The complete mitogenome can provide valuable genetic information to reconstruct the relationship network between species. In this study, we sequenced a freshwater loach, Homatula laxiclathra (Teleostei: Nemacheilidae), which is only distributed in the northern region in Qinling Mountains, China. The size of H. laxiclathra mitogenome is 16,570 bp, contains 37 typical mitochondrial genes including 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and a control region (D-loop) with the total A+T content of 55.8%, which is similar to other Nemacheilidae sequences published in GenBank. Furthermore, the mito-phylogenomic analysis of 46 Nemacheilidae species arranged H. laxiclathra in a robust monophyletic Homatula cluster with other Homatula species. The results may contribute to understand a true phylogeny with large-scale taxonomic samplings and grasp the evolution of fish mitogenomes.

Sample collection and DNA extraction

Adult specimens of Homatula laxiclathra (Gu et Zhang, 2012) were collected from Xinguansi (33.98°N, 109.11°E), Chang’an County, the Dayu River located on the north slope of the Adult specimens of Homatula laxiclathra (Gu et Zhang, 2012) were collected from Xinguansi (33.98°N, 109.11°E), Chang’an County, the Dayu River located on the north slope of the Adult specimens of Homatula laxiclathra (Gu et Zhang, 2012) were collected from Xinguansi (33.98°N, 109.11°E), Chang’an County, the Dayu River located on the north slope of the Qinling Mountains of Shaanxi Province, Central China (Figure 1). All these specimens were preserved in 95% ethanol. The animal processing was approved by the Animal Care and Use Committee of Shaanxi Normal University. Total genomic DNA was extracted from muscle tissues using a TIANamp Animal DNA Kit (Tiangen Biotech, Beijing, China), according to the manufacturer's protocol. Voucher specimens deposited in the Fish Disease Laboratory, Shaanxi Normal University (Accession number: HL20160124).

PCR amplification and sequencing

By using a primer-walking strategy, thirty conseved fish primers were designed to amplify the mitogenome (Miya & Nishida, 1999). PCR amplification were performed with FastPfu Fly DNA polymerase (TransGen biotech, Beijing, China), following the published reaction conditions (Zhu et al., 2018).

Genome annotaton and sequence analysis

Raw sequences were assembled using Staden Package v1.7.0 (Staden et al., 2000). Then, gene predictions were comparing with published mitogenomes of Homatula fishes. PCGs and rRNAs were identified through DOGMA with default settings (Wyman et al., 2004). All of tRNA genes and their secondary structures were verified with tRNA-scan SE (Lowe & Eddy, 1997). The secondary structure of tRNA genes and O_L were drawn by RNAstructure 6.1 and modified by SturctureEditor (Mathews, 2014). MEGA 7 was used to calculate the relative synonymous codon usage (RSCU) and base composition of each gene (Kumar et al., 2016). The nucleotide composition skew value of 13 PCGs were counted by the formulas: (AT-skew = [A-T]/[A+T], GC-skew = [G-C]/[G+C]) (Perna & Kocher, 1995). The complete sequence and the annotation were performed by MitoFish, including a graphic circular map (Iwasaki et al., 2013).

Phylogenetic analysis

A total of 43 GenBank-retrieved mitogenones of species from the Nemacheilidae were used to reconstucted phylogenetic relationship (Table 1). Two species from the Cyprinidae (Hemibarbus labeo GenBank: DQ347953, Hemibarbus longirostris GenBank: DQ347952) was selected as outgroups. The nucleotide sequences of 12 PCGs were aligned separately by MEGA 7 with default setting. ND6 gene was excluded for phylogenetic analysis on account of high heterogeneity (Miya et al., 2003). The 12 PCGs were concatenated to a combination sequence without termination codon due to the degeneracy. Maximum likelihood (ML) and Bayesian inference (BI) analysis were used for phylogenetic relationship among the Nemacheilidae (Ronquist et al., 2012; Kumar et al., 2016). The two phylogenetic trees were modified by FigTree v1.4.3 (Vlad et al., 2008).

GenBank under accession number MK279351