Genetic Diversity Of Sinibotia Superciliaris And S. Reevesae

Genetic diversity is an important component of biodiversity,which reflects the degree of genetic variation among different individuals within the same group or between groups of a species,and indicates the genetic relationship among different groups.Understanding the genetic diversity of species is not only helpful for the relationship of wild fishes,but also lays the foundation for breeding and genetic improvement.It hlep endangered or threatened species to formulate scientific and reasonable protective measures to reduce or avoid the loss of biological genetic diversity by analyzing the influence of living habits,geographical environmental factors on the genetic diversity of species.Sinibotia superciliaris and S.reevesae belong to Sinibotia(Teleostei,Cypriniformes,Botiidae).There are currently six recognized species in this genus,two species(S.superciliaris and S.reevesae)distribute in the Yangtze River,and other species(S.pulchra,S.robusta,and S.zebra)distribute in the Pearl River,and S.longiventralis distribute in Mekong River.Fish from this genus are mostly inhabiting in the flowing water environment with the bottom of sand and gravel,they have different color and spots which makes it very interesting to observe.To date,the studies on this genus are limited,most studies have been restricted to embryonic development histology and reproductive biology among species of this genus are based on the analysis of single molecular markers and do not include all species.There havebeen many studies related to the S.superciliaris and S.reevesae,the studies found that the two species are very similar in morphology,and genetic diversity is very low at molecular level.They cannot be divided by DNA barcode and there might be gene flow between them.Therefore,we treat S.superciliaris and S.reevesae as a whole to analyze their genetic diversity and genetic structure in this study.Based on the above considerations,firstly,we used mitochondrial(COI and Cytb)and nuclear(RH,RAG1,and IRBP)gene sequences as molecular markers to analyze phylogenetic relationships among species of Sinibotia.Secondly,we developed polymorphic microsatellite markers for S.superciliaris based on the Illumina paired-end sequencing technology and further screened for cross-amplification in S.reevesae,compared the genetic diversity of the two species.Finally,combined with microsatellite and mitochondrial Cytb markers,the genetic diversity and genetic structure of the wild populations of S.superciliaris and S.reevesae were analyzed,their genetic diversity and evolutionary potential were evaluated.The main results were as follow:1.The analysis of phylogenetic relationship of the genus Sinibotia based on five molecular markers.A total of 187 sequences were obtained by PCR amplification and sequencing,including 39 Cytb gene sequences,50 COI gene sequences,42 IRBP gene sequences,49 RAG1 gene sequences and 47 RH gene sequences.The phylogenetic relationships of Botiidae were analyzed by using maximum likelihood(ML)and Bayesian inference(BI)methods.Results of concatenated dataset indicated that the family Botiidae was divided into two lineages:(Leptobotia + Parabotia)+((Botia +Chromobotia)+((Sinibotia + Ambastaia)+(Syncrossus + Yasuhikotakia))),they were all monophyletic respectively;S.robusta was located at the basal position in Sinibotia,three species(S.robusta,S.pulchra,S.zebra)were monophyletic group respectively.The S.superciliaris and S.reevesae cluster with each other.2.Genetic diversity of the S.superciliaris and S.reevesae were analyzed based on Cytb gene sequences and 9 SSR markers.(1)A total of 387 Cytb gene sequences were successfully amplified,and 86 haplotypes were isolated.The phylogenetic trees and networks based on haplotypes supported that the populations can be divided into three lineages,the fist was all the populations of S.superciliaris except population from Miyi,the second was the S.reevesae population in Hechuan,the third was the S.superciliaris populations in Miyi and the S.reevesae populations in Baima and Zizhong;(2)Of the66 microsatellite loci,39 loci showed polymorphism in S.superciliaris.Ninepolymorphic microsatellite loci were used to genotype of 12 populations.When k = 3,the results of structure analysis of microsatellite consistent with the results of phylogenetic trees and networks based on haplotypes;(3)The results of genetic diversity based on 86 haplotypes and 9 microsatellite markers showed that the genetic diversity of S.superciliaris and S.reevesae was higher,and the genetic diversity of S.superciliaris was higher than S.reevesae.The results of genetic structure analysis showed that the genetic differentiation between the populations of S.superciliaris was smaller except population from Miyi,and the genetic differentiation between the populations of S.reevesae was larger;(4)The results supported that there were population expansion signals in S.superciliaris recently based on mismatch distribution analyses,neutrality tests,and Bayesian skyline plots,but only a slight signals in S.reevesae.