BackgroundMitochondrial introgression may result in the mitochondrial genome of one species being replaced by that of another species without leaving any trace of past hybridization in its nuclear genome. Such introgression can confuse the species genealogy estimates and lead to absurd inferences of species history. We used a phylogenetic approach to explore the potential mitochondrial genome introgression event(s) between two closely related green pond frog species, Pelophylax nigromaculatus and P. plancyi.ResultsDNA sequence data of one mitochondrial and two nuclear genes from an extensive sampling of the two species were collected, and the genealogies of the three genes were constructed and compared. While the two nuclear genes congruently showed mutual reciprocal monophyly of both species, the mitochondrial phylogeny separated a Korean P. nigromaculatus clade, a paraphyletic central China P. plancyi assemblage, and a large well-supported introgression clade. Within the introgression clade, the mitochondrial haplotypes of the two species were mixed together. This reticulated pattern can be most parsimoniously explained by an ancient mitochondrial introgression event from P. plancyi to P. nigromaculatus that occurred at least 1.36 MYA, followed by multiple recent introgression events from P. nigromaculatus back to P. plancyi within the last 0.63 MY. The re-constitution of previously co-adapted genomes in P. plancyi may be responsible for the recent rampant introgression events. The Korean P. nigromaculatus clade likely represents the only surviving "true" mitochondrial lineage of P. nigromaculatus, and the central China P. plancyi assemblage likely represents the "original" P. plancyi mitochondrial lineage. Refugia in the Korean Peninsula and central China may have played a significant role in preserving these ancient lineages.ConclusionsThe majority of individuals in the two species have either introgressed (P. nigromaculatus) or reclaimed (P. plancyi) mitochondrial genomes while no trace of past hybridization in their nuclear genomes was detected. Asymmetrical reproductive ability of hybrids and continuous backcrossing are likely responsible for the observed mitochondrial introgression. This case is unique in that it includes an ancient "forward" introgression and many recent "backward" introgressions, which re-constitutes the original nuclear and mitochondrial genomes of P. plancyi. This hybrid system provides an excellent opportunity to study cyto-nuclear interaction and co-adaptation.

While comparatively few amphibian species have been described on the North East Asian mainland in the last decades, several species have been the subject of taxonomical debates in relation to the Yellow sea. Here, we sampled Dryophytes sp. treefrogs from the Republic of Korea, the Democratic People's Republic of Korea and the People's Republic of China to clarify the status of this clade around the Yellow sea and determine the impact of sea level change on treefrogs' phylogenetic relationships. Based on genetics, call properties, adult morphology, tadpole morphology and niche modelling, we determined the segregated status species of D. suweonensis and D. immaculatus. We then proceeded to describe a new treefrog species, D. flaviventris sp. nov., from the central lowlands of the Republic of Korea. The new species is geographically segregated from D. suweonensis by the Chilgap mountain range and known to occur only in the area of Buyeo, Nonsan and Iksan in the Republic of Korea. While the Yellow sea is the principal element to the current isolation of the three clades, the paleorivers of the Yellow sea basin are likely to have been the major factor for the divergences within this clade. We recommend conducting rapid conservation assessments as these species are present on very narrow and declining ranges.

Aim Pelophylax frogs in East Asia provide an opportunity to explore the impact of glacial cycling on demographic and genetic dynamics, because it has been suggested that they experienced distribution shifts and subsequent mtDNA introgression from Pelophylax plancyi to Pelophylax nigromaculatus in association with climatic oscillations. However, their evolutionary history, including the pattern of introgression, is incompletely understood. We used phylogenetic analyses based on multiple markers to address their evolutionary history, and palaeodistribution modelling to test whether the predicted distribution can explain the pattern of introgression suggested by molecular phylogenetics.Location East Asia, including far-eastern Russia, mainland China, the Japanese archipelago, the Korean Peninsula and Taiwan.Methods Ninety-nine samples of the Pelophylax nigromaculatus species complex were collected from 75 localities throughout the distributional range of each species. Phylogenetic analyses were performed with the mitochondrial cytochrome b (cytb) gene and six nuclear genes using two European Pelophylax frogs as outgroups. Additionally, palaeodistributions of P. nigromaculatus and P. plancyi were predicted using bioclimatic variables.Results Differences were observed between the mitochondrial and nuclear DNA trees in the phylogenetic position of P. plancyi. Similar phylogenetic discordance was also observed within P. nigromaculatus. Distribution modelling suggested that a considerable distributional shift of P. nigromaculatus occurred in association with glacial cycling and that the distributional range of P. plancyi was relatively narrow during glacial periods. Main conclusionsThe analyses detected five historical mtDNA introgression events and determined their directions, none of which had been deduced in previous studies. In association with glacial cycling, the distributional ranges of P. nigromaculatus and P. plancyi appear to have shifted repeatedly, resulting in multiple complex introgressions. By combining phylogenetic analyses with palaeodistribution modelling, our results supported the hypothesis that the history of mtDNA introgression among Pelophylax frogs was shaped by glacial cycling.

In mice and humans, the androgen receptor (AR) gene, located on the X chromosome, is not known to be involved in sex determination. In the Japanese frog Rana rugosa the AR is located on the sex chromosomes (X, Y, Z and W). Phylogenetic analysis shows that the AR on the X chromosome (X-AR) of the Korean R. rugosa is basal and segregates into two clusters: one containing W-AR of Japanese R. rugosa, the other containing Y-AR. AR expression is twice as high in ZZ (male) compared to ZW (female) embryos in which the W-AR is barely expressed. Higher AR-expression may be associated with male sex determination in this species. To examine whether the Z-AR is involved in sex determination in R. rugosa, we produced transgenic (Tg) frogs carrying an exogenous Z-AR. Analysis of ZW Tg frogs revealed development of masculinized gonads or ‘ovotestes’. Expression of CYP17 and Dmrt1, genes known to be activated during normal male gonadal development, were up-regulated in the ZW ovotestis. Testosterone, supplied to the rearing water, completed the female-to-male sex-reversal in the AR-Tg ZW frogs. Here we report that Z-AR is involved in male sex-determination in an amphibian species.

Systematics of the Water Toad (Bufo stejnegeri) Elucidates the Evolution of Semi-aquatic Toad Ecology and Pleistocene Glacial Refugia.