| Andrew's toad(Bufo andrewsi)is one of the species widely distributed in southwest China,with an altitude of 750?3500 meters,and its habitat types are very diverse.Therefore,this toad was used as the research object in this article,the geographic variation of genetic variation,morphological characteristics(body size,age and organs size)in population were discussed through microsatellite(S SR)molecular markers,paraffin sectioning,and dissection.In the meantime,the correlation between the genetic differentiation and morphological characteristics was be explored,and contacted the environment climate conditions of its habitat,the adaptive mechanism of Andrew's toads to environmental changes be deeply understood,and provide scientific advice for the protection of this specie.The main results of the research are as follows:(1)In this study,we development new SSR loci for Andrew's toad from published transcriptional genome data.30 samples from the population of Qibie Village in Yunnan Province were used to test the polymorphism and stability of the loci.By comparing factors such as the amplification stability of SSR loci,number of allele number(Na)and polymorphic information content(PIC),eight SSR loci that could be used for studies of population genetic diversity and genetic structure of this species were screened out.(2)16 SSR molecular markers were used to analyze 868 Andrew's toads from 30 populations,and the results indicated that the genetic diversity was high at the population level,but relatively low at the population level,and some populations may experience the bottleneck effect recently,and we found that the genetic diversity of population decreases with the increase of altitude.Significant genetic differentiation and clear genetic structure among study populations were revealed by genetic structure analysis.The 30 populations were divided into five clusters:BX,QQ,DCG and HY were clustered into Cluster ?;KG,QB,XGJ,JMK and GC were clustered into Cluster ?;MYC,HB,WX and JC clustered into Cluster ?;SCH,AN,DY,LG,PX,TP,JL,LD and ZJ clustered into Cluster ?,and QLX,SZB,GD,WL,LMS,XYB,LX and MX clustered into Cluster V.The population genetic structure as a whole presented a characteristic of an organized geographic plate structure and random clustering.And the genetic differentiation of pair populations was significantly correlated with the geographic distance between populations.(3)General linear model(GLM)was used to analysis the body size of 435 individuals in 14 populations.The results revealed significant differences in body length(SVL)and weight among populations(F?3.879,P<0.001).When controlling for the effect of age,the difference still existed.But the correlation in altitude and latitude of body size were not significant(P>0.05)based on the analysis of linear mixed models(LMMs).And the toad has obvious sexual size dimorphism with the female significantly being larger than the male,and the allometric growth characteristics of their body sizes are opposite to Rensch's rule according to the reduced major axis regression(RMA)analysis(?RMA<1).Analyzed 596 individuals from 21 populations whose age has been successfully identified,the results indicated significant differences in age between males and females(GLM analysis results:F1,595=25.175,P<0.001).The mean age of females was greater than that of males(female:3.05±0.10 year;male:2.54±0.05 year).The age of both sexes increased as the latitude increased(r?0.309,P<0.001).(4)The organs size of 435 individuals from 14 populations was analyzed by GLM,the results showed significant population variation in the relative size of the heart,lung,gallbladder,liver,spleen,kidney,testis,digestive tract,and brain(F?3.402,P<0.001).LMMs was used to detect the variation of the geographic gradient of organ size at altitude and latitude,and we found only the relative lung size was significantly positively correlated with the altitude gradient(F=5.797,P=0.037),which implied that the high-altitude population of Andrew's toads adapt to the low oxygen environmental conditions.(5)Seven climatic factors,such as average annual temperature and annual rainfall were selected to explore the adaptability of genetic variation and morphological characteristics to environmental climate.The results showed that morphological characteristics(physical condition,age and organs size)changed with the change of climatic factors.Analyzed the correlation between physical condition,age and genetic diversity,we found that the population with better physical conditions and longer lifespan displays higher genetic diversity(r?0.030,P<0.05).And compared the clustering results of population SSR data with this of physical condition and organ coefficients,we discovered that the clustering results overlapped,but they were not completely identical.The above results shown the morphological characteristics are more sensitive to environmental changes,but all the evolution of morphological characteristics and population genetic variation were towards the direction of maximum environmental adaptability and maximum evolutionary potential.In conclusion,we put forward the following two suggestions for the protection and management of Andrew's toads:First of all,considering the high genetic diversity of this toad at species level,relatively low genetic diversity of each population,and significant genetic differentiation among populations,the protection of this species diversity should be implemented to maintain genetic diversity of various populations.The main measure is to effectively protect its habitat.In addition,the population's physical condition and age were positively correlated with genetic variation,and the variation changed with environmental climate factors.So,we could understand the adaptive mechanism and population dynamics of different geographic populations of species by monitoring the body size,age structure,and lifespan of different geographic populations,so thus achieving the purpose of species protection. |
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