Morphological And Genetic Studies Of The Populations Of Pseudocrossocheilus Bamaensis

Pseudocrossocheilus bamaensis belongs to the Labeoninae of Cyprinformes Cyprinidae,which mainly distributed in the Hongshui River of the Pearl River system in China.It is a small-scale economic fish which is common in Tian’e,Bama,Dahua and Du’an 4 counties in Guangxi Zhuang Autonomous Region,and is also occasionally distributed in Pingtang and Luodian county in Guizhou Province.With the improvement of people’s consumption levels,the market demand for the Pseudocrossocheilus bamaensis is increasing.However,as its large-scale artificial breeding has not successfully achieved,the recent overfishing has caused serious degradation of the natural resources of the species,and it is urgent to conduct systematic investigation and evaluation of its germplasm resources.This research combined morphological and genetic studies,detailed comparison of the morphological differences between different geographical populations of the species and the three years old male and female samples,and the key morphological indicators affecting the body weight of the 3-year old male and female samples were scientifically evaluated.Through the mitochondrial D-loop region analyzed the genetic diversity and genetic structure of different geographical populations,and the formation mechanism of the current geographical distribution pattern was systematically revealled.It aims to provide a theoretical basis for the protection and rational development and utilization of the P.bamaensis germplasm resources.1.Morphological study on the population of P.bamaensisIn this study,we conducted a multivariate statistical analysis of the appearance male samples from eight geographic populations of P.bamaensis campestris by combining traditional morphology and frame morphology.Principal component analysis extracted a total of nine principal components（PCA1～PCA9）,of which the top three principal components contributed a higher rate,mainly reflecting the morphological changes in the head,tail and back belly of the fish.The scatter plots of PCA1 and PCA2 showed that some geographical groups could be clearly distinguished,and the difference in PCA1 axis between groups was more significant than that in PCA2 axis.Eighteen standardized character parameters screened by stepwise discriminant analysis had a discriminant accuracy rate of 89.06～97.67%for each geographic population,and a comprehensive discriminant accuracy rate of 92.83%,which could be used to distinguish each population.The results of cluster analysis showed that the geographical populations could be clustered into three populations,the Donglan,Dahua,Tian’e and Heshan four populations first clustered as the first branch,then with the second branch of Pingtang,Luodian and Bama three populations clustered,finally with the third branch of Du’an populations together.The results of the difference coefficient calculation showed that there was subspecies differentiation in some standardized character parameters in all the populations except Bama,but the direction of morphological differentiation among the populations was not consistent,and there was no morphological subspecies differentiation between adjacent populations,so it was impossible to judge that the species had subspecies.In addition,the main scatter plot and cluster analysis results of this study all found that the morphological distance between each population was not significantly consistent with the actual geographical distribution,it was speculated that this may be related to the fact that the P.bamaensis was morphologically susceptible to the local environment and thus produces a large variability.2.Morphological comparison between male and female of P.bamaensis and discussion on the relationship between measurable characters and body weightThis study targeted the male and female individuals of the 3-year old P.bamaensis,and the traditional morphological and frame morphological comparisons based on multivariate statistical analysis were performed.The main scatter plot showed that the morphological differences between male and female were mainly reflected in the abdomen characteristics of fish（PCA2）.The gender discriminant equation of the 3-year old P.bamaensis,which was established by stepwise discriminant analysis,could better distinguish between male and female samples of 2,3,4-year old and above ages.By counting the growth characters of the three years old male and female samples,it was found that the female individuals of this species were significantly larger than the males.The reason for this phenomenon may be related to factors such as the fact that the higher reproductive energy consumption caused by the lower number of mature males in the field and the evolution of fertility selection in females.The relationship between the 14 characters and body weight of the 3-year old male and female was found.The key character parameters affecting male body weight were total length,body thickness and body depth.Females were total length,body thickness,body depth,caudal peduncle depth and trunk length.The key character parameters affecting the quality of male and female were different,it was recommended to classify and select them according to gender in the subsequent breeding process.3.Genetic study on the populations of P.bamaensisA total of 23 haplotypes were detected based on the full length（942 bp）of the mitochondrial D-loop region of the P.bamaensis,and the haplotype diversity（h）was 0.672.The nucleotide diversity（π）was 0.0012 and the total genetic diversity was low.Among the populations with higher levels of genetic diversity were Tian’e（h=0.870,π=0.0022）,Luodian（h=0.705,π=0.0009）and Dahua（h=0.660,π=0.0005）;the lower were Donglan（h=0.239,π=0.0001）,Pingtang（h=0.123,π=0.0001）and Bama（h=0.213,π=0.0000）.In addition,the genetic differentiation index(F_st)of the P.bamaensis population was between-0.02596 and 0.94204,the genetic distance varied from 0.0000 to 0.0037,and the average nucleotide difference(K_xy)ranged from 0.393 to 5.182.From the above three genetic parameters and the inter-group gene flow（N_m）,the exact P test and the haplotype phylogenetic tree,it showed that the P.bamaensis could be divided into two major branches according to geographical distribution.Three geographical groups of Pingtang,Luodian and Tian’e in the upper reaches of the Hongshui River was one,and the five geographical groups of Donglan,Bama,Dahua,Du’an and Heshan in the middle and lower reaches were another branch.There was a large genetic differentiation between the two branches and the upper Pingtang group and the Luodian and Tian’e two populations,and the genetic differentiation between the five downstream geographical groups was small.The molecular variance analysis（AMOVA）of the two branches showed that the genetic differentiation between the populations of the P.bamaensis was mainly from different geographical intervals in the water system and between the various groups within the geographical interval.The haplotype network map showed a distinct pedigree structure corresponding to the geographical distribution.The ancestor haplotypes of the species were inferred to be HAP1and HAP2 according to the network map.Combined with the nucleotide diversity of each population,it estimated that the origin of its ancestors should be Tian’e County of Guangxi Zhuang Autonomous Region.The results of the neutral test of Tajima’s D and Fu’s Fs suggested that the P.bamaensis had experienced recent mass expansion events.Based on the quantitative and spatial expansion model,the nucleotide mismatch distribution curve of the upper reaches of the Hongshui River are obviously single peaks,which were estimated to have experienced habitat expansion about 29,000 years ago,and experienced quantitative expansion in about 23,000 years.The nucleotide asymmetric distribution curve fitting effect of the population was poor,but the sum of the square deviations and the roughness index values in the nucleotide unpaired distribution based on the spatial expansion model were not significant,it was speculated that the species had also experienced habitat expansion around21,000 years ago.In summary,most of the geographical populations of P.bamaensis exhibit low h and lowπphenomenon,which may be related to the shorter migration time of the species and the greater survival pressure of each group in recent years.It is suggested that scientific management and utilization should be carried out according to the status of resources at different sampling points and the distribution of water systems.