Studies On Genetics And Habitat Of Sichuan Snub-nosed Monkey (Rhinopithecus Roxellana)

Sichuan snub-nosed monkey (Rhinopithecus roxellanae) is an unique primates in china, witch is in severe danger now and is mainly distributed in Shanxi, Hubei, Sichuan and Gansu province. In recent years, since the forest has been cut excessively and human activities have become increasingly frequent, habitat loss and habitat fragmentation have caused the population isolation and differentiation, the reduction of genetic diversity in Sichuan snub-nosed monkey. Therefore, it is very important and urgent to study the population genetic structure, genetic diversity and habit area of Sichuan snub-nosed monkey.The population genetic diversity, genetic structure, gene flow, population differentiation of Sichuan snub-nosed monkey in Hubei Shennongjia National Nature Reserve and Shanxi Changqing National Nature Reserve have been studied systematically by using the methods of non-invasive sampling and molecular markers such as microsatellite loci, sex identifying related to Y chromosomes and gene segments of the mtDNA D-loop area. Moreover, the habitat selection and habitat evaluation were researched based on the field investigation of Sichuan snub-nosed monkey in Shanxi Changqing National Nature Reserve by the methods of "3S" techniques, multivariate statistical methods and MAXENT models. Results and conclusions were summarized as follows.1) One hundred and twenty-three faeces samples and 3 muscle samples were collected from two natural populations of Sichuan snub-nosed monkey in Dolongtan and Qianjiaping of Shennongjia National Nature Reserve, and 186 faeces samples were collected from ten observation populations in Changqing National Nature. Thirty-six Shennongjia mitochondrion analysis sequence and 35 Changqing analysis sequence were gained through the amplification of the mtDNA D-loop area segmental sequence for these samples. Among the comparable 397bp sequence obtained, there were 23 and 29 variable sites, the rate of witch were 5.79% and 7.30%, respectively.19 haplotypes was detected in the 36 Shennongjia Sichuan snub-nosed monkey, the haplotype diversity was 0.937 and nucleotide diversity was 0.0097; 20 haplotypes was detected in the 35 Changqing Sichuan snub-nosed monkey, the haplotype diversity was 0.947 and nucleotide diversity was 0.0181. the result shows that Shennongjia and Changqing Sichuan snub-nosed monkey still have a relatively rich genetic diversity at Mitochondrial DNA gene level.2) Twenty-four microsatellite loci selected of Rhesus monkeys were amplified in DNA genome of Sichuan snub-nosed monkey, and nine microsatellite loci including D6S474, D3S1768, D1S533, D8S505, D15S644, D1S207, D7S1830, D17S1290, D5S820 has obvious genetic diversity, and genotyping success rate was 87% and 91% in the gene group of Shennongjia and Changqing Sichuan snub-nosed monkey respectively. The average effective number of alleles was (Ne) 3.85 and 4.67 from Nine pairs of microsatellite loci in the Shennongjia and Changqing Sichuan snub-nosed monkey genomic DNA. Shennongjia Sichuan snub-nosed monkey gene heterozygosity (Ho) ranged from 0.115-1.000, with an average of 0.72, and polymorphism information content (PIC) ranged from 0.165～0.860, with an average of 0.60; Changqing Sichuan snub-nosed monkey gene heterozygosity (Ho) ranged from 0.544～0.892, with an average of 0.76, and polymorphism information content (PIC) ranged from 0.490～0.810, with an average of 0.67. the result shows that at nuclear gene level of Shennongjia and Changqing Sichuan snub-nosed monkey still has a relatively rich genetic diversity, and genetic diversity of Shennongjia Sichuan golden snub-nosed monkeys was slightly lower than that of the Changqing Sichuan golden snub-nosed monkeys.3) By using the Y chromosome labeling method,18 male and 40 female individuals (1:2.2) was identified in 58 special individuals of Shennongjia Sichuan snub-nosed monkey,56 male and 91 female individuals (1:1.7) was identified in 147 special individuals of Changqing Sichuan snub-nosed monkey. This sex identification technique is very simple, reliable, and especially applicable for the faeces DNA test.4) The microsatellite markers and mtDNA D-loop area gene sequence data showed that there is obvious genetic differentiation in the two populations of Shennongjia National Nature Reserve, but the former (Fst=0.0734) is smaller than the latter (Fst=0.4752). Based on whether UPGMA tree constructed from microsatellite data or NJ tree generated from mitochondrial DNA sequence data, and Bayesian clustering analysis model constructed from STRUCTURE software, it was showed that two small population in Shennongjia was differentiated into three large branches, and further showed that some gene exchange still existed among the Qiangjiaping and Dalongtan.5) Both Microsatellite markers and mtDNA D-loop area gene sequence data indicated that there were considerable gene flow in 10 observations populations of Changqing Sichuan snub-nosed monkey, but obvious genetic differentiation and genetic structure were not formed. With GENELAND space model for landscape genetics, it was reasonable that ten observation populations were defined into four genetic populations, and at present there were not population geographical isolation and gene block caused by ecological factors.6) The result of the distribution analysis for haplotype bifurcation points, the neutral test by Tajima's D and Fu and Li's D showed Sichuan snub-nosed monkey in both Shennongjia and Changqing distribution area has experienced recent population expansion events or has relatively complex population history and population structure.7) The principal component analysis of 215 sample sites used in Changqing National Nature Reserve indicated that the distance from the main roads and vegetation types constituted the key factors of habitat selection of Sichuan snub-nosed monkey, and shrub density, coverage of shrub, tree DBH, tree's height, tree's canopy and slope constituted other important factors that affected the habitat selection of Sichuan snub-nosed monkey. Synthetical analysis about the characteristic values of main components and the frequency of habitats factor showed that Sichuan Snub-nosed Monkey prefer to live in areas of low shrub density, moderate cover, tree growing well, gentle slope and anthropogenic interference.8) The MAXENT ecological niche models was used to simulate the spatial distribution of suitable habitat of Sichuan snub-nosed monkey in Changqing National Nature Reserve of Shanxi province, the result showed that the habitat quality of Sichuan snub-nosed monkey in Reserve was relatively good, with good connectivity of the spatial distribution but no obvious patches of habitat fragmentation, which further validated the molecule-biological result that there was no obvious heredity differentiation in the Changqing Sichuan snub-nosed monkey population at the space level. The habitat quality of Sichuan snub-nosed monkey was mainly affected by environmental factors such as the highest temperature of hottest month, mean annual temperature, precipitation range in seasons, altitude, annual precipitation and slope. The suitable habitat are mainly distributed in both sides of the valley far away from the main roads, and in the mid-altitude deciduous broad-leaved forest and coniferous mixed forest. The area of the suitable habitat is 6360.70 hm2 and is 18.12% of the total area of Reserve. What is more, nearly half of the area (45.15%) is in sub-suitable state.Some protection advices were proposed for the species-source saving and population rejuvenation of Sichuan snub-nosed monkey according to protection genetics and landscape genetics, and research achievement above.