Genome-wide Molecular Mechanism Of High-altitude Adaptation Of Snub-nosed Monkeys (Rhinopithecus)

The snub-nosed monkeys (Primates:Cercopithecidae:Colobinae:Rhinopithecus) are the most advanced monkey in the family Cercopithecidae, and occupies a critical evolutionary position between Old World monkeys and anthropoids. Studies on Rhinopithecus speices have important significance for us to understand the differentiation and evolution of human and primates. Rhinopithecus comprises five recently (1.69 million years ago) formed closely related species, including R. bieti, R. roxellana, R. brelichia, R. avunculus and R. strykeri. Because of severe disturbance by human beings and deteriorating habitats of these Rhinopithecus species, they are now confined to very limited areas in southern China (R. bieti, R. roxellan, R. brelichia and R. strykeri) and northern Vietnam (R. avunculus), each with limited population size, and listed as endangered species in the IUCN Red List of Threatened Species, in which giant panda was included in.In addition to high conservation value, Rhinopithecus species offer us an excellent study model to examine the adaptation mechanism to extreme habitat environment. These speices have occupied different elevations soon after respective speciations, compare with R. avunculus and R. brelichia which inhabit lowland (200-1200m) subtropical rain forest of Northern Vietnam and inhabit of the Mount Fanjing Guizhou Province in China (1500-2200m), respectively, R. bieti, R. roxellana and R. strykeri were distributed in higher mountians in China (>2000m). Particularly, R. bieti is found exclusively from high-frigid forest habitate of the border area between Yunnan and Tibet (3400-4600m), and has been thought as the highest altitude-dwelling nonhuman primate. Interestingly, previous phylogenetic analyses based on data of nuclear genes have showed that the high-altitude dwellers R. bieti and R. strykeri are grouped with the low-altitude dwellers R. avunculus, and so are the high-altitude dwellers R. roxellana and the low-altitude dwellers R. brelichi. Accordingly, Rhinopithecus offers an interesting study model not only to investigate non-human primate adaptive mechanism to high-altitude, but also to examine the differences and similarities of adaptive mechanism from different high-altitude regions.Our present study is conducted with newly obtained genome data of R. bieti (genome size is 2.94Gb, coverage is 76.5-fold, contig and scaffold N50 is 20.5kb and 2.2Mb, respectively) by our group and genome data of R. roxellana (genome size is 2.90Gb, coverage is 146-fold, contig and scaffold N50 is 25.5kb and 1.55Mb, respectively) which had published online. To investigate the mechanisms of high-altitude adaptation in Rhinopithecus, we performed a comparative genomic analysis of all snub-nosed monkeys by including three newly re-sequencing genomes for R. brelichia, R. strykeri and R. avunculus (>30-fold coverage on average per species). First, we construct phylogenetic tree for all Rhinopithecus species by different methods (ML method/STAR and ASTRAL species tree methods) based on 69,586,645 SNP sites, using Macaca as outgroup. The phylogenetic tree is identical from different methods, strongly supporting the high-altitude R. bieti and R. strykeri are grouped with the low-altitude R. avunculus, and the high-altitude R. roxellana are grouped with the low-altitude R. brelichi, by high confidence level. Therefore, the genome phylogeny tree consistent with previous nuclear tree which based on twelve nuclear genes, indicating the adaption of high-altitude in R. bieti, R. strykeri and R. roxellana may occur independently. We then identified common amino acid substitutions occurred and positive selection genes (each and all) in the three high-altitude snub-nosed monkeys based on 12,295 orthologous genes. Overall, twenty common amino acid substitutions in eighteen genes were observed in R. bieti, R. strykeri and R. roxellana. Of the common substitutions identified, eighteen occurred in sixteen genes that were also identified as evolving under positive selection. Because of have gene flow between Rhinopithecus species, the analyses of the reason for the generation of the eighteen sites, indicating twelve sites may recurrent mutations and other six sites may gene flow or ancient low frequency variation. Interestingly, further examination of these sixteen genes found that six genes, including ARMC2, NT5DC1, RNASE4, CDT1, RTEL1, and DNAH11, have functional associations with lung function, angiogenesis, DNA repair, or respiratory cilia movement, suggesting a role in high-altitude adaptation of the snub-nosed monkeys, and the eight mutations in the six genes not found in other published mammalian genomes. Five, three and nine PSGs were found from the R. bieti, R. strykeri and R. roxellana, respectively. The gene OR51V1 in R. roxellana and the gene OR51G2 in R. bieti may play a key role in adaption to food resources of different high-altitude regions of Rhinopithecus. In addition, the gene ROCK2 which was under positive selection in R. bieti, was associated with high altitude essential hypertension.Our study extends genomic analysis of the high-altitude adaptation to a branch of the non-primate evolutionary tree that has received little attention. Based on genome-level survey, the analyses find genetic signatures that may reflect molecular adaptations have consistency and diversity of the high-altitude from three snub-nosed monkeys, contributing to a novel and complete understanding of the complex biological features of high-altitude adaptation.