Systematics And Phylogeography Of Genus Cobitis(Teleostei,Nemacheilidae) From North Huai River,China

Fishes of the genus Cobitis（Cypriniformes:Cobitidae）,a small freshwater fish that live in sandy and slow flowing river,widely distributed in Palaearctic and Oriental Regions,excluding the Tibetan Plateau.Studies on the species diversity,genetic diversity,phylogeny and biogeography of Cobitis from North Huai River in China.Classification of the genus Cobitis from North Huai River in China based on morphological characters and DNA sequences of the mitochondrial Cyt b gene.six species of Cobitis have been recorded from the North Huai River in China,including of Cobitis lutheri,Cobitis granoei,Cobitis melanoleuca,Cobitis gracilis,a new species of the genus Cobitis（Cobitis wangfangensis）and a new record species of Cobitis Choii.Phylogenetic tree of the genus Cobitis by BI and ML methods based on the mitochondrial Cyt b gene,Cobitis in China is not clustered according to geographical location,forming two major branches,the six species north of the Huaihe River belong to these two branches,shown that there may be two differentiation centers.Molecular dating time based on the mitochondrial Cyt b gene suggests that the divergence time of Cobitis could be dated back to Early-Middle Oligocene（24.8 Ma）.The genus Cobitis maybe originated from the South China.Mitochondrial Cyt b genes have been used as molecular maker to perform the genetic diversity and phylogeographic analyses of five species from the North Huai River in China.The specific research results are as follows:Based on mitochondrial Cyt b gene,the genetic diversity and phylogeography of C.lutheri were analyzed on the basis of 86 specimens from 5 sampling in 3 basins from North Huai River in China.40 haplotypes were identified,the 3 populations all have high haplotype diversity and nucleotide diversity.The analysis of molecular variance（AMOVA）and genetic differentiation index(F_ST)among the populations showed that the three populations all were highly significant.Phylogenetic analyses and Network analysis showed that the 3 basins were not clustered separated but mixed with each other according to the geographical distribution;population of Suifen River（SFH）is an older group.Neutral test and mismatch analysis showed that overall C.lutheri population experienced population expansion,the recent demographic expansions time of three populations of C.lutheri at 0.201–0.052 Ma.The divergence time of C.lutheri could be dated back to 14.5 Ma,C.lutheri spreaded from thenorthern part of the Korean peninsula to the Suifen River and Ussuri River in China.During the interglacial period,the individuals of Ussuri River spread along the water system to the Songhua River.Based on mitochondrial Cyt b gene,the genetic diversity and phylogeography of C.granoei were analyzed on the basis of 156 specimens from 9 sampling in 7 basins from North Huai River in China.79 haplotypes were identified,except for the population of Xiaoling River（XLH）,all other populations show high nucleotide diversity.The analysis of molecular variance（AMOVA）and genetic differentiation index(F_ST)among the populations showed that 7 populations had different degrees of genetic differentiation,mainly reflected in geographical distance and different water systems,long geographical distance and different water systems have large genetic differentiation.Phylogenetic analyses and Network analysis showed that the 7 basins were not clustered separated but mixed with each other according to the geographical distribution;population of Heilong River（HLJ）is an older group.Neutral test and mismatch analysis showed that the recent demographic expansions time of three populations of C.granoei（the populations of Heilong River,Fuzhou River and Luan River）at0.190–0.029 Ma.The divergence time of C.granoei could be dated back to Early Pleistocene（2.4 Ma）,C.granoei originated in the upper reaches of Heilong River,the population of Liao River spread south with the glacial margin to the Luan River and the water system around the Liao River.Based on mitochondrial Cyt b gene,the genetic diversity and phylogeography of C.melanoleuca were analyzed on the basis of 89 specimens from 5 sampling in 3 basins from North Huai River in China.22 haplotypes were identified,the three populations all have low haplotype diversity and high nucleotide diversity.The analysis of molecular variance（AMOVA）and genetic differentiation index(F_ST)among the populations showed that 3populations have significant differences.Phylogenetic analyses and Network analysis showed that the 3 basins were not clustered separated but mixed with each other according to the geographical distribution;population of Luan River（LH）is an older group.Neutral test and mismatch analysis showed that the recent demographic expansions time of three populations of C.melanoleuca（the populations of Xiliao River and Hai River）at 0.127–0.078 Ma.The divergence time of C.melanoleuca could be dated back to Early Pleistocene（2.4 Ma）,The ancestor of C.melanoleuca may be differentiated with the ancestors of C.granoei.Later,the Luan River as the spread center,C.melanoleuca spread to the Haihe River and then to the Liaohe River.Based on mitochondrial Cyt b gene,the genetic diversity and phylogeography of C.gracilis were analyzed on the basis of 93 specimens from 3 sampling in 2 basins from North Huai River in China.32 haplotypes were identified,the two populations both have high nucleotide diversity,the population of Yellow（HH）River have high haplotype diversity.The analysis of molecular variance（AMOVA）and genetic differentiation index(F_ST)among the populations showed that 2 populations hardly have any differentiation significant.Phylogenetic analyses and Network analysis showed that the 2 basins were not clustered separated but mixed with each other according to the geographical distribution;population of Yellow River is an older group.Neutral test and mismatch analysis showed that the recent demographic expansions time of the populations of Yalu River at 0.076–0.062Ma.The divergence time of C.gracilis could be dated back to Late Miocene（9.7 Ma）,It is shown that C.gracilis was distributed in northern China during the late Miocene.C.gracilis may originate in the lower Yalu River and spread to the Yellow River.Based on mitochondrial Cyt b gene,the genetic diversity and phylogeography of C.wangfangensis were analyzed on the basis of 99 specimens from 5 sampling in 4 basins from North Huai River in China.23 haplotypes were identified,except for the population of Huai River（HH）,all other populations show low haplotype diversity and high nucleotide diversity.The analysis of molecular variance（AMOVA）and genetic differentiation index(F_ST)among the populations showed that 4 populations are more than moderate genetic differentiation.Phylogenetic analyses and Network analysis showed that the 4 basins were not clustered separated but mixed with each other according to the geographical distribution;population of Huai River is an older group.Neutral test and mismatch analysis showed that the recent demographic expansions time of three populations of C.wangfangensis（the populations of Wei River and Fuzhou River）at 0.097–0.041 Ma.The divergence time of C.wangfangensis could be dated back to Early Pliocene（4.0 Ma）,The population of the Huai River first spread to the Yellow River,C.wangfangensis spreads to the Liaodong Peninsula through the land bridge between the Liaodong Peninsula and the Shandong Peninsula forming the population of Fuzhou River.