Isolation Of Microsatellite Markers And Population Genetic Structure Analysis Of Pelophylax Nigromaculatus

Widespread anuran amphibians are regarded as good models for phylogeographic research. And global population decline of amphibians has attracted the attention of conservation biologist all over the world. As the most widespread and common amphibian, black-spotted frog (Pelophylax nigromaculatus) is also suffering from serious population decline. The work on phylogeography and conservation genetics was not only important for basic research, but also provided essential information for conservation strategies of amphibians.We developed a set of microsatellite loci for black-spotted frog which were immediately applied to the research of phylogeography and conservation genetics of black-spotted frog with a fragment of D-loop. The major results were as follow:(1) We isolated 13 polymorphic microsatellite loci according manetic beads enrichment protocol. Analysis of 35 muscle samples collected from Jinhua in Zhejiang showed the number of alleles from 3-14, observed heterozygosity and expected heterozygosity were 0.171-0.941 and 0.422-0.912, respectively. The polymorphic information content of these loci was 0.341-0.891, and combined identity exclusion probability reached 0.9999.(2) We collected 607 samples from 23 populations in Palearctic Realm. There were 321 individuals detected by the primers which could amplify a 630 bp fragment of D-loop. We identified 114 haplotypes which were clustered 2 clades based neighbor-joining method. The two lineages were named Central Plain Lineage and Northeast Lineage according distribution. The Central Plain Lineage was split into two clades based Bayes tree, NeighborNet network and Median-joining network. The two clades were approximate sympatry. It suggested the Central Plain Lineage was polymorphic before dispersal.(3) According to the universal evolutionary rate of anuran D-loop (3.5% per million years), we estimated the occurrence of population expansion began approximately 0.070MYA for Northeast Lineage and 0.025MYA for Central Plain Lineage. But due to lack of fossil calibration, there might be some error in temporal estimation.(4) As high degree of population genetic structure, only 7 microsatellite loci could be amplified among all population. Rnh5 could be amplified in the population belong to Central Plain Lineage. It suggested that high nuclear gene differentiation of two lineages, and it was verified by STRUCTURE analysis. But it was unknown that whether the lineages were two Evolutionarily Significant Units due to lack of evidence from other molecular markers, especially adaptive molecular markers. The population of Northeast Lineage could be divided into two groups, by STRUCTURE analysis. But it could not be verified by mtDNA. Maybe it explained by sex-biased dispersal or higher resolution of microsatellites.(5) There was a significant correlation between geogeraphic isolation and genetic differentiation in Central Plain Lineage (P-values were 0.001 and 0.015, respectively) reflected by both mtDNA and microsatellites. Because the major Northe lineage distributed in flatland of north China, and there was nothing separating populations of different regions. But there was no correlation between geogeraphic isolation and genetic differentiation in Northeast Lineage (P-values were 0.552 and 0.160, respectively). Because the major Northeast Lineage distributed in Changbai mountain range, and elevation could hinder gene flow of different populations. It suggested that the genetic distribution of black-spotted frog were impacted by landscape.(6) There was more genetic diversity in the populations of Central Plain Lineage according both nuclear genes and mtDNA markers. The haplotype diversity and nucleotide diversity of mtDNA were 0.958 and 0.01268 in populations of Central Plain Lineage, while 0.859 and 0.00492 in the populations of Northeast Lineage, respectively. The number of effective alleles, allelic richness, observed heterozygosity and expected heterozygosity of microsatellites in the populations of Central Plain Lineage were 5.451,9.29,0.676,0.734, while 1.883,4.45,0.342, 0.367 in the populations of Northeast Lineage. In the populations of Central Plain Lineage in Palearctic Realm, the genetic diversity of south populations was higher than north populations. In the populations of Northeast Lineage, the populations adjacent to korean peninsula had higher genetic diversity. The pattern of black-spotted frog might be related to population dispersal after glacial period.(7) Many black-spotted frog populations had experienced bottleneck in recent years. These populations were distributed widely, and the adjacent populations did not have unanimous demographic history in recent years. It suggested that the major causes of black-spotted frog might be habitat destruction or/and over exploitation, rather than epidemic or climate change.