Complete mitochondrial genome of Belligobio pengxianensis (Cypriniformes: Gobionidae)

Abstract

Belligobio pengxianensis is a small fish endemic to the upper Yangtze River of China. In this study, the complete mitochondrial genome of B. pengxianensis is determined for the first time, and it should become a reference sequence to aid in species identification, biodiversity monitoring and conservation. The mitogenome has overall length of 16,610 bp and AT content of 55.23%, including 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, and one non-coding control region. The results of phylogenetic analyses show that B. pengxianensis is nested within the genus Hemibarbus.

Keywords:

• Cypriniformes
• Gobionidae
• upper Yangtze River
• Hemibarbus
• Belligobio pengxianensis

1. Introduction

Fishes of the family Gobionidae (Teleostei: Cypriniformes) include about 28 genera and 226 species widely distributed in drainages across East Asia and Europe (Fricke et al. 2022). The gobionid fishes can be divided into three major clades—the Hemibarbus-Squalidus clade, Sarcocheilichthyinae, and Gobioninae—where the Hemibarbus-Squalidus clade is composed of the three genera Belligobio, Hemibarbus, and Squalidus (Tan and Armbruster 2018). Belligobio is comprised of three species: B. eristigma Jordan & Hubbs 1925, B. nummifer (Boulenger, 1901), and B. pengxianensis Lu, Luo & Chen 1977 (Bănărescu 1992; Yue 1998).

B. pengxianensis (Figure 1) is a small (standard length of less than 13 cm) fish (Yue 1998; Li 2020). In the very few studies (Ding 1994; Wang et al. 2017), it was considered to be rare, on the basis of its known records only in the Jianjiang River (a tributary of the upper Yangtze River). In a recent study (Li 2020), B. pengxianensis has been found to also be distributed in the Fujiang River (a tributary of the upper Yangtze River) based on molecular species delimitation using the mitochondrial cytochrome b gene and eight nuclear loci. Li (2020) has also indicated that B. pengxianensis and B. nummifer could not be differentiated by previously defined diagnostic characteristics (Yue 1998), such that prior records on B. nummifer in multiple tributaries of the upper Yangtze River (Ding 1994; Lei et al. 2015; Chen et al. 2017) might be due to misidentification of B. pengxianensis. Therefore, the ambiguities in species identification between B. pengxianensis and B. nummifer, based on morphological characteristics, require molecular evidence to delimit species boundaries.

Figure 1. Female B. pengxianensis from Tongji Town, Pengzhou City, Sichuan Province, China. Photographed by Cuizhang Fu on September 12, 2015.

In this study, the complete mitochondrial genome of B. pengxianensis is determined for the first time. The mitogenome should become a reference sequence to aid in molecular species identification, biodiversity monitoring and conservation, and is also useful in clarifying phylogenetic position of B. pengxianensis.

2. Materials

The specimen of B. pengxianensis was collected in a dead state from Tongji Town, Pengzhou City, Sichuan Province, China (31.1611°N, 103.8394°E) on 12 September 2015. We obtained the sample from the fish catches of a local fisherman. It was placed in the Zoological Museum of Fudan University under the voucher number FDZM-BPPZ20150912-01 (Cuizhang Fu, [email protected]).

3. Methods

Total genomic DNA was extracted from muscle tissues using a high-salt method (Miller et al. 1988). Thirteen primer pairs were used to amplify DNA fragments, with the same PCR system and settings as Chai and Fu (2020). The forward primer of Primer4, reverse primer of Primer6 as well as primers of Primer12 were newly designed in this study (supplemental Table S1 in Appendix I), while the remaining primers have been previously published (Chai and Fu, 2020). A total of 13 segments were obtained through Sanger sequencing using the ABI 3730 platform (Applied Biosystems, Foster City, USA). Using a reference genome from B. nummifer (KJ413052; Wang et al. 2016), contiguous and overlapping sequences were assembled into mitochondrial genome by Sequencher 5.4 (Gene Codes, Ann Arbor, MI), based on the default parameters. Then, the assembled mitogenome was annotated by MitoAnnotator (Iwasaki et al. 2013; http://mitofish.aori.u-tokyo.ac.jp/annotation/input.html). The genome map was drawn using the CGView Server (Grant and Stothard 2008).

Table 1. Characteristics of B. pengxianensis mitogenome.

Element	From	To	Length (bp)	Start codon	Stop codon	Strand*
tRNA^Phe	1	69	69			H
12S rRNA	70	1028	959			H
tRNA^Val	1029	1100	72			H
16S rRNA	1101	2792	1692			H
tRNA^Leu	2793	2868	76			H
ND1	2870	3844	975	ATG	TAA	H
tRNA^Ile	3849	3920	72			H
tRNA^Gln	3919	3989	71			L
tRNA^Met	3991	4059	69			H
ND2	4060	5104	1045	ATG	T--	H
tRNA^Trp	5105	5175	71			H
tRNA^Ala	5178	5246	69			L
tRNA^Asn	5248	5320	73			L
tRNA^Cys	5352	5419	68			L
tRNA^Tyr	5421	5491	71			L
COX1	5493	7043	1551	GTG	TAA	H
tRNA^Ser	7045	7113	71			L
tRNA^Asp	7118	7189	72			H
COX2	7203	7893	691	ATG	T--	H
tRNA^Lys	7894	7969	76			H
ATPase8	7971	8135	165	ATG	TAA	H
ATPase6	8129	8812	684	ATG	TAA	H
COX3	8812	9595	784	ATG	T--	H
tRNA^Gly	9596	9668	73			H
ND3	9669	10017	349	ATG	T--	H
tRNA^Arg	10018	10087	70			H
ND4L	10088	10384	297	ATG	TAA	H
ND4	10378	11760	1382	ATG	TA-	H
tRNA^His	11760	11828	69			H
tRNA^Ser	11829	11897	69			H
tRNA^Leu	11899	11971	73			H
ND5	11972	13807	1836	ATG	TAG	H
ND6	13804	14325	522	ATG	TAA	L
tRNA^Glu	14326	14394	69			L
Cytb	14399	15539	1141	ATG	T--	H
tRNA^Thr	15540	15611	72			H
tRNA^Pro	15611	15680	70			L
D-loop	15681	16610	930			H

*H: gene is transcribed from the heavy strand; L: gene is transcribed from the light strand.

Bănărescu (1992) has treated Belligobio as a sub-genus of Hemibarbus, while Tang et al. (2011) have suggested that Belligobio is a junior synonym of Hemibarbus. Therefore, our phylogenetic analyses included eight mitochondrial genomes from GenBank as follows: B. nummifer (KJ413052; Wang et al. 2016), Hemibarbus medius (KF640234; Chen et al. 2015), H. umbrifer (AP011415; Tang et al. 2011), H. maculatus (JF906109; Li et al. 2012), H. labeo (DQ347953; Kim et al. 2009), H. longirostris (DQ347952; Kim et al. 2009), Squalidus chankaensis (MT767746; Chai and Fu, 2020) and S. multimaculatus (KX495606; Yi et al. 2017). Our phylogeny was inferred using maximum likelihood (ML) and Bayesian (BI) methods based on 13 protein-coding genes or complete mitochondrial genomes, which the analyses of ML and BI were performed on the software IQ-TREE v2.1.3 (Minh et al. 2020) and BEAST v2.6.7 (Bouckaert and Drummond 2017), respectively. The ML phylogeny was reconstructed with 1000 ultrafast bootstrap replicates (Hoang et al. 2018) and the best models of nucleotide substitution searched by ModelFinder in IQ-TREE. The BI phylogeny was reconstructed with 50,000,000 generations sampled per 1000 generations, and the models of nucleotide substitution were automatically acquired through bModelTest Analyzer in BEAST.

4. Results

A reference Image of B. pengxianensis is provided in Figure 1. B. pengxianensis is mainly distinguished from other species in the Gobionidae by the combination of three morphological characteristics, as follows: Three row teeth, soft and slim of the last simple dorsal ray, and the scales on the chest hidden under the skin (Yue 1998).

The complete mitochondrial genome of B. pengxianensis (MW080628) was assembled from PCR products of 13 segments (supplemental Appendixes I and II). This mitogenome is 16,610 bp in length with the AT content of 55.23%. The circular mitogenome consists of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and one non-coding control region (Figure 2). The characteristics of the mitogenome are provided in Table 1. The protein-coding genes start with ATG/GTG and end with TAA/TAG/TA-/T–. Two conserved blocks of the control region (5′-CAAACCCCCCTACCCCCT-3′ and 5′-TGTCAAACCCCGAAACCA-3′) named CSB-II and CSB-III by Lee et al. (1995), are located in 16,370–16,387 bp and 16,412–16,429 bp, respectively.

Figure 2. Mitochondrial genome map of B. pengxianensis.

The reconstructed phylogenetic trees display consistent topologies under the analyses of ML and BI using 13 protein-coding genes (Figure 3) or complete mitochondrial genomes (supplemental Figure S2 in Appendix III). The results show that B. pengxianensis and B. nummifer don’t form the sister relationships, and all of them are nested within the genus Hemibarbus with high support values.

Figure 3. Maximum-likelihood phylogeny between B. pengxianensis and its close relatives based on the 13 mitochondrial protein-coding genes. The bootstrap confidences (1000 replicates) and Bayesian posterior probabilities are shown near nodes.

5. Discussion and conclusion

In this study, we report the mitochondrial genome of B. pengxianensis for the first time. This mitogenome displays the same patterns in the gene compositions, gene arrangements, A + T bias of base compositions, the use of start or stop codon, and conserved sequence blocks of the control region as B. nummifer (Wang et al. 2016) and other gobionid species (see, e.g. Kim et al. 2009; Li et al. 2012; Chai and Fu 2020; Tian et al. 2022). The phylogenetic nesting of B. pengxianensis and B. nummifer in the genus Hemibarbus was revealed in our phylogeny, bringing into question the validity of the genus Belligobio. In summary, the new mitogenome of B. pengxianensis could contribute to reconstructing inter-specific relationships among species of the genera Hemibarbus and Belligobio, and is also very useful as reference molecular information to support species identification, biodiversity monitoring and conservation in the future.

Ethical approval

Field sampling conformed to the Chinese legislation for scientific purposes. All animal experiments followed Laboratory animal—Guideline for ethical review of animal welfare of the National Standard of the People’s Republic of China (GB/T 35892-2018).

Author contributions

FCZ conceived this study; XB and LMY conducted the experiments; XB and NXM analyzed the data, XB wrote the drafting of the paper; FCZ and NXM revised it critically, and that all authors agree to be accountable for all aspects of the work.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at https://www.ncbi.nlm.nih.gov/ under the accession no.MW080628. Sanger sequencing results are provided in supplementary materials.

Additional information

Funding

This study was funded by National Natural Science Foundation of China [31872209].