Genetic Differentiation Of Different Geographical Populations Of Microhyla Fissipes

Recent studies conducted on dogs,rodents and humans have uncovered genetic evidence for physiological adaptations to life along a geographical gradient,however,amphibians have received little attention in this respect.In this paper,we revealed adaptive differentiation along a latitudinal gradient in the Ornate Striped Microhyla(Microhyla fissipes)with restriction site-associated DNA sequencing(RAD-seq).To identify possible signatures of adaptation to latitude,we performed a genome scan with RAD-seq of pooled samples from 10 populations of M.fissipes.All 10 study sites were along a 1398-km latitudinal and 486-m altitudinal transect across the Hainan,Guangxi,and Sichuan provinces in China.A total of 275.3 million reads were obtained by using the Illumina HiSeq X Ten platform,ranging from 19.1(population GH)to 41.8(population XD)million reads depending on the population.We aligned a total of 258.1 million reads to the reference sequences where the number of aligned reads ranged from 17.7(population GH)to 39.6(population XD)million within each population.We validated 20,572 single nucleotide polymorphisms(SNPs)across all 10 populations.The expected heterozygosities were very similar(≈ 0.39)in all populations.The genomewide average nucleotide diversity ranged from 0.410(population CJ)to 0.431(population GH),and the genomewide average population mutation rate from 0.226(population XD)to 0.245(population BH).We discovered significant geographic differentiation among all sites,with an average FST = 0.090 across all SNPs.Apart from clear patterns of isolation by distance,we discovered numerous outlier SNPs showing strong associations with variation in latitude(5 SNPs),altitude(28 SNPs),average annual temperature(69 SNPs),and average annual precipitation(248 SNPs).In addition,636 candidate genes were uncovered and significantly enriched in metabolic and binding process.The overall levels of genome-wide genetic differentiation were moderate across sampling locations(mean= 0.219).Numerous SNPs showed strong associations with variation in average annual precipitation and/or temperature.Levels and patterns of genetic differentiation in these SNPs play an important role for ecology(precipitation and temperature)as a driver of genetic differentiation among local M.fissipes populations.Outlier SNPs were significantly enriched for genes involved in metabolicand binding process,and several genes potentially related to response to stress(HSPA12B and HSPA1B)were identified.However,most genomic targets of selection remain unknown.In general,the results provided strong genomic support for substantial adaptive differentiation along latitudinal and altitudinal gradient in amphibians.