The analysis for mitochondrial genome sequencing of Triplophysa lewanggensis

Abstract Triplophysa lewanggensis revealed that the complete length of its mitochondrial genome was 16,568 bp, composed of A (31.7%), T (27.1%), G (15.9%), C (25.4%), and A + T (58.8%). Its genetic constitution and arrangement were consistent with other Triplophysa, including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control area (D-loop). All genes were encoded by the H-strand, except for one protein-coding gene (ND6) and eight tRNA genes (tRNAGln, tRNAAla, tRNACys, tRNAAsn, tRNATyr, tRNASer(UCN), tRNAGlu, tRNAPro) are encoded by the L chain, and the remaining genes are encoded by the H chain. The phylogenetic tree was divided into two main clades: one just including Triplophysa and the other including Oreonectes, Homatula, Schistura, Nemacheilus, Traccatichthys. T. lewanggensis is distributed in Southwest Guizhou Province attached to Yunnan–Guizhou Plateau, T. xiangxiensis is distributed in Hunan Province, they are near relatives, but their kinship with Triplophysa distributed in Tibetan Plateau is farther.


Introduction
Triplophysa genera affiliated to Cobitidae family, Nemacheilinae subfamily, and it was known that there are about 100 species and subspecies in Triplophgsa, mainly distributed in the Tibetan plateau (Zhu 1989). Triplophysa lewanggensis is distributed in Southwest Guizhou Province attached to Yunnan-Guizhou Plateau. In the present study, the specimen was collected from Lewang town, Wangmo County of China (25 17.7873 0 N, 106 18.7548 0 E) in 2015. Now the specimen is deposited in the animal specimen room of the School of Life Sciences, Guizhou Normal University, Guiyang, China.

Methods
Total DNA was extracted from the fish muscle tissues, and 26 pairs of primers were used to amplify genomic DNA. Then, complete mitochondrial genome was submitted to the GenBank (Accession no. KU987438).
Complete mitochondrial DNA sequences of 30 species of Nemacheilinae were obtained from GenBank, from which Cytochrome-b genes were extracted. The nucleic acids of cytochrome-b genes were then compared using the ClustalW method of MEGA version 6.0 software, the terminal irregular regions were removed manually. Subsequently, the phylogenetic tree was established using the neighbour-joining (NJ) method in MEGA 6.0, while reliability was tested using the Kimma2-Pamameter distance model and bootstrap method by repeating 1000 times.

Results and discussion
The overall length of T. lewanggensis mitochondrial genome is 16,568 bp, including a total of 22 tRNA genes, two rRNA genes, 13 PCGs and a control region (D-loop). Twenty-eight genes are encoded on the H-strand, the remain nine genes including ND6 and eight tRNA genes (tRNA Gln , tRNA Ala , tRNA Cys , tRNA Asn , tRNA Tyr , tRNA Ser(UCN) , tRNA Glu , tRNA Pro ) are encoded on the L-strand. The nucleotide sequence consists of A (31.7%), T(27. 1%), G (15.9%), C (25.4%), A þ T (58.8%) shows a certain degree of A-T bias, the content of A þ T in D-loop area was highest (68.8%).
The phylogenetic tree was divided into two main clades: one just including Triplophysa, and the other including Oreonectes, Homatula, Schistura, Nemacheilus, Traccatichthys (Figure 1). The latter clade was also divided into two sister groups, the first one consisted of Homatula, Schistura, Nemacheilus, Traccatichthys, the other one only contained Oreonectes. Triplophysa divided into two branches, one branch is distributed in the high elevation area of the plateau containing seven Triplophysa fishes, each branch are clustering based on morphological classification relationship; another branch is distributed in the Yunnan-Guizhou Plateau containing two Triplophysa fishes. T. lewanggensis is distributed in Southwest Guizhou Province attached to Yunnan-Guizhou Plateau, T. xiangxiensis is distributed in Hunan Province, they are near relatives, but their kinship with Triplophysa distributed in Tibetan Plateau is farther.
Since mitochondrial genomic information for Triplophysa of Nemacheilinae is relatively rare, it is necessary to obtain mitochondrial genomes of more Triplophysa and other genus of Nemacheilinae in order to clarify the historical development of the Nemacheilinae. To further improve the reliability of the phylogenetic tree, a combination of information on the mitochondrial and nuclear genome is desirable to elucidate the phylogenetic relationships of the Nemacheilinae.

Disclosure statement
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.