The Complete Mitochondrial Genome Sequence and Phylogenetic Analysis of Saurogobio punctatus (Cypriniformes: Cyprinidae: Gobioninae)

Juan Guo, Yu Zeng*, Jin Li, Ling Mao and Yu Shuang Zhao

China West Normal University, Nanchong 637000, People’s Republic of China

ABSTRACT

Saurogobio punctatus is a small-sized freshwater fish, widely distributed in eastern Asia. Presently, eight species are recognized as valid in Surogobio, among which the complete mitochondrial genome information had been known for seven species, except for S. punctatus. The lack of information was partly due to the recent discovery and establish of S. punctatus in the genus Saurogobio only in 2018. In the present study, the complete mitochondrial genome of S. punctatus was determined and phylogenetic of Saurogobio was analyzed using the complete mitochondrial genome sequence. The genome was 16,600 bp in length, with 55.98% of (A + T) content. It consisted of 13 protein-coding genes, 2 ribosomal RNAs, 22 tRNAs and a control region. The gene composition and the structural arrangement of the S. punctatus complete mtDNA were identical to other Saurogobio species. Phylogenetic analyses indicated that S. punctatus was closely related to S. dabryi, with strong statistical support, and was regarded as the sister taxon of the clade formed by S. xiangjiangensis, S. gracilicaudatus and S. dabryi, distinctly separated from all other Saurogobio species. It was inconsistent with previous results; the possible reason could be the different sequence utilized in each study. Moreover, phylogenetic results based on whole mitochondrial genomes were more reliable than partial mitochondrial.

Article Information

Received 30 June 2022

Revised 20 July 2022

Accepted 01 August 2022

Available online 10 January 2024

(early access)

Published 21 April 2025

Authors’ Contribution

YZ conceived and designed the research. JL, LM, YZ and JG were involved in the analysis and interpretation of the data. JG wrote the drafting of the paper and revised the manuscript. YZ critically reviewed the article regarding its intellectual content. All authors agree to be accountable for all aspects of the work.

Key words

Saurogobio punctatus, Mitochondrial genome, Genetic structure and composition, Phylogenetic

DOI: https://dx.doi.org/10.17582/journal.pjz/20220630110656

* Corresponding author: [email protected]

0030-9923/2025/0003-1099 $ 9.00/00

Copyright 2025 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Saurogobio (Cypriniformes: Cyprinidae: Gobioninae) is a small-sized freshwater fish endemic to eastern Asia, including China, Russia, North Korea and northern Vietnam (Yang et al., 2003). Presently, eight species are recognized as valid, i.e., the Saurogobio dabryi (Bleeker, 1871), S. dumerili (Bleeker, 1871), S. gracilicaudatus (Yao and Yang, 1977), S. gymnocheilus (Lo et al., 1998), S. immaculatus (Koller, 1927), S. xiangjiangensis (Tang, 1980), S. lissilabris (B˘an˘arescu and Nalbant, 1973) and S. punctatus (Tang, 2018) (Nelson et al., 2016; Tang et al., 2018). The complete mitochondrial genome information on complete mitochondrial genome of Saurogobio fish had been published for seven species, except for S. punctatus (Xu et al., 2015; Tong and Fu, 2019; Wan et al., 2015). Currently, studies on S. punctatus are quite limited, including the first report of S. punctatus by Tang et al. (2018) and the population genetic structure and geographic differentiation of S. punctatus in the Yangtze River basin by Li et al. (2018a). Moreover, the results were all based on partial mitochondrial genes, while the whole mitochondrial genome of this species is still unavailable. Based on previous result we found differences between partial and complete mitochondrial genome of the results. Therefore, we want to use the mitochondrial genome to explore the status of S. punctatus in the Gobioninae.

Mitochondrial genome has the characteristics of simple genomic structure, small size, rapid evolution, maternal inheritance and low level of recombination. It was widely used in phylogenetics, population genetic variation and species identification (Brown et al., 1979; Moritz et al., 1987). The complete mitochondrial genes of seven species of Saurogobio had been published and could be obtained on NCBI, except S. punctatus. The lack of sequence data has largely hindered the research and conservation of S. punctatus on population genetics and species identification. Thus, the study of whole mitochondrial genome in S. punctatus provides important information for its phylogenetic evaluation and molecular evolution.

In this study, we sequenced and evaluated complete mitochondrial genomes of S. punctatus. We also conducted phylogenetic analyses using the obtained genomes of S. punctatus and 11 complete mitogenomes retrieved for the subfamily Gobioninae. The results helped to clear the phylogenetic position of S. punctatus, and contribute to reconstructing interspecific phylogenetic relationships within the genus Saurogobio.

Materials and Methods

The specimen of S. punctatus was collected from Peng’an section of the Jialing River (31°2′14″N, 106°23′45″E) by cage net. S. punctatus was identified from fish samples according to the morphological descriptions by Tang et al. (2018). The specimen was preserved in 100% ethanol until DNA extraction. DNA was extracted with DNeasy Blood and Tissue kit (Qiagen, Germany) following the manufacturer’s protocol. DNA of S. punctatus then delivered to the Tsingke Biological Technology Company (Chengdu, China) for Illumina sequencing. Methods of sequence assembly, annotation and sequence analysis could refer to Mao et al. (2021).

The outgroup taxa and close relatives of Saurogobio fishes were selected, based on the results of previous studies (Tang et al., 2011; Li et al., 2018b). Pseudogobio esocinus, P. guilinensis, P. vaillanti and Abbottina rivularis were used as outgroups, seven valid Saurogobio species were used as close relatives. Mitogenomes of 11 species belonging to the subfamily Gobioninae were retrieved from GenBank to study the phylogenetic position of S. punctatus and phylogenetic relationships within the Gobioninae (Table I). We used PhyloSuite (Zhang et al., 2020) to conduct, manage and streamline the analyses of 12 sequences, with the help of several plug-in programs. 13 sequences were aligned in batches with MAFFT (Katoh and Standley, 2013) using ‘auto’ strategy and codon alignment mode. The alignments were refined using the codon-aware program MACSE v. 2.03 (Ranwez et al., 2018), which preserved reading frame and allowed incorporation of sequencing errors or sequences with frameshifts. Ambiguously aligned fragments of 13 alignments were removed in batches using Gblocks (Talavera and Castresana, 2007) with the following parameter settings. Minimum number of sequences for a conserved/flank position (7/7), maximum number of contiguous non-conserved positions (8), minimum length of a block (10), allowed gap positions (with half). Model Finder (Kalyaanamoorthy et al., 2017) was used to select the best-fit model using BIC criterion. Maximum likelihood phylogenies were inferred using IQ-TREE (Nguyen et al., 2015) under the model automatically selected by IQ-TREE (Auto option in IQ-TREE) for 20,000 ultrafast (Minh et al., 2013) bootstraps, as well as the Shimodaira Hasegawa like approximate likelihood-ratio test (Guindon et al., 2010). Bayesian Inference phylogenies were inferred using MrBayes 3.2.6 (Ronquist et al., 2012) under GTR+I+G+F model (2 parallel runs, 5000000 generations), in which the initial 25% of sampled data were discarded as burn-in. The complete mitochondrial genome sequence then was submitted to NCBI (GenBank: ON041156). Strand skew values were calculated according to the formulae given by Perna and Kocher (1995): AT skew = (A - T)/ (A + T) and GC skew = (G -C)/ (G + C), where A, T, C, G were the four bases.

 

Table I. List of the 11 Gobioninae species in phylogenetic analysis, with their GenBank accession numbers.

Species	Subfamily	GenBank No	Size (bp)
Saurogobio gymnocheilus	Gobioninae	NC 050400	16,604
Saurogobio lissilabris	Gobioninae	NC 050401	16,594
Saurogobio dabryi	Gobioninae	KF612272	16,601
Saurogobio immaculatus	Gobioninae	NC 033919	16,988
Saurogobio gracilicaudatus	Gobioninae	NC 050398	16,608
Saurogobio xiangjiangensis	Gobioninae	NC 050399	16,600
Saurogobio dumerili	Gobioninae	NC 022187	16,601
Pseudogobio esocinus	Gobioninae	NC_013759	16,609
Pseudogobio guilinensis	Gobioninae	MN883565	16,609
Pseudogobio vaillanti	Gobioninae	NC 032292	16,605
Abbottina rivularis	Gobioninae	NC 023781	16,597

 

Results

Mitogenome organization

The complete mitochondrial genome of S. punctatus was a circular molecule of 16,600 bp and showed a typical teleost mitochondrial order, which included 2 rRNA genes (12s rRNA and 16s rRNA), 22 transfer RNA genes (tRNA), 13 protein-coding genes (PCGs) and the control region (D-loop). The nucleotide composition was: A 30.07%, T 25.91%, G 16.68%, C 27.34%. The genome had an overall AT content of 55.98% and GC content of 44.02%, and it showed a biased A + T ratio. The composition was skewed away from A in favored of T (the AT-skew was +0.074), but was almost balanced for G and C (the GC-skew was -0.242). A negative GC and AT skew value meant more Cs or Ts in coding strand, respectively. To the opposite, a positive GC and AT skew value indicated the coding strand had more Gs or As, respectively. The mitochondrial genome map and gene characteristics were shown in Figure 1 and Table II, respectively.

 

Most genes were encoded on the heavy strand (H strand), except for the ND6 and eight tRNA genes (tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser, tRNA-Glu, tRNA-Pro). Two types of start codons (ATG and GTG) and four types of stop codons (TAA, TAG, TA and T) were used in protein-coding genes. Among the 13 protein-coding genes, 12 PCGs used ATG as regular initiation codon, while only COX1 gene started with GTG. Six genes (ND2, COX1, ATP6, ATP8, ND4L, ND5) made use of the regular stop codon TAA, ND1, ND3 and ND6 used TAG, whereas the remaining 4 genes (COX2, COX3, ND4, CYTB) used incomplete codons TA or T as termination codons (Table II). Four pairs of adjacent PCGs, ATP8-ATP6, ATP6-COIII, ND4L-ND4, and ND5-ND6 had the overlapped size of 7, 1, 7, and 4 bp, respectively. And there were eleven regions of gene overlap totaling 28 bp (ranging from 1 to 7 bp) and 12 intergenic spacer regions totaling 38 bp (ranging from 1 to 13 bp). The two rRNA genes combined were 2648 bp in length. The location of 12S rRNA gene (958 bp) was between tRNA-Phe and tRNA-Val, and 16S rRNA gene (1690 bp) was located between tRNA-Val and tRNA-Leu. In addition, the 22 tRNA genes, ranging from 69 to 76 bp, were distributed across the mitogenome of S. punctatus. The noncoding control region (D-loop), with the length of 926 bp, was located between the tRNA-Phe and tRNA-Pro genes.

Although BI and ML analyses relied on different methods, they produced almost the same phylogenetic trees. Both topological structures indicated the existence of two major clades: One group involved S. dumerili, S. lissilabris, S. gymnocheilus, and S. immaculatus, and another group consisted of S. dabryi, S. gracilicaudatus, S. xiangjiangensis and S. punctatus. But compared with Tang’s findings, the location of S.dabryi is different. In this study, S. dabryi and S. punctatus were closely related, while in Tang et al. (2018) study the two are separated by S. gracilicaudatus and S. xiangjiangensis.

Discussion

The composition and arrangement of the mitochondrial genome of S. puntatus were similar to other Gobioninae species (Mao et al., 2021; Tong and Fu, 2019). The use of codons was also similar to other fishes: start with ATG and GTG, end with TAA, TAG, TA-, T-. Similar patterns in the gene arrangements and codon use had also been observed in published mitochondrial genomes of other Saurogobio and Squalidus species (Wan et al., 2015; Xu et al., 2015; Chai and Fu, 2020). Presumably, TAG and incomplete codons in ND2 would be completed to TAA by post-transcriptional polyadenylation (Ojala et al., 1981). At the same time, the GC content (44.02%) of S. puntatus mitochondrial genomes was lower than AT content (55.98%), which was frequently observed in other Cyprinid species (Yue et al., 2006). The higher GC content of DNA, the more stable was the doublestranded helical molecule. On the contrary, the more unstable (Bhagavan, 2002).

S. punctatus is a bony fish belongs to Cypriniformes, Cyprinidae, Gobioninae, and according to previous results Saurogobio is a monophyletic group. The results of Bayesian analysis and maximum likelihood tree showed a topology with strong posterior probability values, suggesting that the phylogenetic tree was well-supported. The Saurogobio was a monophyletic group which could be divided into two major groups (Fig. 2). S. punctatus was regarded as the sister taxon of the clade formed by S. xiangjiangensis, S. gracilicaudatus and S. dabryi, distinctly separated from all other Saurogobio species. The topology of the phylogenetic tree further supported the results of previous studies, which confirmed the basic relationships in the Saurogobio genus (Tong and Fu, 2019). The phylogenetic tree proved that S. punctatus and S. dabryi were closely related, while Tang et al. (2018) research was not the case. A possible reason could be

 

Table II. Characteristics of the complete mitochondrial genome of S. punctatus.

Gene	Start	Stop	Strand	Length	Codon start/ stop	Anti codon	Intergenic region
tRNA-Phe	1	69	+	69		GAA	0
12S rRNA	70	1027	+	958			0
tRNA-Val	1030	1101	+	72		TAC	2
16S rRNA	1102	2791	+	1690			0
tRNA-Leu	2792	2867	+	76		TAA	0
ND1	2868	3842	+	975	ATG/TAG		0
tRNA-Ile	3847	3918	+	72		GAT	4
tRNA-Gln	3917	3987	-	71		TTG	-2
tRNA-Met	3989	4057	+	69		CAT	1
ND2	4058	5104	+	1047	ATG/TAA		0
tRNA-Trp	5104	5174	+	71		TCA	-1
tRNA-Ala	5177	5245	-	69		TGC	2
tRNA-Asn	5321	5351	+	31		GTT	1
rep-origion	5247	5319	-	73			1
tRNA-Cys	5351	5418	-	68		GCA	-1
tRNA-Tyr	5421	5488	-	68		GTA	2
COX1	5490	7040	+	1551	GTG/TAA		1
tRNA-Ser	7041	7111	-	71		TGA	0
tRNA-Asp	7115	7186	+	72		GTC	3
COX2	7200	7890	+	691	ATG/T		13
tRNA-Lys	7891	7966	+	76		TTT	0
ATP8	7968	8132	+	165	ATG/TAA		1
ATP6	8126	8809	+	684	ATG/TAA		-7
COX3	8809	9593	+	785	ATG/TA		-1
tRNA-Gly	9593	9663	+	71		TCC	-1
ND3	9664	10014	+	351	ATG/TAG		0
tRNA-Arg	10013	10082	+	70		TCG	-2
ND4L	10083	10379	+	297	ATG/TAA		0
ND4	10373	11751	+	1379	ATG/TA		-7
tRNA-His	11755	11824	+	70		GTG	3
tRNA-Ser	11825	11893	+	69		GCT	0
tRNA-Leu	11893	11965	+	73		TAG	-1
ND5	11966	13801	+	1836	ATG/TAA		0
ND6	13798	14319	-	522	ATG/TAG		-4
tRNA-Glu	14320	14388	-	69		TTC	0
CYTB	14393	15533	+	1141	ATG/T		4
tRNA-Thr	15534	15605	+	72		TGT	0
tRNA-Pro	15605	15674	-	70		TGG	-1
D-loop	15675	16600	+	926			0

 

Note: + and - represent heavy strand and light strand, respectively. In the intergenic region column, negative numbers indicate an overlap between two adjacent genes.

 

the different sequence utilized in each study, that Tang et al. (2018) used the partial mitochondrial genome (cytb), whereas this study used the whole mitochondrial genome. Previous research had also confirmed this, Mao et al. (2021) research on phylogenetic analysis with the whole mitochondrial genome were inconsistent with Yang et al. (2006) with cytb. In addition, Arnason and Gullberg (1996) found the phylogenetic relationships based on cytb among the five clades were not resolved in the bootstrap analysis, whereas in Arnason’s later study (Arnason et al., 2004), he used the whole mitochondrial genome to figure out their relationships. These articles demonstrated that the phylogenetic results based on ctyb and the whole mitochondrial genome were different, and the results of the latter were more reliable.

In summary, we reported the complete mitochondrial genome of S. punctatus for the first time. The full-length of S. punctatus mitochondrial genome was 16,600 bp long, and it showed a similar order and characteristic with other Saurogobio fishes. Our findings could provide novel molecular resources for further population genomics, evolution, and conservation studies of S. punctatus.

Acknowledgments

We thank Ling Mao for her help in sample collection and identification.

DECLARATIONS

Disclosure statement

The creators report no irreconcilable circumstance. The writers are liable for the substance and composing of the article.

Data availability statement

The complete mitochondrial genome sequence of Glyphoglossus yunnanensis is deposited in the GenBank database under the accession number ON041156. The associated BioProject number, SRA accession number, and BioSample number are PRJNA846592, SRR19576530, and SAMN28898293, respectively.

Funding statement

This research was supported by the National Natural Science Foundation of China (No. 51,779,210), and the Fundamental Research Funds of Technical Office in Sichuan Province (No. 22YYJC3225).

Ethical approval

The specimen for this paper were approved by the Animal Ethics Committee at China West Normal University. All animals handling and processing by the Law of the People’s Republic of China on the Protection of Wildlife and approved by the Animal Care Committee of CIB, CAS.

Statement of conflict of interest

The authors have declared no conflict of interest.

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