Systematics And Phylogeography Of Genus Barbatula (Teleostei, Nemacheilidae) From North-eastern China

Stone loaches belonging to the genus Barbatula Linck,1970(Teleostei,Nemacheilidae)are small benthic freshwater fish found throughout most of the Palaearctic,with the exception of northern Africa.However,the taxonomy of this genus not entirely free from doubt.At present,26 species of Barbatula as valid: 5 from Europe and 21 from Asia.From these,China has 10 species: only one is known from north-western China and other 9 species are from north-eastern China.Although China has abundant fish resources of Barbatula,many current studies on this group were still in the stage of description of them,a lot of dundamental research has not been carried out,such as the study on genetic diversity and biogeography.Barbatula is an important component of freshwater fish in north China;it has important scientific value in the study of species generation and evolution.Therefore,we first confirm the species validity according to their morphological character,use mitochondrial(COI,Cyt b)and nuclear(RAG1)gene sequences fragments as molecular markers to reconstruct the phylogenetic relationships within genus Barbatula.Then,we use mitochondrial gene sequences fragments(Cyt b,D-loop)as molecular markers to estimate genetic diversity and population demographic history of tow species of Barbatula.Finally,we use mitochondrial gene sequences to reconstruct the phylogenetic relationships of Barbatula to show their systematic position in the Cobitoidea,and estimate the divergence time of the major Barbatula lineages,investigate the origin and evolution of it.The main results were as follows:1.Six valid species of genus Barbatula from North-eastern China has been re-described: B.nuda,B.toni,B.kirinensis,B.pechiliensis,B.potaninorum and B.gibba.Seven new species has been described: B.emuensis,B.liaoyangensis,B.linjiangensis,B.zhangwuensis,B.cheni,B.mulingensis and B.suifensis.At present,there are 13 species of Barbatula in North-eastern China.2.Mitochondrial DNA Cyt b,COI and nuclear DNA RAG-1 genes have been used as molecular maker by Bayesian inferences(BI)and Maximum Likelihood(ML)method to perform the phylogenetic analysis of genus Barbatula from North-eastern China.Cyt b and COI genes support these 11 species(except B.gibba and B.potaninorum)can constitute different branch and the genetic distance among species greater than 1.9% in COI,greater than 2.4% in Cyt b.RAG1 cannot support some constitute different branch,as the low evolutionary rate.may be related to the low evolutionary rate of RAG1 gene,or imply these species had gene flow recently.3.Mitochondrial DNA Cyt b and D-loop genes have been used as molecular maker to perform the genetic diversity and phylogeographic analyses of three populations of B.toni from north China.57 haplotypes were identified for 119 individuals of B.toni.No haplotype shared among different populations.Relatively high haplotype diversity(h=0.805�0.926)and low nucleotide diversity(? =0.00095�0.00458)were detected in three populations of B.toni.Haplotype Bayesian tree showed B.toni from three river systems were separately clustered as three clades.According analysis of molecular variance(AMOVA),the variation among populations(accounting for 79.45%)was the main source of variation.Differentiation index(FST)showed the three populations have significant differences,with FST >0.25(P<0.01).Neutrality tests and mismatch distribution indicated the recent demographic expansion of B.toni.The divergence time of three populations of B.toni could be dated back to 1.082�0.669 Ma,recent demographic expansions time at 0.071�0.047 Ma,Pleistocene climatic changes could be a key factor of the population dynamics.4.Mitochondrial DNA Cyt b gene have been used as molecular maker to perform the genetic diversity and phylogeographic analyses of three populations of B.kirinensis from north China.49 haplotypes were identified for 93 individuals of B.kirinensis,only 1 haplotype shared among different populations.Relatively high haplotype diversity(h=0.911�0.966)and low nucleotide diversity(? =0.00269�0.00571)were detected in three populations of B.kirinensis.Haplotype Bayesian tree showed B.kirinensis from tow river systems were separately clustered as two groups,group A and group B.According analysis of molecular variance(AMOVA),the variation among groups(accounting for 31.36%)was the main source of variation.Differentiation index(FST)showed the three populations have significant differences,with FST >0.25(P<0.01).Neutrality tests and mismatch distribution indicated the recent demographic expansion of these two groups.The recent demographic expansions time at0.29�0.23 Ma of this species,0.10�0.08 Ma of group A,0.20�0.16 Ma of group B.Some individuals of Songhua Rivers passing the interconnected rivers and migrate to Tumen Rivers during the demographic expansions and formed the distribution pattern.5.Molecular dating time suggests that the lineage leading to the Barbatula and Triplophysa diverged approximately at 24.8 Ma,Asian and European Barbatula groups diverged approximately at 11.9Ma.The genus Barbatula maybe originated from the Siberia.Northeastern China Barbatula originates from the Heilongjiang drainage and then spread to the east and the south regions.The current distribution pattern of the genus Barbatula in the Northeastern China have been formed under the influence of Quaternary glaciations.