Study On The Genetic Diversity And Conservation Of Sichuan Golden Monkey In Shennongjia Nature Reserve

Golden snub-nosed monkey (Rhinopithecus roxellana), which is endemic to China, belongs to the Colobinae subfamily, Cercopithecidae family in Primate Order. It is categorized as endangered species that just distributes in three areas now:Sichuan/Gansu (SG), Shanxi Qinlin (QL) and Hubei Shennongjia (SNJ). Three populations are isolated from each other and the population in Shennongjia is the smallest one in the eastern border of the distribution of Sichuan golden monkey in China. It is showed that the population in Shennongjia has uniqueness in genetic and has an important role in their evolutionary history with the conservation studies about Sichuan golden monkey. However, Shennongjia population has a fragmentation habitat deforested by large-scale commercial logging since 1950’s. Habitat fragmentation and small population may increase the extinction risk of golden monkey in Shennongjia.The provisioned group, a special group in Shennongjia population formed by food supplied, stays stability in a small range. With the dominate male’s change in an A-Male-Unit (AMU) family, the family pedigree has been vague in relationship of father-offspring. And how to avoid inbreeding and how to keep genetic diversity are the urgent issues of this group to make out.Therefore, molecular biology and landscape genetics were used in this study to analyze the genetic structure and endangered mechanism of the population in Shennongjia for keeping its genetic diversity. Results and conclusions were summarized as follow.(1) To ensure the experiment of fecal DNA successfully, a technique system of fecal DNA storage, extraction and PCR for Sichuan golden monkey in Shennongjia was optimized. Three storage methods (freezing, drying and freezing-drying) in three different periods were compared, and the freezing-drying is the best one for its feasibility in field and highest success ratio of PCR in laboratory. With the process of the experiments, DNA extraction and PCR were improved. Meantime,16 loci were picked out for genetic study of Sichuan golden monkey in Shennongjia with 42 microsatellite loci. And 12 of them can be well amplified with the fecal DNA. Combined PID and Combined PIDsib of such 12 loci were 2.99E-8 and 3.80E-4, and there was no null allele detected. Such loci were suitable for study of individual identification and genetic diversity on samples of Sichuan golden monkey in Shennongjia.(2) Combined with the data of microsatellite loci and the information of GIS, It was to evaluate the genetic diversity of population in Shennongjia and the gene flow between different groups in the population.455 fecal samples were collected from 11 sites in Dalongtan area, Qianjiaping area and Jinhouling area. And 316 different individuals were identified. With 12 loci, a total of 62 alleles were detected, and 4-7 alleles were distributed in different loci. The high frequency of alleles had relatively the same distribution among different sites and different group. Mean observed heterozygosity(Ho), mean expectedheterozygosity (He), and mean polymorphism information content (PIC) ofpopulation in Shennongjia were 0.626、0.559 and 0.650. The difference of value of genetic diversity among groups and among sites was not significant. With the result of STRUCTURE (K=4), it was indicated that the provisioned group had or would have differentiation with other groups, and DLT group and JHL group had their own differentiation. The isolation-by-distance model showed that the genetic distance of different sites was not relative with their geographic distance. With the information from GIS, the fragment habitat may have some effect on the monkeys’ spreading, such as the road crossedthrough the forest, or shrub and grassland. With the value of Fst is 0.042, it was suggested that the population in Shennongjia should be protected as a unit, the habitat of the monkey groups should be repaired as soon as possible, and the provisioned group should be guided to meeting other groups to avoid inbreeding and to even the difference between them.(3) To provide useful reference information for genetic management of provisioned group in Shennongjia National Reserve, the microsatellite markers were used in this study to evaluate the genetic status and to correct the pedigree.15 microsatellite loci were showed good amplification and assayed in the 51 individuals. A total of 56 alleles were detected, and 2-5 alleles were distributed in different loci. Mean observed heterozygosity (Ho) was 0.621, mean expected heterozygosity (He) was 0.595, and polymorphic information content (PIC) of 15 loci was 0.533. Relatedness analysis showed that these 11 units with certain parents and offspring units had not been suffered from inbreeding yet (relatedness index< 0.1875). The inbreeding coefficient (Fis) of this population was-0.041. However, the average relatedness index of this group was0.1108, and 21.64% of individual pairs were possible relatives (relatedness index< 0.1875), which was 2 times higher than theoretical data in the wild animal population (10%). Additionally,2 candidate parent pairs in present OMU (One Male Unit) were found to be possible relatives (>0.1875). The results from both parent identification and relatedness suggested that this group was in high risk of inbreeding. Simulation results showed that population size and sex ratio had an impact on genetic diversity, and lose of genetic diversity would be slowed down by increasing the population size and optimizing the sex ratio. With the results above, it is suggested that the essential task to the provision group is to build a whole of accurate family pedigree and to check out the genetic background of individuals, especially candidate parents, based on molecular markers such the study used. And to establish a larger and more effective breeding group, with much greater genetic diversity, the roup needs to get the individuals with many relatedness pairs out of group and to recruit suitable ones from other groups.(4) 53 alleles were detected in 16 loci, and it had 61.29% of alleles of population in Shennongjia (SSR01-SSR12). A batch of 15 microsatelliteloci (except SSR07) was used to conduct parentage identification and to analyze relatedness among individuals analysis. GM1 and GM5, G12 and GM6, GM10 were detected to certain relationship of father-offspring. And relatedness index of the parent pairs was 0.000. With three relative pairs (GM03 and GM12:0.3145, GM05 and GM08:0.3040, GM01and GM08:0.2773), there was 10 relative pairs in breeding group. And average relatedness index of this group is 0.0883. According to the result of Simulation, number of this group should be 30-50 to keep genetic diversity. The manager of this group should maximize the effective population number, keep male and female individuals balance, and keep relative the same number of offspring, when choose the family pairs. Combined the rule mentioned above and the fact of present breeding group, it was suggested that the breeding group set up 4 families:GM1 & GM6, GM12& GM11, GM8 & GM10 and GM03,GM2&GM5 and GM4. However, If some suitable individuals can move into the group, the one who has much more relatives than others should remove out, such as GM5、GM6、GM10 and GM12. And new pairs would be chosen to set up families again with the rule.