Phylogeography And Conservation Genetics Study Of Ichthyophis Bannanicus(Gymnophiona,Ichthyophiidae),Based On Microsatellite And MtDNA Cytb Gene Data

In this study,tetranucleotide ATAG-motif microsatellite loci in Ichthyophis bannanicus were isolated successfully.We used the mitochondrial gene sequence of Ichthyophis bannanicus Genebank published to design the Cytb amplification primers successfully.And then we used these microsatellite loci and mtDNA date to study population phylogeography and conservation genetics study on Ichthyophis bannanicus of 158 individuals in seven sample points from Yunnan province,Vietnam,Guangxi and Guangdong provinces.The frist part:the development of molecular of Ichthyophis bannanicus.(I)We used the FIASCO(Fast Isolation by AFLP of Sequences Cotaining repeats)protocol to isolate tetranucleotide ATAG-motif microsatellite loci in Ichthyophis bannanicus.After digestion and ligation,hybridization with biotinylated probe,magnetic field separation,we got DNA sequences with SSR.And then complex fragments were ligated into vector and transformed into competent cells(DH5?),cloning and sequencing,designed primers,PCR amplification,genotyping and data analysis,we got 15 microsatellite loci.The number of alleles ranged from 3-13;observed heterozygosity(Ho)ranged from 0.036-0.935,and expected heterozygosity(He)ranged from 0.105-0.892.Only four loci deviated from Hardy-Weinberg equilibrium.Finally,there was no evidence of linkage disequilibrium among pairs of loci in the sample.(II)The primers of mtDNA Cytb gene designed and PCR amplification and sequencing.In this study,we designed three primers under the forward L14014(forward primer),L14500 and H15198(reverse primer),and L14500 was sequencing primer.The second part:Phylogeography and conservation genetics study of Ichthyophis bannanicus(Gymnophiona,Ichthyophiidae),based on microsatellite and mtDNA Cytb gene data.(?)Population genetic diversity analysis.One thousand and one hundred forty-three base pairs of mtDNA Cytb sequence were obtained from 157 individuals after alignment,sixteen different haplotypes were identified,haplotype diversity(h = 0.657)and nucleotide diversity(? =0.0070)were detected.For microsatellites,genotypes at 15 microsatellite loci from 158 individuals were successfully genotyed.The mean number of alleles per locus(MNA)ranged from 3.71 to 8.29,the values of observed heterozygosity(HO)ranged from 0.462 to 0.666 and expected heterozygosity(HE)ranged from 0.479 to 0.715.Test for Hardy-Weinberg equilibrium showed that significant departure expectations was found in 5 of 75 tests prior to Bonferroni correction and there were no linkage disequilibrium occurred for any loci in any population.(?)Genetic structure and phylogeography analysis.Genetic differentiation among all populations was relatively strong and mtDNA and microsatellite date produced similar results:significant FST values were detected between population and the BN population being more differentiated than other populations.Estimates of pairwise FST ranged from 0.0004 to 0.8964 based on mtDNA data,and FST ranged from 0.0273 to 0.3478 based on microsatellite.Moreover,the result based on the STRUCTURE also showed a clear maximum.When K=2,there are two clusters were identified:(?)individuals from Xishuangbanna,Yunnan provlnce(BN)and(?)Ichthyophis bannanicus from Guangxi province(BL),Guangdong province(YC/DQ)and north of Vietnam(VN).At K=3,clustering analysis identified three genetic clusters:(?)individuals from BN also predominantly formed a separate cluster,(?)VN,BL and YC formed a private component,and(?)a new evident cluster(DQ)was found separated from other populations.The median-joining network showed a clear pattern with obvious phylogeographic structure that two clades were distinguished from the network analysis:Xishuangbanna Yunnan(BN)and anther big group of Guangxi,Guangdong and Vietnam(BL,YC,DQ and VN).(?)Population demographic analysis.About population demographic,there was no significant support for population expansion by Fu's Fs test and Tajima's D test based on mtDNA data.Based on microsatellite data,BOTTLENECK indicated not significant signs of recent bottleneck in all populations under the infinite alleles(IAM)and two-phase mutational(TPM)models and in one population under the stepwise mutational model(SMM)as well.However,Bayesian coalescent simulation provided compelling evidence for a more long-term population decline for all populations.Conclusion:through the above analysis,we assumed the current distribution of Ichthyophis bannanicus is the recent sudden expansion across Indo-China peninsula to southern China.In the study,two obvious ESU(Evolutionarily significant units)were distinguished from netwoerk analysis and clustering analysis.In addition,considering the DQ population and other populations to be separated by the Pearl River,we suggested three protection units should be established:BN population,Guangdong/Guangxi-Vietnam population and DQ population.