Confused geographical-structure of population and mito-nuclear discordance have been shaped by a combination of rapid population demographic changes and ecological shifts. In this study, we generated a time calibrated phylogeny of Scutiger boulengeri, an endemic Tibetan alpine toad occurring in mountain streams in Qinghai-Tibet Plateau, based on three mitochondria (mtDNA) genes, in which eight clades were assigned into three deeply divergent lineages. While nuclear DNA (nuDNA) revealed three distinct clusters without geographical structure indicating significantly high rates of gene flow by population structure analysis. The coalescent theory framework (approximate Bayesian computation model DIYABC and Migrate-N) suggested that the intraspecific main clusters divergence was the result of hybridization after secondary contact in Holocene around 0.59 Ma, with population size change. The ratio of mtDNA divergence to nuDNA divergence was 2.3, less than 4, failed to test a sex-biased dispersal. Geographic cline analysis showing a wide hybrid zone initially established in southwestern China, without significant reproductive isolation but a strong influence of introgression in S. boulengeri, suggested high fitness of hybrids. Moreover, mtDNA exhibited isolation by distance (IBD) while nuDNA showed significant isolation by environment (IBE). Our results suggested such mito-nuclear discordance might have been first caused by geographic isolations, followed by hybridization mediated by precipitation, and produced a wide hybrid zone, which contributed to confused geographical-structure of the nuDNA in S. boulengeri. This study unveiled the complicated historical process that might have led to specific genetic pattern and specific climate factor facilitating phenotypic diversity in this system.

The studies of climatic-niche shifts over evolutionary time accompanied by key morphological innovations have attracted the interest of many researchers recently. We analyzed the realized niche dynamics across clades within Scutiger boulengeri using ecological niche models (ENMs), ordination method (environment principal component analysis; PCA-env), and correspondingly key morphological innovations combined phylogenetic comparative methods (PCMs) and phylogenetic generalized least squares (PGLS) regression methods throughout their distributions in Qinghai-Tibet Plateau (QTP) margins of China in Asia. Our analyses reveal that there is obvious niche divergence caused by niche expansion across S. boulengeri clades, especially in E. A, E. B and partial of E. C clades. Moreover, niche expansion is more popular than niche unfilling into novel environmental conditions. Annual mean temperature and Annual precipitation are the most important contributors in E. A and E. B clades, while Precipitation of driest month is most likely to be the leading limited factor in these two regions according to jackknife test of variable importance. In addition, we identified several key ecological and morphological traits that tend to be associated with niche expansion in S. boulengeri clades correspondingly. Specifically, we found that Elevation, Isothermality, Mean diurnal range and Max temperature of warmest month are significantly negative predictors of snout–vent length (SVL) under phylogenetic models, while the S. boulengeri toads from warmer and more arid environments tend to be larger. There seems to a trade-off strategy by trait covary of locomotor performance combined with enlarged SVL, which provide us a potential pattern of how a colonizing toad might seed a novel habit to begin the process of speciation and finally adaptive radiation. It is worth noting that we should not overlook that the Tibet continuously growing and moving northward over millions of years has laid the foundation for early divergence of clades within S. boulengeri.