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

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.

INTRODUCTION

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 (OL 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 is only distributed in the northern region in Qinling Mountains. The genome structure and gene characterization of H. laxiclathra are compared with those reported for other Homatula species. To assess the deeper phylogenetic relationships of Nemacheilidaes, we reconstructed the tree using Maximum Likelihood (ML) and Bayesian inference (BI) methods. The investigation of the H. laxiclathra mitogenome may provide valuable evidence about teleost evolution as well as aid in species identification.

MATERIALS AND METHODS

Sample collection and DNA extraction

Adult specimens of Homatula laxiclathra Gu & 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). Specimens were preserved in 95% ethanol. 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), according to the manufacturer's protocol. Voucher specimens deposited in the Fish Disease Laboratory, Shaanxi Normal University (Accession number: HL20160124).

FIGURE 1

Map of sampling localities of Homatula laxiclathra. The map was downloaded from the Wikimedia Commons with slight modification (https://commons.wikimedia.org/wiki/File:East_Asia_topographic_map.png)

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 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 OL 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: (AT‐skew = [A − T]/[A + T], GC‐skew = [G − C]/[G + C]) (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 (Table 1). Two species from the Cyprinidae (Hemibarbus labeo GenBank: DQ347953, Hemibarbus longirostris GenBank: DQ347952) were selected as outgroups. Nucleotide sequences of 12 PCGs were aligned separately by MEGA 7 using the default setting. The ND6 gene was excluded for phylogenetic analysis due to a high degree of heterogeneity (Miya et al., 2003). The 12 PCGs were concatenated to a combination sequence without termination codon due to a high degree of degeneracy. Maximum likelihood (ML) and Bayesian inference (BI) analyses were used to define phylogenetic relationships among the Nemacheilidae (Kumar et al., 2016; Ronquist et al., 2012). The phylogenetic trees were modified by FigTree v1.4.3 (Vlad, Balaji, Vikas, Ramani, & Larry, 2008).

TABLE 1

Species mentioned in this study with GenBank accession number

Family	Species	Size (bp)	Whole genome composition				Accession number
			A%	C%	G%	T%
Nemacheilidae	Acanthocobitis botia	16,660	30.5	26.5	15.9	27.1	AP012138
	Acanthocobitis zonalternans	16,642	30.1	27.1	16.5	26.4	AP012140
	Barbatula barbatula	16,630	28.5	27.1	18.2	26.2	KP715096
	Barbatula nuda	16,619	28.4	27.2	17.9	26.5	KF574248
	Barbatula toni	16,617	28.5	27.3	17.8	26.4	AB242162
	Homatula potanini	16,569	30.1	26.9	16.7	26.3	KM017732
	Homatula variegata	16,571	29.5	27.1	17.3	26.1	JX144893
	Homatula laxiclathra	16,570	29.6	27.0	17.2	26.1	MK279351
	Lefua costata	16,579	29.9	26.5	16.8	26.9	KT943751
	Lefua echigonia	16,559	30.7	24.8	16.1	28.4	AB054126
	Lefua nikkonis	16,589	29.9	26.3	16.7	27.1	AP011300
	Oreonectes furcocaudalis	16,569	31.1	29.5	12.9	26.5	KX778472
	Oreonectes platycephalus	16,580	30.2	26.9	16.1	26.8	AP011296
	Schistura balteata	16,564	31.7	27.0	15.3	26.0	AB242172
	Schistura corica	16,572	29.7	26.6	17.3	26.4	AP011445
	Schistura fasciolata	16,560	30.9	26.9	16.2	26.1	KY404236
	Schistura geisleri	16,819	30.0	28.2	17.0	24.9	AP013295
	Schistura jarutanini	16,594	30.3	28.3	16.9	24.4	AP011307
	Schistura kaysonei	16,575	30.6	28.2	16.5	24.8	AP011297
	Schistura notostigma	16,568	29.8	27.9	17.1	25.2	AP011308
	Schistura pridii	16,576	30.8	28.4	16.3	24.5	AP011443
	Schistura reticulofasciata	16,603	30.8	27.7	16.5	25.0	KY379150
	Schistura scaturigina	16,585	30.8	27.0	16.4	25.8	KU380330
	Schistura sikmaiensis	16,581	33.8	21.1	13.5	31.6	KY379151
	Triplophysa anterodorsalis	16,567	27.4	25.7	18.4	28.6	KJ739868
	Triplophysa bleekeri	16,568	27.1	25.8	18.5	28.6	JX135578
	Triplophysa dorsalis	16,572	26.9	26.1	16.1	30.9	KT241024
	Triplophysa lixianensis	16,570	27.8	25.4	18.4	28.5	KT966735
	Triplophysa orientalis	16,562	27.4	25.5	18.7	28.5	KJ631323
	Triplophysa pappenheimi	16,572	28.2	25.4	18.1	28.3	KY419201
	Triplophysa robusta	16,570	28.2	25.3	18.0	28.4	KM406486
	Triplophysa rosa	16,585	31.8	25.3	15.6	27.3	JF268621
	Triplophysa siluroides	16,574	28.8	25.0	17.5	28.7	KJ781206
	Triplophysa stenura	16,569	27.8	25.4	18.4	28.4	KX354975
	Triplophysa stewarti	16,567	27.8	25.4	18.4	28.4	KJ631324
	Triplophysa stoliczkai	16,571	28.1	25.2	17.9	28.8	JQ663847
	Triplophysa strauchii	16,590	28.3	25.4	17.8	28.5	KP297875
	Triplophysa tenuis	16,571	27.5	25.7	18.6	28.2	KT224363
	Triplophysa tibetana	16,574	26.9	25.6	19.1	28.3	KT224364
	Triplophysa venusta	16,574	27.8	26.9	18.4	26.9	KT008666
	Triplophysa wuweiensis	16,681	28.0	25.7	18.1	28.2	KT224365
	Triplophysa xiangxiensis	16,598	30.8	26.3	16.0	26.8	KT751089
	Triplophysa xichangensis	16,570	28.6	25.3	17.6	28.6	KT224366
	Triplophysa yarkandensis	16,574	31.9	30.4	17.4	20.3	KP050360
Cyprinidae	Hemibarbus labeo	16,612	29.7	27.1	17.2	26.0	DQ347953
	Hemibarbus longirostris	16,608	27.7	27.2	18.7	26.3	DQ347952

RESULTS AND DISCUSSION

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 (Table 2). Nearly, all the genes are transcribed on the heavy strand, whereas ND6 and eight tRNA genes are located on the light strand. The structure and composition of H. laxiclathra is identical to other mitogenomes of nemacheilids to date (Vlad et al., 2008). The nucleotide composition of the H. laxiclathra mitogenome has a gently biased A + T content for 55.7%. The overall base composition of H. laxiclathra is the following: A, 29.6%; T, 26.1%; C, 27.0%; G, 17.2%. The overall AT‐ and GC‐skew of H. laxiclathra mitogenome are −0.013 and −0.233S. The nucleotide frequency of each protein‐coding gene is A + T > C + G, respectively, showing a strong AT bias (Table 3). For analyses within the genus, the same information from H. potanini and H. variegata was calculated. H. potanini showed the highest A + T frequency at 56.4% with H. variegata and H. laxiclathra having the most robust AT‐skew. The whole mitogenome base composition of H. variegata is highly similar to H. laxiclathra with A for 29.5%, T for 26.1%, C for 27.1%, and G for 17.3%, suggesting they share a deep homology.

FIGURE 2

Mitogenome map of Homatula laxiclathra, generated from MitoFish (Iwasaki et al., 2013)

TABLE 2

Annotation of mitochondrial genome of Homatula laxiclathra

Gene	Position From‐to	Length (bp)	Intergenic length	Strand	Start codon	Stop condon
tRNAPhe	1–69	69		H
12SrRNA	70–1,020	951	0	H
tRNAVal	1,021–1,092	72	0	H
16SrRNA	1,093–2,764	1672	0	H
tRNALeu(UUR)	2,765–2,839	75	0	H
ND1	2,840–3,814	975	0	H	ATG	TAA
tRNAIle	3,822–3,893	72	7	H
tRNAGln	3,892–3,962	71	−2	L
tRNAMet	3,964–4,032	69	1	H
ND2	4,033–5,077	1,045	0	H	ATG	T
tRNATrp	5,078–5,147	70	0	H
tRNAAla	5,150–5,218	69	2	L
tRNAAsn	5,220–5,292	73	1	L
tRNACys	5,323–5,388	66	30	L
tRNATyr	5,388–5,456	69	−1	L
COI	5,458–7,008	1551	1	H	GTG	TAA
tRNASer(UCN)	7,009–7,079	71	0	L
tRNAAsp	7,083–7,155	73	3	H
COII	7,169–7,859	691	13	H	ATG	T
tRNALys	7,860–7,935	76	0	H
ATP8	7,937–8,104	168	1	H	ATG	TAA
ATP6	8,095–8,777	683	−10	H	ATG	TA
COIII	8,778–9,561	784	0	H	ATG	T
tRNAGly	9,562–9,634	73	0	H
ND3	9,635–9,983	349	0	H	ATG	T
tRNAArg	9,984–10,053	70	0	H
ND4L	10,054–10,350	297	0	H	ATG	TAA
ND4	10,344–11,725	1,382	−7	H	ATG	TA
tRNAHis	11,726–11,795	70	0	H
tRNASer(AGY)	11,807–11,862	56	11	H
tRNALeu(CUN)	11,864–11,936	73	1	H
ND5	11,937–13,775	1839	0	H	ATG	TAA
ND6	13,772–14,293	522	−4	L	ATG	TAG
tRNAGlu	14,294–14,362	69	0	L
Cytb	14,367–15,507	1,141	4	H	ATG	T
tRNAThr	15,508–15,579	72	0	H
tRNAPro	15,578–15,647	70	−2	L
D‐loop	15,648–16,570	923	0	H

TABLE 3

The AT‐ and GC‐skew in the PCGs of Homatula laxiclathra

Gene	Nucleotide frequency (%)				A + T (%)	AT‐skew	CG‐skew
	A	T	C	G
ATP6	28.7	30.2	27.1	14.1	58.9	−0.025	−0.316
ATP8	31.0	26.8	26.2	16.1	57.8	0.073	−0.239
COI	24.6	29.4	25.9	20.2	54.0	−0.089	−0.124
COII	29.2	25.8	28.4	16.6	55.0	0.062	−0.262
COIII	26.8	26.0	28.8	18.4	52.8	0.015	−0.220
ND1	27.9	27.7	29.6	14.8	55.6	0.004	−0.333
ND2	31.3	24.0	31.0	13.7	55.3	0.132	−0.387
ND3	25.2	30.1	27.2	17.5	55.3	−0.089	−0.217
ND4	27.8	26.9	29.2	16.1	54.7	0.016	−0.289
ND4L	22.6	29.0	31.0	17.5	51.6	−0.124	−0.278
ND5	30.1	27.4	28.2	14.4	57.5	0.047	−0.324
ND6	40.2	16.1	30.8	12.8	56.3	0.428	−0.413
Cytb	28.0	28.4	27.4	16.1	56.4	−0.007	−0.260
12SrRNA	29.9	19.9	27.3	22.9	49.8	0.201	−0.088
16SrRNA	35.8	20.5	23.2	20.5	56.3	0.272	−0.062
D‐Loop	32.5	33.6	19.5	14.4	66.1	−0.017	−0.150
Total	27.3	28.1	26.9	16.8	55.8	−0.013	−0.233

Protein‐coding genes

The 13 PCGs of H. laxiclathra are similar in component and length to other familial fishes, ranging from 168 bp for ATP8 to 1839 bp for ND5. All these PCGs are coded by the heavy strand except ND6 which is coded by the light strand (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 (Table 4). Almost all codons are present in H. laxiclathra except for the specific mammals stop codons AGA and AGG. The most common amino acids in protein‐coding genes are leucine (463), alanine (331), and threonine (298). Leucine was coded by CUA (213) in H. laxiclathra PCGs, the same as in H. variegata and H. potanini. GCC (147) and GCA (129) are shared equally, coding for alanine, and the same trend is shown by threonine: ACC (120) and ACA (129).

TABLE 4

Relative synonymous condon usage (RSCU) in all proteins of Homatula laxiclathra

Codon	n (RSCU)	Codon	n (RSCU)	Codon	n (RSCU)	Codon	n (RSCU)
UUU(F)	108 (1)	UCU(S)	32 (0.81)	UAU(Y)	41 (0.78)	UGU(C)	8 (0.64)
UUC(F)	108 (1)	UCC(S)	61 (1.55)	UAC(Y)	64 (1.22)	UGC(C)	17 (1.36)
UUA(L)	108 (1.1)	UCA(S)	85 (2.16)	UAA(*)	0 (0)	UGA(W)	89 (1.53)
UUG(L)	16 (0.16)	UCG(S)	7 (0.18)	UAG(*)	0 (0)	UGG(W)	27 (0.47)
CUU(L)	94 (0.96)	CCU(P)	38 (0.73)	CAU(H)	25 (0.48)	CGU(R)	8 (0.45)
CUC(L)	94 (0.96)	CCC(P)	67 (1.28)	CAC(H)	79 (1.52)	CGC(R)	10 (0.56)
CUA(L)	213 (2.18)	CCA(P)	88 (1.68)	CAA(Q)	78 (1.58)	CGA(R)	47 (2.65)
CUG(L)	62 (0.63)	CCG(P)	16 (0.31)	CAG(Q)	21 (0.42)	CGG(R)	6 (0.34)
AUU(I)	175 (1.24)	ACU(T)	35 (0.47)	AAU(N)	41 (0.74)	AGU(S)	8 (0.2)
AUC(I)	107 (0.76)	ACC(T)	120 (1.61)	AAC(N)	70 (1.26)	AGC(S)	43 (1.09)
AUA(M)	118 (1.4)	ACA(T)	129 (1.73)	AAA(K)	61 (1.61)	AGA(*)	0 (0)
AUG(M)	50 (0.6)	ACG(T)	14 (0.19)	AAG(K)	15 (0.39)	AGG(*)	0 (0)
GUU(V)	53 (1.03)	GCU(A)	43 (0.52)	GAU(D)	21 (0.58)	GGU(G)	34 (0.61)
GUC(V)	37 (0.72)	GCC(A)	147 (1.78)	GAC(D)	51 (1.42)	GGC(G)	47 (0.85)
GUA(V)	92 (1.8)	GCA(A)	129 (1.56)	GAA(E)	69 (1.41)	GGA(G)	85 (1.53)
GUG(V)	23 (0.45)	GCG(A)	12 (0.15)	GAG(E)	29 (0.59)	GGG(G)	56 (1.01)

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 (Lee & Kocher, 1995). In addition, some tRNAs showed mismatched pairs in stems (e.g. U‐G and A‐C in the acceptor arm of tRNA‐Arg for three Homatula species). These conserved mismatched pairs may be similar to the molecular synapomorphy for the genus. The length of 12S rRNA and 16S rRNA of H. laxiclathra were 951 bp and 1672 bp, respectively. The values were similar with H. potanini and H. variegata, falling well into the size range in fishes. The A + T contents of the 12S rRNA and 16S rRNA of H. laxiclathra were 49.8% and 56.3%, respectively, thus indicating some diversity in nucleotide distribution. Both 12S rRNA and 16S rRNA had a positive AT‐skew (0.201 and 0.272), and a negative GC‐skew (−0.088 and −0.062) at the same locations on the heavy strand. Similar to other Nemacheilidae species, 12S rRNA was located between tRNA‐Phe and tRNA‐Val and the 16S rRNA was located between tRNA‐Val and tRNA‐Leu.

FIGURE 3

Secondary structures of transfer RNA genes in Homatula laxiclathra, generated from RNAstructure 6.1 and SturctureEditor (Mathews, 2014)

Noncoding regions

The mitogenome of H. laxiclathra has two noncoding regions, the D‐loop and OL. The 923 bp D‐loop is located between tRAN‐Pro and tRNA‐Phe with 66.1% A + T content (Figure 4). The OL is 30 bp in length, located in the WANCY region between tRNA‐Asn and tRNA‐Cys with a putative hairpin structure (Figure 5). The D‐loop region is complex and highly variable and can determine the replication pattern of the mitogenome (Liu, Zhang, Tang, Yu, & Zhou, 2010).

FIGURE 4

Sequence of D‐loop in Homatula laxiclathra with CSBs marked

FIGURE 5

The stem‐loop secondary of OL of Homatula laxiclathra, generated from RNAstructure 6.1 and SturctureEditor (Mathews, 2014)

By comparing with other Nemacheilidae, the D‐loop can be divided into three functional segments, the termination associated sequence (TAS‐1 and TAS‐2), the central conserved sequence block (CSB‐D, CSB‐E, and CSB‐F) and the conserved sequence block (CSB‐1, CSB‐2, and CSB‐3). The termination associated sequence varies markedly among different lineages, although it can play vital roles in determining the fate of the heavy strand. The core conserved sequence TACAT and complementary sequence ATGTA were detected in TAS, folded into a stable hairpin structure. Two poly‐T stretches and a conserved motif (TA)5 were found by comparing against other fishes. Significant tandem repeats were not recognized in the H. laxiclathra D‐loop.

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). Both phylogenetic trees consistently showed three major clades, including (I) Acanthocobitis and Schistura, (II) Oreonectes and Lefua, (III) Homatula, Barbatula, and Triplophysa. All the congeneric species represented a single cluster for each genus (Acanthocobitis, Homatula, Barbatula, Lefua, Oreonectes, Schistura, and Triplophysa), and, the relationship of the Nemacheilidae was consistent with other phylogenetic and morphological studies on these species (Prokofiev, 2010; Stout, Tan, Lemmon, Lemmon, & Armbruster, 2016). Thus, Homatula was shown to be valid as an inherent Asian fish group according to where the genus falls out on both trees. Further, Homatula shares a close ancestor with Oreonectes and Lefua making it a sister group. 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.

FIGURE 6

Phylogenetic relationships among Nemacheilidae, generated from MEGA 7 and MrBayes 3.2.7 (Kumar et al., 2016; Ronquist et al., 2012)

CONCLUSIONS

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.

CONFLICT OF INTEREST

The authors declare that they have no competing interests.

AUTHOR CONTRIBUTION

Mengfei Cao: Data curation (equal); Formal analysis (equal); Investigation (equal); Resources (equal); Software (equal); Writing‐original draft (equal); Writing‐review & editing (equal). Ling Tang: Data curation (equal); Investigation (equal); Resources (equal). Juan Chen: Data curation (equal); Formal analysis (equal); Investigation (equal); Resources (equal). Xiaoyu Zhang: Data curation (equal); Resources (equal). Russell H. Easy: Writing‐review & editing (equal). Ping You: Conceptualization (equal); Funding acquisition (equal); Investigation (equal); Project administration (equal); Supervision (equal); Writing‐original draft (equal); Writing‐review & editing (equal).