Characteristics and phylogenetic analysis of the complete mitochondrial genome of Glyptothorax pallozonus (Siluriformes, Sisoridae)

Abstract

Glyptothorax pallozonus Lin, 1934 is a small benthic fish belonging to the Sisoridae family that is distributed in the Dongjiang and Rongjiang Rivers of China. In the present study, we sequenced and characterized the complete mitochondrial genome of G. pallozonus for the first time. The complete mitogenome of G. pallozonus is 16,542 bp in length and includes 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), two ribosomal RNA (rRNAs), and a control region (CR). The mitogenome architecture was identical to that of other teleosts. Maximum likelihood (ML) phylogenetic analysis strongly supported the monophyly of Glyptothorax, which contains two clades. These results advance our understanding of the molecular phylogeny of the genus Glyptothorax.

Keywords:

• Glyptothorax pallozonus
• mitochondrial genome
• Sisorida
• phylogenetic analysis

Introduction

Glyptothorax is the most species-rich and widely distributed genus in the Sisoridae family and currently comprises 101 recognized species (Bánki et al. 2023). Glyptothorax pallozonus is a small benthic fish (Figure 1) that is endemic to the eastern region of Guangdong Province, China, and is characteristically found in the Dongjiang and Rongjiang Rivers (Zheng 1991). The species, like other sisorids, live in fast-moving streams, where they have adapted to using an adhesive apparatus on their underside to attach themselves to rocks and prevent themselves from being washed away (Ng and Rachmatika 2005). Over the past three decades, G. pallozonus has experienced a decline in population size due to environmental degradation and habitat changes. To date, this species is only occasionally caught in the wild; therefore, studies of this species are limited. Previous studies have mainly focused on the morphological, distributional, and systematic characteristics of G. pallozonus (Zheng 1991; Chu and Mo 1999; Jiang et al. 2011), and genetic information on this species is scarce. In the present study, the complete mitochondrial genome and the phylogenetic relationships of G. pallozonus were determined for the first time.

Figure 1. Specimen of Glyptothorax pallozonus was collected from the Fengshun county, Guangdong Province, China. The photo was taken by shujie liu on Mar 9, 2018.

Materials and methods

A live specimen of G. pallozonus was collected from Fengshun, Guangdong Province, China (23°44′57ʺN, 116°9′26ʺE). The specimen was morphologically identified as described by Zheng (1991) and Chu and Mo (1999). In particular, it could be distinguished from its congeners by the presence of a bright white triangular patch on the base of the dorsal fin (Figure 1). The specimen was deposited at the ichthyological museum of Freshwater Fisheries Research Institute of Jiangsu Province, China (Dr Liqiang Zhong, e-mail: [email protected]) under the voucher number JSFFRI-18003.

Methods

Genomic DNA was extracted using the Ezup Column Animal Genomic DNA Kit (Sangon, China) following the manufacturer’s protocol. The mitogenome was amplified using 30 sets of fish universal primers (Miya and Nishida 1999), and gaps were filled with self-designed primers based on the mitogenomes of other sisorids (Supplementary material, Table S1). The PCR products were sequenced by Sanger sequencing using the same primers.

Raw sequences were manually checked and assembled. The assembled sequences were annotated and visualized using MitoAnnotator (Zhu et al. 2023). Based on the concatenated sequences of 13 protein-coding genes (PCGs) from 23 fish (Table 1), a maximum likelihood (ML) phylogenetic tree was constructed with 1000 bootstrap assemblies and the GTR + G + I model using MEGA 11 (Tamura et al. 2021).

Table 1. Species and GenBank accession number of mitogenomes used in this study.

No.	Species	Accession ID	References
1	Creteuchiloglanis gongshanensis	KP872697	(Ma et al. 2015)
2	Creteuchiloalanis kamengensis	MN396886	(Ma et al. 2020)
3	Creteuchiloalanis macropterus	KP872682	(Ma et al. 2015)
4	Exostoma gaoligongense	MW256713	(Gong et al. 2021)
5	Exostoma labiatum	JQ026255	(Ma et al. 2015)
6	Exostom tenuicaudatum	ON641841	(Gong et al. 2022)
7	Exostoma tibetanum	ON641840	(Gong et al. 2022)
8	Glvptothorax annandalei	MN396887	(Lin et al. 2021)
9	Glyptothorax cavia	KY230517	(Li 2017)
10	Glyptothorax fokiensis	JQ917224	(Zhou et al. 2012)
11	Glyptothorax granosus	KP872684	(Li et al. 2023)
12	Glyptothorax lanceatus	KP872685	(Li et al. 2023)
13	Glyptothorax laosensis	KP872686	(Huang et al. 2017)
14	Glyptothorax longinema	KP872687	(Li et al. 2023)
15	Glyptothorax macromaculatus	MH213458	(Lv et al. 2018)
16	Glyptothorax pallozonus	OP723308	This study
17	Glyptothorax sinensis	KJ739617	(Yan et al. 2016)
18	Glyptothorax trilineatus	JQ026262	(Ma et al. 2015)
19	Glyptothorax zainaensis	KU212205	(Li et al. 2016)
20	Oreoglanis immaculatus	KP872690	(Ma et al. 2015)
21	Oreoglanis jingdongensis	KP872691	(Ma et al. 2015)
22	Oreoglanis macropterus	JQ026261	(Ma et al. 2015)
23	Odontobutis haifengensis	MF383619	(Zhong et al. 2018)

Results

The complete mitochondrial genome of G. pallozonus (GenBank accession number: OP723308) is 16,542 bp long and includes 13 PCGs, two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and one control region (CR) (Figure 2). The mitogenome architecture and gene-coding strands are similar to those of the teleost consensus (Ma et al. 2015). We observed a small AT-rich characteristic (59.2%), with a nucleotide composition of 27.1% T, 25.9% C, 32.1% A, and 14.9% G. The ND6 gene and eight tRNAs (tRNA^Ala, tRNA^Asn, tRNA^Cys, tRNA^Gln, tRNA^Glu, tRNA^Pro, tRNA^Ser, and tRNA^Tyr) were encoded on the light strand, whereas the other components were located on the heavy strand. Twelve PCGs started with ATG, while the COI gene began with GTG. Six genes ended with the complete stop codon TAA (ND1, COI, ATPase8, ND4L, ND5, and ND6), whereas the remaining genes had incomplete stop codons TA (ATPase 6) or T (ND2, COII, COIII, ND3, ND4, and Cytb). The origin of light-strand replication was 32 bp in length and located in the WANCY cluster between tRNA^Asn and tRNA^Cys. The CR was 890 bp in length and contained a termination-associated sequence (TAS), three central conserved sequence blocks (CSB-D, CSB-E, and CSB-F), and three conserved sequence blocks (CSB-1, CSB-2, and CSB-3).

Figure 2. Gene map of the mitochondrial genome of Glyptothorax pallozonus (GenBank accession number: OP723308), with 13 protein coding genes, 22 tRNAs, 2 rRNAs, and a control region. Genes encoded on light strand and heavy strand were shown on the inner and outer sides of the ring, respectively.

The ML phylogenetic tree indicated the monophyly of the genus Glyptothorax with high support scores (Figure 3). Two clades were identified within the genus Glyptothorax; G. pallozonus is grouped with G. zanaensis, G. longinema, G. granosus, G. lanceatus, G. trilineatus, G. laosensis, and G. annandalei, and eventually forms a clade. The remaining four species, G. macromaculatus, G. cavia, G. sinensis, and G. fokiensis, form a separate clade.

Figure 3. Maximum-likelihood (ML) phylogenetic tree was reconstructed based on the concatenated 13 protein-coding genes of Glyptothorax pallozonus and other 22 fishes. Accession numbers were indicated after the species name. Numbers at the nodes indicated bootstrap support values from 1000 replicates.

Discussion and conclusion

The structure of the complete mitochondrial genome of G. pallozonus is similar to that of other teleosts (Miya et al. 2003). In particular, the mitogenome structures of the genus Glyptothorax are highly conserved, suggesting that the mitochondrial genome is a perfect tool for population genetics and phylogenetic studies of these fishes. The ML phylogenetic tree strongly supported the monophyly of Glyptothorax, which was consistent with the results of a previous study based on the single gene sequences of RAG2, COI, and Cyt b (Jiang et al. 2011). Previous phylogenetic studies have included only four to seven species in the genus Glyptothorax and thus, have generated only a limited phylogeny of these fishes. Based on phylogenetic analysis, these fishes can be divided into two main clades: G. zanaensis and G. trilineatus, and G. macromaculatus, G. sinensis, and G. fokiensis (Huang et al. 2017; Li 2017; Lv et al. 2018). The new mitogenome discovered in this study supports the placement of G. pallozonus in the Glyptothorax genus. To the best of our knowledge, this is the first report on the evolutionary position of this species. At the same time, in the present study, the phylogenies of all 12 available fishes of Glyptothorax with complete mitogenomes were reconstructed, which would be useful in understanding the relationships within the Glyptothorax genus. However, the complete mitochondrial genome of most fishes in Glyptothorax (approximately 89 species) is still unavailable, and to elucidate the phylogenetic relationships of the Glyptothorax genus, extensive sampling and additional molecular information is necessary.

Author contributions

QFG and ZLQ conceived this study; QFG and YS conducted the experiments, LAP and WQ analyzed the data; QFG wrote the drafting of the paper; QFG and ZLQ revised it critically. All authors contributed to the article and approved the submitted version.

Ethical approval

The species used in this study is not protected under CITES or wildlife laws in China, and its status is data deficient by the IUCN. Therefore, no specific permission or license is required for research sampling according to Regulations of the People’s Republic of China. The live samples were obtained in accordance with the guidelines of the animal care and Ethical Committee of Freshwater Fisheries Research Institute of Jiangsu Province and Animal Experiments of Jiangsu Agri-animal Husbandry Vocational College.

Acknowledgments

We are grateful to Shujie Liu, Binbin Zhan and Huiwen Xiao for help in field assistance and the species reference image.

Data availability statement

The mitochondrial genome sequence is available on GenBank of NCBI at www.ncbi.nlm.nih.gov with the accession number of OP723308.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This study was supported by the Earmarked fund for Jiangsu Agricultural Industry Technology System [JATS(2022)517] and the research project of Jiangsu Agri-Animal Husbandry Vocational College (NSF2023TC05).