| As historical processes such as climate change and urbanization continue,the unique multiplicity of patchy habitats has led to the isolation of continuously distributed populations and the formation of local isolated population.Long-term isolation hinders the migration of individuals and gene flow between populations,leading to inbreeding and inter-population genetic drift.In addition,the restriction of gene flow will accelerate genetic differentiation between populations,which in turn creates the distinct genetic structure that affect the course of population evolutionary history.In the long term,populations are at risk of altered genetic structure,reduced genetic diversity,reduced ability to cope with unexpected emergencies(e.g.epidemics),population declines,and even local extinction.Alpine musk deer(Moschus chrysogaster),a timid and vigilant ruminant,is listed as key protected wildlife of class Ⅰ in China and served as an endangered(EN)species by IUCN.It is a typical western highland species,now patchily distributed in the montane forests of the Qinghai-Tibet Plateau and surrounding areas.Due to the limitations of ecological barriers such as the Yellow River,cities,and deserts,as well as the impact of many factors(natural and nonnatural elements),the Alpine musk deer population in the Helan mountains is a typically isolated population with a narrow range and extremely low population sizes,and its current situation is not optimistic.Therefore,it is urgent to understand the dynamic changes and genetic characteristics of this population in order to provide scientific reference for the rescue,protection,and management of this endangered species.From November to May 2020 and 2021,field surveys were conducted on the population size and distribution range of Alpine musk deer in the Helan mountains using the line transectfeces pile count method and infrared camera survey method.Fresh fecal samples were collected,and the genetic diversity and structure of the Alpine musk deer population were analyzed using mitochondrial and microsatellite markers combined with molecular biological methods to evaluate the genetic status of this population in the Helan mountains.Meanwhile,potential dispersal patterns of Alpine musk deer in the region were analyzed and discussed.The main results were as follows:1.Population size and distribution of Alpine musk deer in Helan mountainsThe estimated Alpine musk deer population size in Helan mountains was 174(± 45)by line transaction method,and 192(± 26)by infrared camera method,indicating that the population was still at the lowest level in history and the recovery was weak.In terms of distribution,Alpine musk deer were distributed in the middle and southern sections of Helan mountains in patches and mainly distributed in three areas on the west slope of Helan mountains,namely Halawu,Zhenmuguan,Nansi and surrounding areas of Gangouliang,with an altitude of 2 100~3 200 m and a distribution area of 121.16 km2,accounting for only 4.4% of the whole area of Helan mountains.Structure equation model(SEM)analysis revealed that Alpine musk deer population was affected by many factors,and although the coefficient of the internal modular path did not reach a significant level(P > 0.05),there was a significant positive correlation between the influence of sympatric Blue sheep(Pseudois nayaur),Red deer(Cervus elaphus),Red fox(Vulpes vulpes),Badger(Meles meles)and Blue eared-pheasant(Crossoptilon auritum)(P < 0.05).2.Mt DNA-based population genetics of Alpine musk deer in Helan mountainsThe 78 samples successfully amplified in this study were derived from six geographic populations.A total of 6 haplotypes were detected in the mitochondrial Cyt b sequence,the haplotype diversity(Hd)was 0.051,and the nucleotide diversity(π)was 0.000012.A total of 12 haplotypes were obtained from the mitochondrial D-loop sequence,the Hd was 0.716,and the π was 0.00156.A total of 16 haplotypes were detected from the combined sequence of both methods,and the Hd and π were 0.728 and 0.00100,respectively.Compared with other musk deer species,the genetic diversity of Alpine musk deer in Helan Mountains was at a lower level based on mitochondrial markers.Bayesian clustering analysis,haplotype network,and phylogenetic tree showed that the geographic populations of Alpine musk deer in Helan mountains were interleaved and staggered with each other,and no significant genealogical geographic structure was formed.Calculation of genetic differentiation value(Fst)and analysis of molecular variance(AMOVA)for the three sequences showed that there were different degrees of genetic differentiation among the geographical groups,and that most of the genetic differentiation originated within the geographical groups.The analysis of the population dynamic suggested that Alpine musk deer population in Helan mountains had experienced a history of population expansion.3.Microsatellite-based population genetics of Alpine musk deer in Helan mountainsA total of 149 fecal samples were collected from 12 geographic populations,with a female-to-male ratio of 1.13.Using 10 pairs of microsatellite loci with high polymorphism,the overall allele number(Na)was 1.900,the effective allele number(Ne)was 1.549,the observed heterozygosity(Ho)was 0.447,and the expected heterozygosity(He)was 0.297.The low genetic diversity of Alpine musk deer in Helan mountains was detected by microsatellite markers.A total of 85 multi-locus genotypes(MLG)were detected,with a high genotype diversity(0.979).The results of the inbreeding analysis showed that although the sex ratio of musk deer in different geographical populations varied greatly,no inbreeding was found.The results of discriminant analysis of principal components(DAPC),minimum spanning network(MSN),phylogenetic tree,and cluster analysis indicated that Alpine musk deer did not form a special cluster structure and did not show an evident phylogeographic pattern in Helan mountains.The genetic differentiation value and analysis of molecular variance revealed that there were different levels of genetic differentiation among the geographic populations,and most of the variation came from within the geographic populations.At the same time,there was no significant correlation between the geographic distance and the genetic distance.Analysis of the population dynamics history suggested that overall Alpine musk deer in Helan mountains did not experience the bottleneck effect,but some geographic populations(such as ZMG and MDZ)might exist and have experienced the genetic bottleneck effect.4.Dispersal pattern of Alpine musk deer in Helan mountainsSpatial autocorrelation analysis showed that females tended to spread more than males.The male Alpine musk deer demonstrated significant positive correlation r values at 3.5 km,8 km,11 km,and 12 km,respectively,showing significant spatial structure and genetic similarity at relevant spatial scales.Whereas the females showed alternating positive and negative r values,and only at 15.5 km and 17.5 km,respectively,indicating that there was no significant spatial structure within close distances.The assignment test yielded a negative value for females(AIc =-0.997),and individuals with long tails were distributed between females and males,but the difference between the sexes did not reach a significant level(Z =-1.963,P = 0.050),and the results only suggest a potential for dispersal of both females and males in Alpine musk deer population in this area.Recent dispersal events were detected in a total of four individuals,all females,with dispersal distances ranging from 6.532 to 24.301 km,and no male dispersal events were detected,generally indicating a higher probability of female-biased dispersal phenomenon in Alpine musk deer.Neither the relatedness analysis nor the Fst comparison between the two sexes showed a clear pattern of dispersal.In general,our results showed that both female and male Alpine musk deer are likely to disperse in Helan mountains,but with a preference for female dispersal.The results obtained still needed ongoing attention and validation.In summary,the results indicated that the population size and genetic diversity of Alpine musk deer in Helan mountains were at a low level,with a narrow distribution range.There was a certain degree of genetic differentiation among different geographic populations,but no obvious phylogeographic structure had been formed.In view of the urgent situation of this endangered population,we suggested that 1)stricter human management measures should be implemented,especially for the geographical populations that are closer to the scenic areas with a high level of human disturbance,such as Binggou(BG)and Xuelingzi(XLZ),2)efforts should be made to protect germplasm resources and establish captive populations and germplasm banks,3)priority conservation areas or identification of networks that need to be defined,such as the clear identification of potential dispersal corridors between the geographic populations of Qingshuwan(QSW)and Huangtuliangzi(HTLZ),can effectively improve the quality of conservation and management and promote population recovery and stability. |
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