Genetic Pattern Of The Eastern Golden Frog (Pelophylax Plancyi) In The Megacity,Shanghai

Urbanization is developing rapidly across the world,which causes severe threats to wildlife.Because of the limited dispersal ability and sensitivity to environmental changes,amphibian has been one of the most threatened taxonomic groups in cities.In return,amphibian is frequently used as an indicator to evaluate ecological impacts of urbanization.Therefore,the study of how urbanization influences amphibian species can help us comprehensively understand the mechanisms of urbanization impacts on amphibian species,and more effectively guide the wildlife conservation in urban environments.In this research,we studied the aboriginal eastern golden frog(Pelophylax plancyi)in Shanghai,which is a generalist distributed across the city.As the third biggest megacity in the world,Shanghai represents a typical rapid urbanization mode in developing countries and areas,and it possesses both a low-level of historical urbanization dominated by agriculture for thousands of years followed by a rapid and intensive modern urbanization since the Reform and Opening-up started in 1978.The urbanization expands outward from the urban area to the peripheral area,which has formed an urban-to-rural urbanization gradient.We selected 15 sampling sites across the urbanization gradient,and collected a total of 407 samples of P.plancyi.Using mitochondrial DNA(mt DNA),microsatellite(SSR),and single nucleotide polymorphism(SNP),population genetic analyses were conducted including demographic history,genetic diversity,genetic differentiation,population structure,and gene flow.Through this study,we aim to identify the population status of P.plancyi in Shanghai,and discuss the influencing mechanisms of different urbanization stages to P.plancyi considering the landscape data,which can effectively guide the wildlife conservation during the Eco-City Construction in Shanghai City.The main results were as the following.1.The demographic history analysis indicated that all of 15 local populations experienced bottlenecks at least 300–500 years ago during the historical urbanization.The SSR data showed that the ancestral maximum effective population sizes(N_max)were 3082.15±904.74(±SD),while the contemporary effective population sizes(N_e)decreased to 368.77±98.6.The SNP data identified additionally that after a historical bottleneck,all local populations experienced either moderate increases of N_e which still smaller than ancient effective population sizes(N_anc)(population recovery)or even a great increase of N_e which larger than N_anc(population expansion).The N_anc were 5550±894.3.The bottleneck effects happened resulting in minimum effective population sizes(N_bot)of 262.1±164.2,and followed by recovering N_e of 1114.6±736.2.All local populations still maintained comparative genetic diversity even in urban areas according to the analyses of the three kinds of molecular marker.For mt DNA,the number of haplotypes(N_H)was 7.93±2.84,the haplotype diversity(h)was 0.73±0.13,and the nucleotide diversity(?)was 0.003±0.001.For SSRs,the number of mean alleles per locus(N_A)was 7.8±0.775,and the observed heterozygosity(H_O)was 0.607±0.024.For SNPs,the H_O was 0.214±0.024,and the?was 0.249±0.01.3.Based on the principal component analysis(PCA),the urbanization index(UI)was calculated at the local scale using the landscape configuration in the range of the local populations.The linear regression model(LRM)indicated that the UI significantly decreased across the urbanization gradient from urban to rural areas.Moderate,but significant negative relationships between genetic diversity and UI were detected at the local scale(N_H of mt DNA:RC(Regression Coefficient)=–4.986,R²_adj=0.207,P=0.050;H_O of SSR:RC=–1.480,R²_adj=0.259,P=0.031;?of SNP:RC=–0.020,R²_adj=0.320,P=0.016).Only N_e calculated by SNPs showed significantly negative relationship with UI(RC=–1812.0,R²_adj=0.529,P=0.002).No significant relationship was found between genetic variations and urbanization level at the cityscape scale.4.Pairwise F_ST calculated by SSR and SNP data identified that the local populations within or near to urban areas and the two island local populations showed higher differentiation compared with the others(SSR:0.017–0.078;SNP:0.026–0.107).It should be noticed that the differentiation of the local populations in heavily urbanized areas can be even stronger than those in islands caused by geographical factors.5.The phylogenetic analysis by Cyt-b indicated that there was no significant divergence between local populations at the mitochondrial level,which means all P.plancyi in Shanghai still belong to one single population.However,SSRs and SNPs showed a differentiated genetic structure.Combining a series of results by different algorithms,the 15 local populations were divided into eight genetic clusters.The local populations in urban areas,some suburban areas,and islands formed distinct clusters,respectively,while the local populations in peripheral areas still belonged to a uniformed cluster,which was called“peripheral cluster”.It is striking that the population structure showed a significantly consistent pattern with the urbanization gradient in the mainland part.6.The gene flow between different clusters was calculated according to the results of population structure.Results by SNP data indicated moderate gene flow(0.165–0.204)from the peripheral cluster to most of the other genetic clusters,except for lower rates to GQ and CJ(0.037 and 0.074,respectively).On the contrary,the gene flow between the other seven genetic clusters or to the peripheral cluster were extremely low(0.004–0.051).The analysis of SSR data showed a consistent result with that of SNPs,with stronger gene flow from peripheral cluster to the other clusters than from the opposite direction.In conclusion,our research indicated that both terms of urbanization showed different mechanisms to influence P.plancyi in Shanghai.The historical urbanization disrupted the population by consuming effective population sizes,while the main consequence caused by heavily modern urbanization might be the strong isolation between local populations.Although the local populations still maintained comparative genetic diversity in urban areas,the urbanization already began to influence the genetic variations at the local scale.Genetic differentiation also emerged at the cityscape scale within a short term,and the gene flow decreased in urban areas.Therefore,core area preservation for improving local habitat quality is the primary step for conserving such kind of species with limited dispersal ability.With healthy local populations,cityscape-scale habitat restoration such as creating corridors and increasing habitat connectivity could further improve the living conditions of the species.As the urbanization will continue to develop rapidly in Shanghai,genetic monitoring for P.plancyi and other aboriginal amphibian species would be required for long-term conservation.