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Species identification is one of the most basic yet crucial issues in biology with potentially far-reaching implications for fields such as conservation, population ecology, and epidemiology. The widely distributed but threatened frog  Paa spinosa  has been speculated to represent a complex of multiple species. In this study, 254 individuals representing species of the genus  Paa  were investigated along the entire range of  P. spinosa : 196 specimens of  P. spinosa , 8 specimens of  P. jiulongensis , 5 specimens of  P. boulengeri , 20 specimens of  P. exilispinosa , and 25 specimens of  P. shini . Approximately 1333 bp of mtDNA sequence data (genes 12S rRNA and 16S rRNA) were used. Phylogenetic analyses were conducted using maximum parsimony, maximum likelihood and Bayesian inference. BEAST was used to estimate divergence dates of major clades. Results suggest that  P. spinosa  can be divided into three distinct major lineages. Each major lineage totally corresponds to geographical regions, revealing the presence of three candidate cryptic species. Isolation and differentiation among lineages are further supported by the great genetic distances between the lineages. The bifurcating phylogenetic pattern also suggests an east–west dispersal trend during historic cryptic speciation. Dating analysis estimates that  P. spinosa  from Western China split from the remaining  P. spinosa  populations in the Miocene and that  P. spinosa  from Eastern China diverged from Central China in the Pliocene. We also found that  P. exilispinosa  from Mainland China and Hong Kong might have a complex of multiple species. After identifying cryptic lineages, we then determine the discrepancy between the mtDNA and the morphotypes in several individuals. This discrepancy may have been caused by introgressive hybridization between  P. spinosa  and  P. shini .

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Phylogeographic Analyses Strongly Suggest Cryptic Speciation in the Giant Spiny Frog (Dicroglossidae: Paa spinosa) and Interspecies Hybridization in Paa