Genetic Structure Of Giant Spiny Frogs Inferred From Mitochondrial DNA Markers

The distribution of Giant spiny frogs(Paa spinosa)in China is the most extensive in Paa.Because of its higher medicinal and food value, human's serious capture,the number of wild giant spiny frog has been declining in the last years.For strengthening the protection of giant spiny frog,the International Union for Conservation of Nature(IUCN)Species Survival Commission(SSC)considered it as vulnerable species in the year 2007.China Species Red List also ordered it in VU level.In order to design effective conservation strategies,there is need for us to study genetic diversity of the giant spiny frog population from different geographies and population genetic structure.We did research on genetic diversity and population genetic structure of the giant spiny frog population from different geographies by using mitochondrial DNA as genetic markers.The major results are as follows:Sequences of the mitochondrial Cyt b for 122 individuals of Paa spinosa from thirteen populations in Chinese mainland and Vietnam were obtained.The aligned 562bp fragments of the Cyt b include 218 variable sites.Fifty-one haplotypes were identified.Most of the haplotypes are confirmed to local populations,and only 7 of the 51 are shared among a few local populations.The overall species haplotype diversity (h=0.968±0.006)and the nucleotide diversity(π=0.1168±0.00680)are all quite high.We found that Yangshan population's genetic diversity was the highest.The analysis of pairwise Fst and migration rates(Nm)showed a significant genetic population subdivision between Anpei population and the other populations.Constructed phylogenetic relationships among haplotypes based on neighbor joining,maximum parsimongy and minimum evolution algorithms indicated that there were three clusters among thirteen populations of giant spiny frog.Namely,cluster AⅠ: Pingbian-Yongfu-Longsbeng-Yangshan population,cluster AⅡ: Huangshan-Jinhua-Lishui-Jianyang-Wuyishan-Xingan-Jinggangshan-Pin gjiang population and cluster B:Anpei populafin.Between the clusters, the nucleotide sequence divergence was from 21.8%(AⅠvs AⅡ)to 27.6%(B vs AⅡ).Within the clusters,the nucleotide sequence diversity was from 0.011(B)to 0.123(AⅡ).The results of relativity analysis of pariwise Fst and geographic distance among the thirteen populations indicated the Fst highly significant positive relativity from geographic distance among the thirteen populations(R=0.285,P=0.012).Genetic distributing structure was accord with Isolation-by-distance model and Stepping-stone model.Interspecific variations of sequence of 5' end of the mitochondrial control region(5'CR)of giant spiny frog were studied.601bp sequences of partial 5'CR of 59 individuals were obtained.The results showed that between Pingbian and Anpei populations genetic distance was little.We merged the two sites of data,namely Yueping population.Yongfu population was separated from the west region.Yongfu country is the common living area for giant spiny frogs from Yueping and the mideast populations:mtDNA control region might be more useful for evaluating genetic diversity and population genetic structure of frogs.Based on our data,we established three protection management units of giant spiny frog:The west unit(Anpei-Pingbian-Yongfu population), the central(Yangshan-Longsheng-Jinggangshan-Wuyishan-Xingan-Pingjiang population)and the east units(Huangshan-Jinhua-Lishui-Jianyang population).The west unit with high genetic diversity should be protected priority.Guangdong and Guangxi to Yunnan provinces of our country should be focus on protected to conserve wild resource of giant spiny frogs.