Conservation Genetics Of Triplophysa Rosa (Teleostei:Cypriniformes)

The Rose Blind Plateau Loach, Triplophysa rosa (Teleostei, Cypriniformes, Nemacheilidae), is a cave-dwelling fish species distributed in the karst landform. At present, it is only found in subterranean streams at Wulong County, Chongqing of Southwestern China. Due to its restricted distribution and the impact of severe drought in southwestern China in recent years, T. rosa is confronted with a possible decline in population size. But so far, the study on its basic biology is still wanted. In this regard, the study on its conservation genetics is of vital importance in terms of conservation biology. The main studies are threefold:(1) the phylogenetic position of T. rosa was analyzed based on mitochondrial DNA (mtDNA) cytb sequences; (2) the genetic diversity of T. rosa was assessed based on microsatellite and mtDNA cytb sequences; (3) the adaptive evolution of T. rosa was investigated based on mclr gene. The main results were as follows:1. The phylogenetic trees were constructed using Bayesian inference and maximum likelihood methods based on mtDNA cytb sequences with 13 newly determined for Triplophysa species plus 50 species of Cobitoidea and 2 species of Cyprinidae retrieved from GenBank. The results show that:(1) each family of the superfamily Cobitoidea forms a monophyletic group; (2) Species from genus Triplophysa forms a monophyletic group, which has a close relationship with Barbatula nuda; (3) T. rosa clustered with two cave-dwelling Triplophysa species, which clade located in the basal position within the genus. However, there is a larger genetic distance between the three cave fish species and non-cave Triplophysa species.2. Polymorphic microsatellites were developed in the genome of T. rosa using 454 sequencing, and cross-species amplification was tested in Triplophysa moquensis. Polymorphic microsatellite and mtDNA cytb markers were used to study the population genetic diversity of T. rosa with 35 individuals. The results show that:(1) of the 145 loci screened,106 were amplified successfully and 11 showed polymorphic patterns; (2) the population have a moderate level of genetic diversity (the mean number of alleles (NA), mean observed (Ho) and expected (HE) heterozygosity, mean polymorphic information content (PIC) were 3.4,0.349,0.551 and 0.486, respectively; of the 11 polymorphic loci,4 loci (TR1, TR5, TR6 and TR8) with no deviation from Hardy-Weinberg equilibrium (HWE), no null alleles and no evidence of linkage equilibrium (LD), which were suitable for further population genetic studies of T. rosa); (3) cross-species amplification was tested for the 106 loci in T. moquensis,81 were amplified successfully and 12 showed polymorphic patterns; (4) the genetic diversity of the population was low based on mtDNA cytb markers (V (variation site)=23, H (haplotype)=3, Hd (haplotype diversity)=0.339, Pi (nucleotide diversity)=0.00672).3. The melanocortin 1 receptor (mc1r) gene of T. rosa was cloned, and its adaptive evolution was analyzed combined with mclr gene of 12 other Triplophysa species. Main results were:(1) the full-length DNA of mclr gene is 1492 bp, containing an open reading frame (ORF) of 987 bp, a 5’un-translated region of 210 bp and 3’-UTR of 295 bp, this gene encodes 328 amino acids; (2) the deduced amino acid sequence of T. rosa Mclr showed the highest similarity (87%) with Carassius auratus and Cyprinus carpio, and 86% similarity with Danio rerio. The phylogenetic tree shows that T. rosa clustered with Triplophysa sp., and they are located in the basal position within the genus; (3) the Mclr protein is a hydrophobin with 7 transmembrane regions and no signal peptide, mainly consists of a-helix and relatively few β-sheet, and consists of a highly conservative and functional domain rhodopsin-like G-protein-coupled receptors between 65 to 305; (4) only one mutation site (V/M) was identified among 23 Mclr amino acids sequences of T. rosa, owing to being found at the fourth transmembrane domain, it contributes to the structural integrity of the protein; compared with other non-cave Triplophysa fishes, four mutation sites (S5, N8, R40, K121) was identified in the two cave-dwelling Triplophysa species (T. rosa and Triplophysa sp.) based on Mclr amino acids sequences of 13 Triplophysa species; (5) the results showed that mclr gene of these Triplophysa species was given priority to strong purifying selection and neutral evolution in the course of evolution by PAML and Datamonkey analysis, respectively. No amino acid site of positive selection was detected by the two methods.