The Phylogeny Of Odorrana Graminea Species Complex Based On Microsatellites

The large odorous frogs?Odorrana graminea?in China are not a single species but species complex which includes 3 valid species?O.chloronota,O.graminea and O.leporipes?based on the results from the morphological data and phylegenetic analyses of mitochondrial12S and 16S gene data.Three highly divergent phylogeographic clades were revealed in the O.graminea species complex and they corresponds to Western?Clade A?,Eastern?Clade B?,and Southern?Clade C?,respectively.However,the relusts from the analyses of 16 nuclear genes contradict with those from mitochondrial data.To further clarify the species composition and the taxonomy of the O.graminea species complex,it is necessary to detect the gene flow and introgression among the different clades,different populations and individuals in O.graminea species complex and explore the speciation and evolutionary history of different clades?or species?.A total of 268 individuals from 22 populations were collected based on extensive sampling in the present study.Seven specific pairs of microsatellite primers were successfully screened from the transcriptome data of the O.graminea from Huangshan population.Two isolated microsatellite markers and the other four primers from the literature were used to explore the phylogeoraphical patterns of O.graminea species complex.The genetic differentiation coefficient and gene flow were estimated using Popgene,genetic structure were revealed based on the clustering analysis using Structure,and bottleneck events were also detected by the analysis of Bottleneck software.The aims were to determine the species composition,genetic differentiation,gene flow and population dynamics in O.graminea species complex.The results are shown as follows:?1?Seven screened microsatellite primers can be used to explore the genetic diversity and structure of O.graminea species complex.?2?The mean polymorphism information content?PIC?of the 22 populations in O.graminea species complex was 0.510 which is higher than 0.5.The percentage of population with PIC higher than 0.5 was 63.64%,and it shows the high genetic diversity in this species complex.?3?The mean F_ST between the two populations in O.graminea species complex was0.1818,and the percentage of populations with FST higher than 0.25 was 80.09%,which suggested the high genetic differentiation in O.graminea species complex.?4?The O.graminea species complex includes three clades,i.e.,Clade A,Clade B and Clace C.Clade A are mainly distributed in the Southeastern Sichuan and Northeastern Guangxi province;Clade B are mainly distributed in Guangdong,Fujian,and Hunan provinces,which includes the type locality Shaoguan of O.leporipes;Clade C are mainly distributed in the East of Yunan-Guizhou Plateau,Westsouthern of Guangxi,and Hainan island,which includes the type locality Wuzhishan of O.graminea.?5?Significant bottleneck events were detected in populations of Meixian in Guangdong province,Jinxiu in Guangxi,and Xingyi in Guizhou based on the Bottleneck analyses?P<0.05?.And population of Wuming were also suggested to experience significant or extremely significant bottleneck event.In addition,highly significant bottleneck events were also detected in the the Shuangpai population?P<0.01?.Conclusion:?1?Based on SSR markers,Odorrana graminea species complex includes 3 clades.?2?The Odorrana graminea species complex of 3 clades lack of obvious geographic barrier,there is greater gene flow between groups.?3?The Odorrana graminea species complex has high genetic diversity,and the genetic differentiation of populations between the same family and the different clades present the polymorphic phenomenon of medium differentiation,high genetic differentiation and undifferentiated coexistence.?4?Guangxi Jinxiu,Guangdong Meixian,Guizhou Xingyi,Guangxi Wuming and Hunan populations experience significant or extremely significant bottleneck event.