Bioacoustics of Kaloula nonggangensis

Discussion Fei et al. (2009) divided Chinese Kaloula species into two groups: the K. pulchra group (consisting of K. pulchra ) and the K. verrucosa group (consisting of K. borealis , K. rugifera and K. verrucosa ), being consistent with our phylogenetic trees. K. nonggangensis sp. nov. embedded within the K. verrucosa group. K. nonggangensis sp. nov. , K. borealis , K. rugifera and K. verrucosa formed a monophyletic group with high supported values ( Figure 4 ). Preliminary molecular data indicated that K. nonggangensis sp. nov. and K. borealis were sister species. However, due to weak inter-nodes support values (<60%), their phylogenetic positions must be considered as unresolved. We found that the genetic distances among lineages in the K. verrucosa group are lower than the values usually representing differentiation at the species level in frogs (Vences et al. 2005; Fouquet et al. 2007) (Table 2). According to the results of these authors, in most cases species are differentiated by uncorrected p -distances in 16S rRNA of 3% or higher. If the criterion was strictly applied in this group, then K. nonggangensis sp. nov. , K. borealis , K. rugifera and K. verrucosa could also be considered as conspecific populations. However, the morphological differences separating these four taxa, including those in larval morphology, are quite distinct ( Figure 2 , 3 and Table 3), and extend to characters that do not usually show intraspecific variability in anurans. For instance, K. borealis differs by lacking osseous tubercles on the upper surface of the tips of fingers in male, and the finger not being dilated. The digital tips of K. verrucosa are not dilated either. Tadpoles of K. borealis , K. rugifera and K. verrucosa have relatively dark colored in tail muscles and tail fins (vs. having some yolk-yellow spots in K. nonggangensis sp. nov. tadpole) (Fei et al. 2009). Besides, although the diploid number among K. borealis , K. rugifera and K. verrucosa was of 2n=28 chromosomes, their karyotypes are different. The K. borealis karyotypes consisted of eleven metacentric (1, 2, 4–11, 13) and three submetacentric (3, 12, 14) pairs. The K. rugifera karyotypes consisted of eleven metacentric (1, 2, 4–12), one submetacentric (pair 3), one subtelocentric (pair 13) and one telocentric (pair 14) pairs. The K. verrucosa karyotypes consisted of eleven metacentric (1, 2, 4–7, 9–11, 13, 14) and three submetacentric (3, 8, 12) pairs (Wu & Yang 1981; Zeng & Wu 1990; Zhang et al. 2006). Despite the low genetic distances among group, considering their distinct morphology and karyotype, we suggest that they are separated species. In fact, in Asian microhylid genera, a low genetic differentiation among some species has also been reported by Matsui et al. (2011). For instance, the uncorrected p -distance of Micryletta inornata (GenBank no.: AB611968 ) and Micryletta steinegeri (GenBank no.: AB634696 ) in 16S rRNA sequence (~540 bp) is 2.8%. Some 16S rRNA sequences of Asian microhylid (data mainly coming from the paper of Matsui et al . (2011) (Appendix II) together our data indicate that the branches in Kaloula are relatively short (figure no showed). This may either indicate that Kaloula are very young and evolved in a recent rapid radiation, or that mitochondrial substitution rates have become decelerated in this genus. To conclude, a re-evaluation of the species boundaries and the phylogenetic relationships within the genus, using larger samples and bioacoustics is warranted.