Genetic Diversity Of Dybowski's Frog (Rana Dybowskii) And The Impacts Of Anthropogenic Introgression On The Genetic Structure

The Dybowski's frog (Rana dybowskii) is an importnat economic species mainly distributing in Lesser Khingan Mountains and Changbai Mountains in northeast China, the oviducts of female Dybowski's frog has been used as traditional Chinese medicine for thousands of years and now still being heavily harvested. In order to protect the wild populations, local people cultivate this species in closing hillsides field in their natural habitats. As a measure, frogs are frequently translocated amongst allopatric populations. This potentially alters the genetic structure of local populations.Fourteen microsatellite loci were used to investigate genetic diversity, genetic structure and variation level of 10 populations under human disturbance. Allele frequency, private allele, heterozygosity (H), polymorphic information content (PIC), genetic differentiation coefficient (FST) and gene flow (NM) were calculated, NJ phylogenetic trees was constructed based on Nei's DA genetic distance. The genetic structure was further estimated using analysis of molecular variance (AMOVA), principal components analysis (PCA) and Bayesian analysis by STRUCTURE program. Some major results and conclusions were as follows:1. We selected 14 polymorphism microsatellite loci with three or four nucleotide repeat, which could steadily amplified 300 samples of Dybowski's frog from 50 loci published in Rana, the proportion was 28%. A total of 87 alleles were observed. The number of allele per locus ranged from 2 to 12 with an average of 6.2, and mean PIC was 0.57. The Ho of these loci ranged from 0.156 to 0.603 (average=0.319) and HE ranged from 0.315 to 0.856 (average=0.596). Genetic diversity was high in 10 populations with estimation of mean expected heterozygosity was from 0.568 to 0.621, which of JMS population was highest, while the lowest was JH population. In addition, one private allele in TH and TL populations respectively were found.2. Results of Hardy-Weinberg equilibrium tests showed that loci Rpi107, RsyC52, RsAAT23, RP23 and Rpi100 were in Hardy-Weinberg equilibrium (P>0.05) in entirely or most populations; another five loci in 3 to 8 populations showed deviation from significant Hardy-Weinberg equilibrium (P<0.01); besides, the other four loci RsyC41, RsyD25, RsyD40 and RsyD88 presented signifieant deviation from Hardy-Weinberg equilibrium in all populations (P<0.01). In our study, some frogs were translocated in different populations, the deviations from Hardy-Weinberg equilibrium of these loci were probably selected by mankind and null alleles maybe exist.3. According to pair-wise FST, we could see that population differentiation was significant between Lesser Khingan Mountains and Changbai Mountains, except the known allopatry introduced populations. Low gene flow was between 3.17-8.28, DA genetic distance varied from 0.087 to 0.175, gathered into two different clades, and genetic structure of them was obviously differed from each other, all of these demonstrated that genetic differentiation were highly significant between populations in two distribution areas. Based on the principles of conservation geneties, we identified two distinctive management units (MUs) to protect and manage Dybowski's frog.4. The results suggested that two local populations (JMS and BX) were dramatically introgressed due to introduction from allopatric populations. Meanwhile, a hybrid breeding population AS was established outside the natural range of this species, and was consisted of genetic resources from allopatric populations in resently two or three decades. Introduction from allopatry had threatened the genetic structure of Dybowski's frog, because of, and the effect was irreversible, that can easily induce population degradation, even affact the existence and proliferation of species.5. Although the consequence of such genetic alterations was not definitely confirmed, we proposed conservation recommendations according to the results above those dentifying two distinctive management units (MUs), namely Lesser Khingan Mountains and Changbai Mountains, to avoid frogs translocated between them. From viewpoint of gene polymorphism conservation, especiaily conservation of high quality gene pools that TH, HB and TL populations should be preserved prior to the others. Introduction from allopatry should not be performed unless these populations were proved genetically homologous, and the extent of introduction should be restricted to a level not impacting the genetic structure. To reduce the potential impacts on local gene pool, the AS population outside natural range should be strictly isolated from natural populations.