Genetic Diversity And Geographic Isolation Analysis Of Rhesus Macaque Along The Southeast Tibetan Plateau Based On Mitochondrial ATP6 And ATP8 Genes

Rhesus macaque(Macaca mulatta)is the most widely distributed non-human primate in the world,and it is also the only non-human primate widely distributed from low altitude to high altitude in China.Mitochondrial DNA(mtDNA)has the characteristics of rapid variation rate,strict matrilineal inheritance and limited recombination,so it has been widely used to study the genetic diversity of species in recent decades.MtDNA ATP6 gene is a protein coding gene that mutates rapidly under natural conditions among the 13 protein coding genes in the mitochondrial genome,and its ATP6 is an important part of ATP synthase.Therefore,the variation of ATP6 gene may lead to changes in the efficiency of oxidative phosphorylation of mitochondria,and then affect the adaptability of species to different altitude environments.In this study,non-invasive sampling was used to collect feces samples from 19geographical populations in 8 provinces(autonomous regions)with an elevation span of5-4000m,focusing on the southeast edge of the Qinghai-Tibet plateau.Our aim was to explore the molecular mechanisms by which rhesus macaque adapt to different environments,especially extreme ones at high altitudes.On the other hand,previous studies on the genetic diversity of rhesus monkeys were mostly based on the mitochondrial control region(D-loop),microsatellite or mitochondrial cytochrome b(Cyt b).In this study,we attempted to verify whether ATP6 could also be used as a marker gene for the systematic geographical study of rhesus monkeys.The specific results are as follows:1)In this study,the complete mtDNA ATP6 gene sequence of 334 rhesus macaques with length of 681bp was successfully amplified,and a total of 95 nucleotide variation sites and 36 amino acid variation sites were detected.A total of 50 mtDNA ATP6haplotypes were defined in this study,among which Hap1 was widely distributed in 7populations from the Qinghai-Tibet plateau,and Hap1 was also the haplotype with the highest proportion(N=137,41.02%).Besides Hap12 and Hap17,the remaining 47haplotypes were all unique to each population.By comparing the ATP6 nucleotide sequences,it was found that the ATP6 variation of rhesus macaques showed obvious elevation correlation,and some of the variation sites were common in the elevation range.In particular,the 7 haplotypes on the Qinghai-Tibet plateau showed high similarity,with only 6 mutation sites detected.By translating nucleotide sequences into amino acid sequences,it was found that among the seven haplotypes in the qinghai-tibet plateau,only one amino acid mutation site 52L was detected in Hap3,and the other six haplotypes all detected 52F and encoded the same amino acid sequence(all are synonymous mutations).2)Rhesus macaque ATP6 showed high genetic diversity:nucleotide diversity(Pi)=0.02332±0.00226,haplotype diversity(Hd)=0.802±0.022,mean nucleotide difference(K)=14.982.However,the genetic diversity within each population was extremely low,and because only a single haplotype was detected in Xizangjiacha(XZJC),Xizangleiwuqi(XZLWQ),Yunnanjianchuan(YNJC)and Sichuanpanzhihua(PZH),the above three indexes were all 0.By calculating three indicators of genetic differentiation:differentiation coefficient(F_ST),gene flow(Nm),and genetic distance(D),we found that the population of rhesus macaques had more distinct differentiation characteristics.First of all,from the Tibetan nonhuman five levels(including YNJC),the differentiation between populations was extremely low,we thought this was because the Hap1 in widely distributed in Tibetan areas.However,the populations of SCBY and QHYS,which were both high altitude populations and shared haplotype Hap1 with the populations in Tibet,showed higher differentiation levels,which we speculated was caused by geographical isolation.Secondly,populations outside the qinghai-tibet plateau showed higher differentiation levels,and the F_ST values between populations were significant(P<0.01).3)Through the neutral test(Tajima s D)and population mismatch distribution analysis,we conducted a population history dynamic analysis of rhesus monkeys.We found that in addition to Sichuan muli(Tajima's D=-1.86311,P<0.05)and Guangxi population(Tajima s D=1.00927,P<0.05),the neutral test of the rest of the population is not significant.We divided the 19 rhesus macaque population into 7 geographical haplogroups and analyzed the mismatch distribution of each haplpgroup.The results showed that the rhesus macaque overall was unimodal indicate that there has been a history of population expansion;and alone,except for Hainan Lingshui(HNLS)and Henan(HN),which were multimodal,indicating that these two groups are relatively stable in structure,the remaining five haplogroups were all unimodal,shows the history of population expansion.4)By constructing a phylogenetic tree of 50 haplotypes of rhesus macaque ATP6,analysis of molecular variance and the calculation of divergence time,we found that geographic isolation(rather than the altitude factor we speculated earlier)was the most important factor in the genetic differentiation of rhesus monkeys.The results showed that the rhesus macaque populations from the southwest of Daxue Mountain,east of the Qinghai-Tibet Plateau and populations from western Sichuan and east of Daxue Mountain were respectively grouped into two obvious branches in the phylogenetic tree,and the divergence time of these two branches was 0.92Ma.Therefore,we speculated that there are two subspecies of western Sichuan rhesus monkeys,namely M.m.lasiotus and M.m.vestita.The former is distributed in the Dadu River Basin and northwestern Sichuan.The latter is distributed in the Yalong River Basin and the Qinghai-Tibet Plateau in western Sichuan,and we believe that the Daxueshan Mountains in western Sichuan are important geographical barriers for the differentiation of these two subspecies.5)We used the Mixed Effects Model of Evolutionary(MEME)to perform a selection pressure analysis on the rhesus macaque ATP6 gene,and tried to find the evidence of positive selection on ATP6 gene.The results showed that the rhesus macaque mtDNA ATP6 gene amino acid sequence No.5,6 and 24 non-synonymous mutation sites were subjected to a significant positive selection effect,but there was no significant positive selection site in the widely distributed Hap1 on the Tibetan Plateau.We speculate that this is due to the short differentiation time of Hap1 but the conclusion of this study is comprehensive We still think that Hap1 is the result of rhesus monkeys'extensive adaptation to high altitude extreme environments.In this study,the genetic diversity of wild rhesus macaque populations in China was analyzed for the first time using mitochondrial coding genes.We found that the ATP6 gene is a good molecular marker.This result also provides basic genetic data for the protection of rhesus monkeys in southwest China.