| The habitat fragmentation caused by natural or human factors is increasing in a global context.Habitat fragmentation will influence the patterns of genetic differentiation and impede the gene flow between populations,while gene flow often represents a key factor in determining and maintaining genetic diversity,and is critical to the fitness of species,especially for the survival of endangered wildlife.The Alashan red deer(Cervus elaphus alashanicus)is currently only distributed in the Helan Mountains of China,it is an isolated population with the least number and the narrowest distribution range compared with other red deer subspecies in China.Studies have shown that the genetic differentiation among alpine ungulate populations situated on "sky islands" was more pronounced than within continuous landscape.However,studies on the genetic diversity of this subspecies are not comprehensive,and the status of genetic differentiation within population is still blank,which poses serious constraints to the sustainable management and scientific protection for the endangered subspecies.In this study,fresh fecal samples were collected from the distribution areas of Alashan red deer in Helan mountains by using the line-intercept and track observation methods,from 2016 to 2017.Mitochondrial DNA(mtDNA)of maternal inheritance and microsatellite markers of parental inheritance were used,combined with molecular biology methods,to study the genetic diversity and genetic structure of Alashan red deer,and to evaluate the status of genetic differentiation for this subspecies.In addition,the dispersal pattern of Alashan red deer was analyzed by means of molecular ecology and gender identification.Besides,the landscape genetics was used to explore the behavior characteristics and landscape factors,that we could analyze the factors that might cause the genetic differentiation of this subspecies from the internal and external aspects,in order to provide the scientific foundation for the effective protection of this subspecies.The results were as follows:1.In this study,a total of 329 individuals of Alashan red deer were identified among the 413 feces samples.The ratio of females to males was 1.69:1.Sixteen haplotypes were defined with the mean haplotype diversity of 0.150 and nucleotide diversity of 0.00019 based on mtDNA Cyt b sequence from 215 individuals.Twenty-eight haplotypes were defined with the mean haplotype diversity of 0.318 and nucleotide diversity of 0.00047 based on mtDNA CR sequence from 153 individuals,and twenty-eight haplotypes were defined with the mean haplotype diversity of 0.476 and nucleotide diversity of 0.00036 based on Cyt b and CR gene united sequence from 110 individuals.The average number of alleles,expected heterozygosity and observed heterozygosity were 4.689,0.596 and 0.792 detected by 11 pairs of highly polymorphic microsatellite loci from 329 individuals.In general,all genetic markers indicated that the genetic diversity of Alashan red deer was at the lowest level among all red deer subspecies in China.The Fis value was negative,indicating that there was no inbreeding risk of Alashan red deer at present.2.STRUCTURE and Geneland analysis were conducted on 12 geographic populations based on microsatellite data,indicating that the optimal grouping number was 3.the neighbor-joining tree,discriminant analysis of principal components(DAPC),phylogenetic trees and haplotype networks indicated the lack of geographic pattern in Alashan red deer.Compared to mtDNA,microsatellites detected more genetic structure.The results of molecular variation analysis(AMOVA)of all markers showed that the population genetic variations mainly produced within geographic populations,but the analysis results of different molecular markers were different.AMOVA of the three mtDNA showed that the genetic differentiation between the two groups of Inner Mongolia and Ningxia reached a significant level(P<0.05),while the microsatellite markers showed that the genetic differentiation within and between geographic populations reached an extremely significant level(P=0.000),and the genetic differentiation between these two groups was not significant(P=0.363).We speculate that the difference between the two markers may be caused by male-biased dispersal.Fst values showed that there were more significant(74.2%,P<0.001)genetic differentiations between groups detected by microsatellite than by mtDNA,indicating that the population of Alashan red deer underwent a gradual differentiation process.3.We found that this subspecies might undergo the bottleneck effects detected by microsatellite data.The estimates of Fu and Li's D and Tajima's D were significantly negatives from the neutral selection test for deer population,and the analysis of mismatch distribution map showed a single peak,all indicated that this subspecies had experienced population expansion.4.The comparison results of Fst values between nuclear markers and standardized mitochondrial markers showed that a slight difference in those two values between most geographical populations,and there was no obvious sex-biased dispersal pattern.The relatedness analysis showed that the genetic relationship(R)value for females(0.158)within most geographic populations was larger than that of males(0.115),showing a male-biased dispersal pattern.In the method of Fst value comparison,the level of genetic differentiation between geographic populations for females was significantly higher than that for males(mean Fst value for females was 0.072,mean Fst value for males was 0.048,P<0.01),which also showed a pattern of male-biased dispersal.The assignment test indicated that the corrected assignment indices(Alc)value for females was 0.375,while that for males was-0.663.Moreover,and most of the individuals in the long tail position were males,also supported male-biased dispersal.Spatial autocorrelation analysis indicated that females showed significant positive correlation and significant spatial genetic structure within a distance of 0 m to 3 000 m,while males showed no significant spatial autocorrelation.Local spatial autocorrelation analysis showed that the 38 individuals which had a significant positive correlation with the four closest individuals.32 were females and 6 were males,showing that males were more likely to dispersal.The local spatial autocorrelation analysis for females and males were performed separately.The mean correlation coefficients(lr)of female and male were-1.6 and-1.9,respectively,indicating that there might be some females that exhibit dispersal behavior,but the dispersal rate was less than that of males.The female deer did not exhibit the spatial genetic pattern expected by the rose-petal model,the unrelated female deer overlapped in space with others.Detection of first-generation migrants showed that 9(8.0%)males and only 1(0.52%)female were detected as first-generation migrants.The dispersal distance of males ranged from 3.33 km to 37.88 km,while that of females was 0.89 km.The dispersal distance and rate of males were significantly higher than those of females.The comprehensive analysis showed that there was a male-biased dispersal in Alashan red deer,which conformed to the characteristics of the dispersal pattern in general mammals.Comprehensive analysis implied that like most mammals,Alashan red deer exhibited a male-biased dispersal pattern.5.The analysis of landscape genetics showed that the genetic differentiation of the Alashan red deer population was in accordance with the pattern of isolation by distance(IBD).In the range of 3 000 m,there was a significant positive correlation between genetic distance and geographical distance.Reciprocal causal modelling analysis showed that the topographic complexity was the most important landscape variables which caused the genetic differentiation of the deer population.Because of vegetation distribution and snow cover,the sunny slope was the most suitable for the activities of red deer.Analysis of linear mixed models found that the combination of topographic complexity,aspect,altitude and slope,affected the spatial distribution pattern of genetic variation of Alashan deer.The current map of Alashan red deer represented that the channels of Suyukou and Chaqikou were important for gene flow communication between Ningxia and Inner Mongolia.In conservation planning,emphasis should be placed on the protection of these areas to reduce the increase in genetic differentiation caused by human disturbance. |
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