The mitogenome of freshwater loach Homatula laxiclathra (Teleostei: Nemacheilidae) with phylogenetic analysis of Nemacheilidae

Abstract The complete mitogenome can provide valuable genetic information to reconstruct relationships between species. In this study, we sequenced a stone loach, Homatula laxiclathra (Teleostei: Nemacheilidae), which is found in the northern region of the Qinling Mountains in China. The size of the H. laxiclathra mitogenome is 16,570 bp, which contains 37 typical mitochondrial genes including 13 protein‐coding genes, 22 transfer RNAs, two ribosomal RNAs, and a control region (D‐loop) with a total AT content of 55.8%. This is similar to other Nemacheilidae sequences published in GenBank. Furthermore, a mito‐phylogenomic analysis of 46 Nemacheilidae species places H. laxiclathra in a robust monophyletic Homatula cluster with other Homatula species. Our results contribute toward a better understanding of a true phylogeny of these species based on large‐scale taxonomic samplings as well as to help grasp the evolution of fish mitogenomes.


| INTRODUC TI ON
Mitochondrial DNA can provide valuable taxon information to reconstruct evolutionary relationships between species. The fish mitogenome is circular, 15-19 k bp in size, and comprises 13 protein-coding genes (PCGs), two ribosomal RNA genes (12S rRNA and 16S rRNA), 22 transfer RNA genes (tRANs) and two noncoding control regions (O L and CR) (Miya, Kawaguchi, & Nishida, 2001).
Mitogenomes are widely used for molecular systematics, phylogeography and taxa identification due to their small and simple structure, rapid evolution, maternal inheritance, and high gene conservation (Boore, 1999). In addition, molecular data for mtDNA, such as secondary structure of tRNAs and rRNAs, amino acid sequence, and codon usage can provide additional data for phylogenetic analyses (Boore, 1999;Zhu, Yan, Song, & You, 2018).
Loaches are small-bodied freshwater fishes, which are widely distributed across Eurasia, Africa, and North America. They are popular in China due to their distinctive flavor and diverse body color.
From a commercial fisheries and ornamental trade value, it is crucial to identify mtDNA mutations to avoid genetic diseases in these fish (Kipp et al., 2010). Partial mtDNA genes from the Nemacheilidae have been used for species identification and systematics (Liu et al., 2012). Unfortunately, partial mitochondrial genes do not contain complete phylogenetic information to accurately define a phylogeny (Cunha, Grande, & Zardoya, 2009;Lee, Conroy, Howel, & Kocher, 1995;Parhi, Tripathy, Priyadarshi, Mandal, & Pandey, 2019).
An effective solution is to conduct comparisons of whole mtDNA from representative species of each genus (Betancur et al., 2017;Shi, Xing, Chen, Yang, & You, 2014). So far, 207 complete mitogenomes of teleostean species have been published in the GenBank database, but only 56 species from Nemacheilidae are available.
In this study, we sequenced the complete mitogenome of Homatula laxiclathra Gu & Zhang, 2012, which

| PCR amplification and sequencing
Using a primer-walking strategy, thirty conserved fish primers were designed to amplify the mitogenome (Miya & Nishida, 1999). PCR amplifications were performed with FastPfu Fly DNA polymerase (TransGen Biotech), following published PCR reaction conditions (Zhu et al., 2018).

| Genome annotation and sequence analysis
Raw sequences were assembled using the Staden Package v1.7.0 (Staden, Beal, & Bonfield, 2000). Gene predictions were compared with published mitogenomes of Homatula fishes. PCGs and rRNAs were TA B L E 1 Species mentioned in this study with GenBank accession number identified through DOGMA using default settings (Wyman, Jansen, & Boore, 2004). All tRNA genes and their secondary structures were verified with tRNA-scan SE (Lowe & Eddy, 1997). The secondary structure of tRNA genes and O L was 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, Stecher, & Tamura, 2016). Nucleotide composition skew values of 13 PCGs were counted by the formulas: (Perna & Kocher, 1995). The complete sequence and annotation were constructed using MitoFish, including a graphic circular map (Iwasaki et al., 2013).

| Phylogenetic analysis
A total of 43 GenBank-retrieved mitogenomes of species from the Nemacheilidae was used to reconstruct phylogenetic relationships (

| Mitochondrial genomic structure and composition
The complete mitogenome of H. laxiclathra is a circular molecule of 16,570 bp ( Figure 2) and is deposited in the GenBank database under accession numbers MK279351. It consists of 37 typical genes, including 13 protein-coding genes (PCGs), 22 transfer RNA genes, two rRNA genes, and a noncoding region (

| Protein-coding genes
The 13 (Miya & Nishida, 2000). Similar to other loaches, the COI gene has a GTG start codon, whereas other twelve PCGs start with ATG. Five PCGs end with complete termination codon TAA and others with T-or TA-. The total length of 13 PCGs is 11,441 bp, which contain 12 intergenic spacers, the smallest spacer is only 1 bp in size, whereas the longest spacer can be up to 30 bp located between tRNA (Asn) and tRNA (Cys). There are six overlaps ranging from 1 to 10 bp, and the longest region is located between ATP8 and ATP6.
Among the 13 protein-coding genes, ATP6 showed the highest A + T content with 58.9% and COIII at the lowest A + T content with 52.8%.
Codon usage and relative synonymous codon usage (RSCU) of the H. laxiclathra mitogenome is summarized (

TA B L E 3
The AT-and GC-skew in the PCGs of Homatula laxiclathra GCC (147) and GCA (129) are shared equally, coding for alanine, and the same trend is shown by threonine: ACC (120) and ACA (129).

| Transfer RNA genes and ribosomal RNA genes
All 22 tRNA genes are found in the mitogenome of H. laxiclathra.
Comparative analysis on potential secondary structures of H. laxiclathra tRNAs is shown (Figure 3). Fourteen tRNAs were located on the heavy strand whereas the other tRNAs were on the light strand. The length of all tRNAs was similar, ranging from 56 bp to 75 bp. Nearly, all tRNA genes were predicted to have typical cloverleaf structures, with the exception of tRNA-Ser (AGN) which lacked a stable DHU stem (Figure 2). This missing stem occurs in most teleost mitogenomes as previously reported . In addition, some tRNAs showed mismatched pairs in stems (e.g. U-G and A-C in the acceptor arm F I G U R E 3 Secondary structures of transfer RNA genes in Homatula laxiclathra, generated from RNAstructure 6.1 and SturctureEditor (Mathews, 2014) of tRNA-Arg for three Homatula species). These conserved mis-

| Noncoding regions
The mitogenome of H. laxiclathra has two noncoding regions, the

| Phylogenetic analysis
Phylogenetic relationships of the Nemacheilidae were reconstructed using two methods, Bayesian inference (BI) and maximum likelihood (ML) (Figure 6). Twelve PCGs from 41 nemacheilid species were concatenated to a matrix and used for phylogenetic analyses; two Cyprinidae species were selected as the outgroups.
The phylogenetic trees generated a similar topology that confirmed the findings from a previous study for loach classification (Sgouros, Page, Orlofske, & Jadin, 2019 The topology also demonstrated monophyly of three Lefua species (Miyazaki et al., 2011). This molecular information provides a more robust data set to support fish classification and identification. In addition, several related articles adapt various standards to classify species, such as phylogeny based on single mitogenome genes or nuclear genes (Liu et al., 2012;Powell, Barker, & Lanyon, 2013;Tang, Liu, Mayden, & Xiong, 2006). Our results are based on the highest coverage of Nemacheilidae mitogenomic data to date and provide an updated view of Nemacheilidae phylogeny.

| CON CLUS IONS
In this study, we present the complete mitogenome of Homatula laxiclathra and provide a comparison of this sequence against other Homatula species to examine the architecture of mitogenome structure, the location of coding genes, and codon usage. The results integrate updated mitogenomic information of the Nemacheilidae and generate a new phylogeny and relationship among different genera of these fishes. However, many genus-level taxonomy studies lack robust molecular data and thus the true phylogeny of the loach remains unresolved.

ACK N OWLED G M ENTS
The authors would like to thank Fe Ye, Jun Yan, and Tao Chen

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.