) sequence were employed to identify 74 zokor samples collected from Ping’an District, Haidong City, Qinghai Province, on the eastern of the Qinghai-Xizang Plateau. Based on skull morphology, individuals with sparsely coated short white hair on the tail (Pac) were identified as Eospalax cansus, whereas those with densely haired tail (Pab) were identified as Eospalax baileyi. A total of 25 Cyt b haplotypes were defined among the 74 specimens. Phylogenetic analysis based on Cyt b gene sequences of eight zokor species from GenBank showed that each species formed a well-supported monophyletic clade. The 16 haplotypes of Pab and the eight haplotypes of Pac from Ping’an District clustered within the clades of E. baileyi and E. cansu, respectively. The genetic distance between Pac and E. cansus was significantly smaller than that between Pac and other species (P E. baileyi was smaller than that between Pab and other species (P Cyt b identification results were consistent with those from skull morphology, confirming that the mitochondrial Cyt b is an effective DNA barcode candidate for Myospalacinae. Morphological and DNA barcoding results indicate that, two species, E. baileyi and E. cansus, are distributed in Ping’an District. Geographically, E. cansus occurs mainly at relatively low altitudes in the north, closer to the Loess Plateau, while E. baileyi is found at higher altitudes in the south, nearer the Qinghai-Xizang Plateau. This study improves our understanding of zokor species composition and distribution in Ping’an District, and provides a solution to the challenges of morphological identification in zokors through DNA barcoding.