Genetic Diversity And Taxonomic Status Of Gymnocypris Chilianensis Based On Cyt B Gene And D-loop

Gymnocypris chilianensis belongs to genus Gymnocypris, subfamily Schizothoracinae, order family Cyprinidae. It is only restricted to the Shiyang River, Heihe River and Shule Rivers, inland river basin of Hexi Corridor of Gansu province. In recent years, influenced by double factors both natural and artificial, distribution area of G. chilianensis is going down sharply, and the wild resources decreased dramatically. Moreover, there are always disagreements on taxonomic status of G. chilianensis, and the taxonomic results based on morphology by several researchers were not exactly the same.We collected 25 samples of G. chilianensis from Heihe River and Shule River, respectively, and we also collected 25 samples of G. przewalskii from Qinghai Lake. Extract the sample DNA and amplify cytochrome b gene(Cyt b) and D-loop control region. Combined with 25 Cyt b sequences and 25 D-loop sequences of another affinis species G. eckloni which were downloaded from Gen Bank, analysis on genetic diversity and sequence differences were conducted, in order to study the genetic diversity and taxonomic status of G. chilianensis on molecular level. The major results are as follows:(1) A total of 100 individuals of four populations were measured, and the complete Cyt b gene of 1140 bp was collected. There were no insertion or deletion loci in these sequences. A total of 267 polymorphic sites and 94 haplotypes were detected. Haplotype diversity of the four populations was 0.9870 to 1.0000, nucleotide diversity was 0.0039 to 0.0063. The genetic diversity leve of Heihe River G. chilianensis(HH) was the highest(h = 1.0000, π = 0.0063), Shule River G. chilianensis(SL) was the lowest(h = 0.9870, π = 0.0039).The genetic distance between populations and intrapopulation were 0.0070-0.0840 and 0.0040-0.0060 by using Kimura-2-parameter model, this result indicated that the genetic distance between populations were greater than intrapopulation; genetic distance of Heihe River G. chilianensis and Shule River G. chilianensis between G. eckloni was 0.0810 and 0.0780, respectively, between G. IV przewalskii was 0.0840 and 0.0810, respectively. All the genetic distance was higher than 0.0600, indicating that the differentiation between G. chilianensis and G. eckloni, G. przewalskii has reached to species level, G. chilianensis is an independent species of Gymnocypris.(2) A total of 100 individuals of four populations were measured, and the D-loop of 747 bp was collected. There were no insertion or deletion loci in these sequences. A total of 116 polymorphic sites and 78 haplotypes were detected. Haplotype diversity of the four populations was 0.9431 to 0.9970, nucleotide diversity was 0.0043 to 0.0082. The genetic diversity leve of Heihe River G. chilianensis(HH) was the highest(h = 0.9970, π = 0.0082), Shule River G. chilianensis(SL) was the lowest(h = 0.9431, π = 0.0043), which consistent with the results of Cyt b gene.The genetic distance between populations and intrapopulation were 0.0084-0.0350 and 0.0050-0.0080 by using Kimura-2-parameter model, this result indicated that the genetic distance between populations were greater than intrapopulation; genetic distance of Heihe River G. chilianensis and Shule River G. chilianensis between G. eckloni was 0.0320 and 0.0280, respectively, between G. przewalskii was 0.0350 and 0.0300, respectively. Which verified the result of genetic distance based on Cyt b gene.(3) G. pachycheilus(Gen Bank accession number of Cyt b and D-loop: JQ082349 and FJ601172) and P. dipogon(Gen Bank number: JQ082345 and FJ601178) were taken as the out-group. NJ phylogenetic tree based on Cyt b and D-loop haplotypes was constructed using Mega 5 0. The results showed that two trees are composed by two branches, one main branch contains two groups of G. chilianensis, the other branch contains G. eckloni and G. przewalskii, which show that the genetic relationship between G. eckloni and G. przewalskii is closer than with G. chilianensis.(4) 75 sequences each of Cyt b gene and D-loop region from G. chilianensis(HH and SL) and G. przewalskii(75 sequences each of Cyt b gene and D-loop region from the same of population) were analyzed. The results show that polymorphic sites percentage of Cyt b gene(21.9%) was significantly higher than D-loop region(14.3%)(P < 0.01); the number of nucleotide substitution per site in Cyt b(0.0221) was greater than D-loop(0.0163). The result show that rate of evolution in Cyt b gene is faster than D-loop region, Cyt b gene is more suitable for the study of genetic differentiation of closely related species in Gymnocypris. The result based on Cyt b gene sequence analysis indicated that the estimated divergence time between G. chilianensis and G. przewalskii was about 2.4 Ma BP in Early Pleistocene, which was in conformity with the uplift of Qinghai-Tibet Plateau.