Accurate species delimitation and identification is crucial for species conservation, providing a foundation for studies on evolutionary biology, ecology, and essentially all biological disciplines. The subfamily Cricetinae (Cricetidae, Rodentia), known as hamsters, is widely distributed in the Palearctic region. At present, there are nine genera and 18 species of hamsters are recognized worldwide, although the taxonomic status of certain taxa remains unclear. In this study, we collected 146 hamster specimens representing 14 species and generated new mitochondrial genomes and nuclear genome-wide single nucleotide polymorphisms (SNPs) to explore their relationships among these hamsters using multiple species delimitation approaches. Results showed: (1) strong phylogenetic support for the classification of Urocricetus, Nothocricetulus, and Cansumys as separate genera; (2) Urocricetus contained two separate species, U. kamensis and U. lama, with U. alticola and U. tibetanus considered synonyms of U. lama; (3) U. kamensis and U. lama are separated by the Nujiang River, with the matching divergence time suggesting that the formation of the river was the primary evolutionary factor driving the species differentiation, and (4) genetic differentiation occurred within the Tscherskia genus, which included two cryptic species.
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