Biogeography Of Polypedates And Phylogenetic Evolution Of Rhacophorus Sensu Lato

Rhacophoridae belong to Amphibia and Anura.Rhacophoridae has 22 genera and it distributed in Subsaharan Africa,Sri Lanka,Nepal,India,Japan,the Philippines and Sulawesi(Indonesia).Rhacophorus and Polypedates are two important genera in the Rhacophoridae.Currently,the genus Polypedates comprises 26 species distributed in South,Southeast,and East Asia.Because of their relatively low dispersal capability and intolerance to seawater,this genus is ideal for the study of terrestrial range evolution that extends into the island archipelagos of southeastern Asia.In this study,15individuals were collected in the field for DNA extraction and PCR amplification,combined with other individual data downloaded from NCBI,we used 3 mitochondrial fragments(12S rRNA,16S rRNA,tRNA^Val)and 3 nuclear gene fragments(POMC,TYR,RHOD)to perform systematic biogeographical analysis.The results showed that the frog originated from Sundaland at the end of Oligocene and showed northward dispersal into mainland Southeast Asia and Asia through the Kra Isthmus in the middle Miocene,and then spread to the south of East Asia through the Red River fault zone in the late Miocene,which coincided with the timing of paleoclimatic change from the Oligocene to Middle Miocene.Climate fluctuations had a profound impact on species diversification within the genus Polypedates.Alternatively,the lineage of widespread insular P.leucomystax strongly supports the hypothesis of terrestrial connection between island archipelagos of Southeast Asia during the Mid-Pleistocene paleoclimate fluctuations.Our biogeographical analysis also supports the recent introduction of P.leucomystax to the Philippines and Ryukyus,as previously suggested.The genus Rhacophorus sensu lato is one of the most diverse genera of the family Rhacophoridae,which contains 94 species and they widely distributed across China,Japan,India,Philippines and Sulawesi.The research on the genus Rhacophorus sensu lato faces major challenges due to their complex interspecies relationships,and the phylogenetic status of many species are still unclear,which hampered the taxonomy and protection of these species.In this study,we investigated the molecular phylogenetic status of R.laoshan and Z.yinggelingensis using mitochondrial DNA(12S rRNA,tRNA^Val,16S rRNA,Cyt b)fragments and nuclear DNA(RAG-1,RHOD,TYR)fragments.Our results revealed that R.laoshan is closely related to R.verrucopus,R.orlovi and R.calcaneus,and Z.yinggelingensis should indeed be placed under Zhangixalus.The genetic distance between Z.yinggelingensis and other species are between 3.0%and 7.0%,among which the closest genetic distance is Z.duboisi and the farthest genetic distance is Z.achantharhena.This research clarified the phylogenetic positions of two species within the genus Rhacophorus sensu lato,and it also has an impact on the protection and biogeographic analysis of these species.At the same time,this study describes the evolution of the geographical distribution range of the species of Polypedates,which provides a reference for the study of the biogeographic evolution process in combination with geoclimatic changes and species diffusion patterns.