The complete mitochondrial genome of Homatula pycnolepis (Teleostei, Cypriniformes) in Lantsang River and its phylogenetic analysis

Abstract Homatula pycnolepis is a small-sized benthopelagic fish species in the family Nemacheilidae, superfamily Cobitidea. In this study, the entire mitogenome sequence of the H. pycnolepis has been sequenced by using sanger sequencing method. The circular mitochondrial genome was 16,570 bp in length and contained of 13 protein-coding genes, two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, an origin of light-strand replication (OL), and one displacement loop locus (D-loop). The overall base composition was 30.1% A, 26.4% T, 26.8% C, and 16.7% G with 56.6% AT content. Most genes were encoded on the heavy strand except for ND6 and eight tRNA genes. There were 11 regions of gene overlaps totaling 29 bp and 7 intergenic spacer regions totaling 37 bp. Its structure type was similar to the mitogenome of Cobitidea fishes. Phylogenetic analysis with 33 complete mitogenomes of Cobitidea and 2 Macrognathus fishes in the family of Mastacembelidae suggested that Homatula species clustered as one monophyletic clade and Homatula was closely related to Schistura. Mitogenome information from this study could be a useful basis for conservation and phylogenetics of this species.

In loach genus Homatula, there were 16 valid species including 2 new species named in recent years (Gu and Zhang, 2012;Min et al. , 2013Endruweit 2015;Yang et al. 2017). They are widely distributed in five major drainages in southwestern China . Homatula pycnolepis was mainly distributed in the Lantsang River (the upper Mekong drainage) and Nujiang River (the upper Salween River basin) as well as its tributaries (Hu and Zhang 2010). It is a small-sized benthopelagic fish species in the family Nemacheilidae, superfamily Cobitidea, and can be developed as an ornamental fish due to its brightly colored appearance. However, its natural populations declined rapidly in recent years due to overfishing, dam construction in the habitat (Yue et al. 2013;Liu et al. 2016).
Four mitochondrial genomes of 3 Homatula fishes have been reported (Shi et al. 2014;Que et al. 2016), but the mitogenome of H. pycnolepis has not been reported. Here, complete sequences of mitochondrial DNA of H. pycnolepis was determinated by using sanger sequencing method. The samples of H. pycnolepis was collected from Jidu River, a littler branch of the Lantsang River, Tue Town, Lanping County, Yunnan Province, China (E99 8 0 42 00 , N26 13 0 55 00 , 1450 m). The voucher specimens was deposited at Neijiang Normal University (accession number 20080819953).
The total genomic DNA was extracted from the fin tissue using the salt-extracted method (Aljanabi and Martinez 1997) with some modifications. Eighteen pairs of PCR primers were designed to amplify and sequence the complete mitochondrial genome. Four new middle primers were designed for sequencing if entire target segments are not obtained by using the aforementioned primers. The overlapping segments were analyzed using the software Lasergene V11 (DNASTAR) and Mega X (Kumar et al. 2018). The mitgenome was annotated with the MITOS (http://mitos2.bioinf.uni-leipzig.de/ index.py) (Bernt et al., 2013), combined with manual corrections.
The H. pycnolepis circular mitogenome was 16,570 bp in length and was submitted to GenBank database under accession No. MT783421. It contained 13 protein-coding genes, two ribosomal RNA genes (12s rRNA and 16s rRNA), 22 transfer RNA (tRNA) genes, and 2 main non-coding regions: origin of light-strand replication (OL) and displacement loop locus (D-Loop). The overall base composition of the genome was as follows: A (30.1%), T (26.4%), C (26.8%), and G (16.7%) with a slight AT bias of 56.5%. It was an average AT bias rich feature of teleost mitochondrial genomes.
Its structure type was similar to the mitogenome of Cobitidea fishes (Shi et al. 2014;Ye et al. 2014;Wang et al. 2016;Wei et al. 2016). Most mitochondrial genes were encoded on the heavy strand except for ND6 and eight tRNA genes (tRNA Gln , tRNA Ala , tRNA Asn , tRNA Cys , tRNA Tyr , tRNA Ser(UCN) , tRNA Glu , tRNA Pro ).
Twenty-two tRNA genes were interspersed among the rRNA and PCGs, ranged in size from 66 bp (tRNA Cys ) to 76 bp (tRNA Lys ). There were two forms of tRNA Ser (UCN and AGY) and two forms of tRNA Leu (UUR and CUN). 12S rRNA (954 bp) and 16S rRNA (1676 bp) were located between tRNA Phe and tRNA Leu and separated by the tRNA Val gene.
There were two major noncoding regions. The origin of light-strand replication, between the tRNA Asn and tRNA Cys , was 31 bp fragment and has the potential to fold into a stem-loop secondary structure. The displacement loop locus (control region) was 916 bp sequence located between the tRNA Pro and tRNA Phe genes, with a high AT content of 65.07%. There were lesser AT repeats than Cyprinidae fishes in D-Loop. There were 11 regions of gene overlaps totaling 32 bp (varying from 1 to 13 bp) and 12 intergenic spacer regions totaling 47 bp (varying from 1 to 13 bp).
To confirm the phylogenetic location of H. pycnolepis within the superfamily of Cobitidea, Thirty-two complete mitogenomes of Cobitidae were obtained from GenBank, and two Macrognathus fishes in the family of Mastacembelidae was used as out-group. Phylogenetic analysis of 35 complete mitogenomes was conducted based on maximum likelihood (ML) analyses implemented in Mega-X (Kumar et al. 2018). Phylogenetic analysis results supported that 4 Homatula species (5 sequences) clustered as one monophyletic clade and Homatula was closely related to Schistura (Figure 1).
The mitogenome sequence data of H. pycnolepis presented in this work would provide the fundamental genetic data for inferring phylogenetic relationships of Homatula, further study on conservation genetic studies for this species.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the Projects of Sichuan Provincial Science and Technology Department [No. 2019ZYZF0099, 2019YJ0498, and 2019YJ0397].

Data availability statement
The data that support the findings of this study are openly available in GenBank of NCBI at https://www.ncbi.nlm.nih.gov, reference number MT783421.