Morphological evidence for introgressive hybridization between Feirana quadranus and Feirana taihangnica in Qinling Mountains, China

Background. Feirana quadranus and Feirana taihangnica, two species of frogs inhabiting in waterbodies in the Qinling Mountains, China, are believed to be sister species that diverged 4.6 million years ago. In their sympatric area, morphological variations found between the two species imply that the two species had inter-bred. Additionally, F. taihangnica’s polyandrous breeding behavior, without amplexus, would not hinder the potential hybridization. Methods. To verify the hybridization, 117 specimens of F. quadranus and F. taihangnica were collected from eight sampling sites in their sympatric area, and 110 of the specimens were classified morphologically into VV, vw&wv, and ww, representing the putative parental and suspected hybrid types. Their maternal bloodlines were identified using a phylogenetic tree based on a region of the mitochondrial 16S rRNA gene. In total, 34 morphometric indices were selected to analyze the morphological variation between 16S-types or among morphotypes. A principal component analysis (PCA) and linear discriminant analysis (LDA) were conducted on total or partial indices for females, males, and total specimens, as well as simulated populations with falsified morphotypes. The most important indices for differentiation among morphotypes were revealed with the assistance of heat-maps. Results. In the mitochondrial DNA tree, most of the VV were in the same clade as the reference F. quadranus, labeled as Q, while most of the ww and vw&wv were grouped with the reference F. taihangnica, labeled as T. According to the PCA, there was a clear differentiation between VV and ww, while vw&wv specimens were in the middle area close to ww. According to the LDA, VV, vw&wv, and ww were clustered into three separate groups. An ambiguous differentiation between Q and T was shown both in mtDNA tree and in multivariate analyses. Seven of the specimens with conflicting classifications blurred the morphological boundary between Q and T. In both the PCA and LDA, indices that were based on the extent of bumps and skin coloration discriminated VV, vw&wv, and ww better than ratio indices that were derived from measurements. Discussion. The distribution of VV, vw&wv, and ww in multivariate spaces, especially vw&wv being scattered between VV and ww, demonstrated an introgressive hybridization pattern. The extents of bumps in the shape of an inverted "V" between the shoulder blades, spot pattern on the back, and large bumps above the anal region were the most important characteristics for differentiating between three morphotypes or between F. quadranus and F. taihangnica.