Population Genetic Structure And Molecular Phylogenetic Analysis Of Wuchuan Odorous Frog Based On MtDNA And Microsatellite Markers

The Wuchuan Odorous Frog?Odorrana wuchuanensis?is endemic to karst regions in the southwest of China.The name of this frog came from the fact that the model specimen was firstly found in Wuchuan Gelao and Miao Autonomous County?Wuchuan County?,Guizhou Province.The Wuchuan Odorous Frog is one of the eight most endangered species in China,and was listed as a vulnerable species by the International Union for Conservation of Nature?IUCN?in 2016,but not yet included in the level of Chinese species protection.Currently,the Wuchuan Odorous Frog was mostly found in the northeast and south of Guizhou with a large central dubious area.The actual distribution of this frog was difficult to determine due to the limited environmental conditions and rare habitats.In this study,firstly we used 20 occurrence records and fifteen environmental variables with resolution of 200 m × 200 m to predict the potential distribution of Wuchuan Odorous Frog by the ecological niche model,MaxEnt,and analyze the relationship between this species with the environmental factors in Guizhou.Secondly,the mitochondrial genome sequence of Wuchuan Odorous Frog was determined and its organization was analysed after noninvasive sampling in the field.To explore the systematic status of this species,the phylogenetic trees were constructed based on 28 complete mitochondrial genomes of ranid species.Last but not least,we analysed the genetic relationship among the Wuchuan Odorous Frog populations from northeast and south of Guizhou based on the mitochondrial markers?complete mitochondrial COI,Cytb genes and partial control region?and six microsatellite loci,with a total of 31 samples from five sites in Guizhou.The main results are following:?1?The potential distribution of the Wuchuan Odorous Frog.The result of MaxEnt analysis showed that the potential distribution?HSI > 0.08?of Wuchuan Odorous Frog was mostly located in the northeast and south of Guizhou,in which the suitability?HSI > 0.24?was mainly located in counties of Wuchuan and Yanhe in the northeast,Libo and Congjiang in south of Guizhou while the sub-suitability?0.08 < HSI ? 0.24?was located in counties of Daozhen,Zheng'an,Dejiang and Dushan?Fig.2-2?.The result of Jackknife test showed the total annual radiation was the main environmental factor affecting the distribution of Wuchuan Odorous Frog for 31.8%,the altitude for 19.9%,then January average relative humidity for 16.0% and slope with a value of contribution 10.4%,respectively?Table 2-1?.Temperature and precipitation had little impact.Our result indicated that Wuchuan Odorous Frog was quite constrained in ecological niche and habitats,which appeared spatially discontinuous.?2?The complete mitogenome and phylogeny of the Wuchuan Odorous Frog.The complete mitochondrial?mt?genome of the Wuchuan Odorous Frog was 18256 bp in length,including 13 protein-coding genes,two ribosomal RNA genes,22 transfer RNA genes,and one control region,as is typical to that of typical vertebrates.The base composition was 27.89% A,29.00% C,15.34% G,and 27.78% T.All genes were encoded on the H-strand,except the ND6 and eight tRNA genes(tRNA^Pro,t RNA^Gln,tRNA^Ala,tRNA^Asn,tRNA^Cys,tRNA^Tyr,tRNA^Ser,and tRNA^Glu),which were encoded on the L-strand.The new mt genome manifested the typical neobatrachian-type arrangement that the LTPF t RNA cluster(tRNA^Leu?CUN?,tRNA^Thr,tRNA^Pro and tRNA^Phe)appeared between the control region and the 12 SrRNA gene.Together with other congeneric species,the O.wuchuanensis shared the translocation of the tRNAHis gene from a typical position between the ND4 and tRNACys genes to the downstream of the control region.Phylogenetic analyses based on the Bayesian inference and Maximum likelihood showed that O.wuchuanensis and O.margaretae,together with O.schmackeri and the newly determined O.hainanensis,were grouped as sister clades.?3?Analysis of population genetic structure of the Wuchuan Odorous Frog.A low population genetic differentiation was found within the five populations in Guizhou based on the mitochondrial and microsatellite markers.The combined mitochondrial data set showed that a total of 3624 bp contained 45 polymorphic sites and 11 potentially parsimony informative sites.While the observed heterozygosity?between 0.00-1.00?and expected heterozygosity?between 0.00-0.89?were observed from the analysis of microsatellite data set.Besides,the haplotype diversity?h = 0.869?and nucleotide diversity?? = 0.002?showed a relatively low genetic diversity.The haplotype networks indicated that the population of Baicun?BC?showed a connection with Dashuigou?DSG?instead of Sucun?SC?in Wuchuan County of Guizhou,and the Laizidong?LZD?population appeared to lack gene flow with other populations.Phylogenetic analysis based on the combined mitochondrial data set implied a southward dispersing trend in the LZD population,from which a secondary contact could be found.Furthermore,a shallow genetic structure between populations from northeast and south of Guizhou Province could be identified resulted from the phylogenetic analysis,whereas no genetic clustering among individuals was found in the Structure analysis based on the microsatellite data set.We concluded that the changes of the tributaries of the Yangtze River played an important role in driving the phylogeographic pattern of O.wuchuanensis.