Analysis Of Genetic Diversity Of Four Leptobotia Species In The Upper Yangtze River Based On Mitochondrial DNA

Leptobotia is one of important genus of the freshwater fish in the upper Yangtze River.At present,four species,L.elongata,L.rubrilabris,L.microphthalma and L.taeniops,are common in the upper reaches of the Yangtze River.Although the four fishes have similar characters,such as benthic living,feeding on aquatic animals or small fish,torrent adaptability and producing drifting eggs,they are located in different regions.L.rubrilabris mainly distributed in the upper reaches of the Yangtze River,the tributary of the Minjiang River and the Black River.L.elongata is found in the middle and upper reaches of the Yangtze River,the tributary of Minjiang River,Jialing River and has a wider distribution than L.rubrilabris.The distribution of L.microphthalma is limited in the Minjiang River and section from Yibin to Chongqing of the Yangtze River.L.taeniops widely disperse in the Yangtze River below Yibin.Recently,with cascade hydropower stations been built in the upper reaches of the Yangtze River,the dams would cut off the fish migration and succedently change the genetic structure.In this study,samples of those four species were mainly collected from locations in the upper reaches of the Yangtze River Panzhihua,Yibin（Min Jiang）,Nanxi,Hejiang,Jiangjin,Banan and then extended to the middlestream of Yichang,Jianli,Jiayu,Jiujiang and from May 2016 to September 2017.The mitochondrial cyt b sequence as well as the control region were used to analyze the genetic diversity and structure.The main results were as following:1.A total of 120 individuals of L.rubrilabris were collected from four sampled sites as Nanxi,Hejiang,Jiangjin,Banan in the upper reaches of the Yangtze River.Fifty-five polymorphic sites and 43 different haplotypes were identified and haplotype diversity and nucleotide diversity were 0.901 and 0.0032,respectively,in cyt b sequence of L.rubrilabris.Fifty-four polymorphic sites and 72 different haplotypes were identified in control region sequence of L.rubrilabris and haplotype diversity and nucleotide diversity were 0.972 and 0.0070,respetively.Network diagram and phylogenetic NJ tree of haplotype showed that haplotypes were not clustered as consistent with geographical positions.However,two distinct haplotype lineages were identitied in all sampled sites,which indicated that t sympatric genetic divergence had occurred in population of L.rubrilabris.Molecular variance analysis（AMOVA）showed that there was no genetic differentiation between the populations.Data of samples collected in 2012 were compared with present samples and it didnot show significant genetic differentiation between them.However,phylogenetic tree revealed two genetic lineages within population.The neutral test,nucleotide mismatch distribution and Bayesian skyline plot（BSP）analysis showed that the population of the L.rubrilabris had occurred population selection or expansion,and the expansion time was deduced in about 90 kya.2.A total of 194 individuals of L.elongata were collected from six sampled sites as PanZhihua,Nanxi,HeJiang,Jiangjin,BaNan in the upper reaches of the Yangtze River and YiChang in the middle reaches of the Yangtze River.Thirty-eight polymorphic sites and 34 different haplotypes were identified and haplotype diversity and nucleotide diversity were 0.625 and 0.0010,respectively,in cyt b sequence of L.elongata.Fifty-seven polymorphic sites and 55 different haplotypes were identified in control region sequence of L.elongata and haplotype diversity and nucleotide diversity were 0.925 and 0.0040,respetively.Network diagram and phylogenetic NJ tree of haplotype showed that haplotypes were not clustered as consistent with geographical positions and neither lineage differentiation.Molecular variance analysis（AMOVA）showed that there was no genetic differentiation between the populations of sample sites of L.elongata(F_ST<0.05).Data of samples collected in 2012 were used to test of AMOVA with samples in this study and showed that there was no significant genetic differentiation between them.The neutral test,nucleotide mismatch distribution and Bayesian skyline plot（BSP）analysis showed that the population of the L.elongata had occurred population selection or expansion,and the expansion time was deduced in about 35 kya.3.A total of 104 individuals of L.microphthalma were collected from two sampled sites as Jiangjin in the upper reaches of the Yangtze River and MinJiang River.Thirty-five polymorphic sites and 37 different haplotypes were identified and haplotype diversity and nucleotide diversity were 0.899 and 0.0040,respectively,in cyt b sequence of L.microphthalma.Fifty polymorphic sites and 65 different haplotypes were identified in control region sequence of L.microphthalma and haplotype diversity and nucleotide diversity were 0.959 and 0.0043,respectively.Network diagram and phylogenetic NJ tree of haplotype showed that haplotypes were not clustered as consistent with geographical positions and neither lineage differentiation.Molecular variance analysis（AMOVA）showed that there was no genetic differentiation between the populations of sample sites of L.microphthalma(F_ST<0.05).The neutral test,nucleotide mismatch distribution and Bayesian skyline plot（BSP）analysis showed that the population of the L.microphthalma had occurred population selection or expansion,and the expansion time was deduced in about 30kya.4.A total of 215 individuals of L.taeniops were collected from seven sampled sites as Nanxi,HeJiang,Jiangjin,BaNan in the the upper reaches of the Yangtze River and JianLi,JiaYu,JiuJiang in the middle reaches of the Yangtze River.Twenty-one polymorphic sites and 22 different haplotypes were identified and haplotype diversity and nucleotide diversity were 0.643 and 0.0011,respectively,in cyt b sequence of L.taeniops.Twenty polymorphic sites and 22 different haplotypes were identified in control region sequence of L.taeniops and haplotype diversity and nucleotide diversity were 0.656 and 0.0023.,respectively.Network diagram and phylogenetic NJ tree of haplotype showed that haplotypes were not clustered as consistent with geographical positions and neither lineage differentiation.Molecular variance analysis（AMOVA）showed that there was no genetic differentiation between the populations of sample sites of L.taeniops(F_ST<0.05).The neutral test,nucleotide mismatch distribution and Bayesian skyline plot（BSP）analysis showed that the population of the L.taeniops had occurred population selection or expansion,and the expansion time was deduced in about 10 kya.5.Comparison of genetic structure of the four Leptobotia species:Among the four Leptobotia species,the genetic diversity of the L.rubrilabris is the highest,followed by L.microphthalma and L.elongata,and that of L.taeniops is lowest.Such situation is coincident with their population expansion time.No geographical genetic differentiation occurred in populations of all the four Leptobotia species,but L.rubrilabris appeared sympatric genetic divergence.Data before and after the construction of the Xiangjiaba dam were analyzed using AOMVA and no significant divergence were revealed,which suggested that the impact of the dam construction on the genetic structure of these fishes has not yet emerged right now.