The Complete Mitochondrial Genome of a Rare Cavefish (Sinocyclocheilus cyphotergous) and Comparative Genomic Analyses in Sinocyclocheilus

Xiaoping Gao1, Yanping Li2*, Renyi Zhang3, Yunyun Lv2, Yongming Wang2, Jinrong Shi2, Jiang Xie2, Chiping Kong1 and Lekang Li1

1Jiujiang Academy of Agricultural Sciences, Jiujiang, 332101, China

2Key Laboratory of Sichuan Province for Fishes Conservation and Utilization in the Upper Reaches of the Yangtze River, College of Life Sciences, Neijiang Normal University, Neijiang, 641100, China

3Key Laboratory of National Forestry and Grassland Administration on Biodiversity Conservation in Karst Mountainous Areas of Southwestern China, School of Life Science, Guizhou Normal University, Guiyang, Guizhou, 550001, China

Xiaoping Gao and Yanping Li contributed equally to this project.

ABSTRACT

The Sinocyclocheilus cyphotergous, belonging to the family Cyprinidae, is endemic to the Karst area of the Yunnan-Guizhou Plateau. Here, we determined the complete mitogenome of S. cyphotergous using an Illumina Hiseq X Ten sequencer. This mitogenome’s structure is typical circular with 16,611 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a control region. The overall base composition of S. cyphotergous is 31.33% A, 25.89% T, 26.49% C, and 16.29% G with a slight AT bias of 57.22%. Most mitochondrial genes except ND6 and eight tRNAs were encoded on the heavy strand. All tRNA genes fold into the typical cloverleaf secondary structures, except for tRNA-Ser (AGY) that lacked the dihydrouracil arm. 15 of 22 tRNA genes were found to have 29 G-U mismatches in their secondary structures, which formed a weak bond. In addition, mismatches of A-C, C-C, U-U, and A-A were also found in their tRNA secondary structures. Result of substitution rate estimation among the mitochondrial protein coding genes (PCGs) showed ATP8 had the largest average Ka and Ka/Ks, while COI had the lowest, which implied that ATP8 might evolve more quickly than the other mitochondrial protein coding genes. Phylogenetic analyses based on Bayesian inference (BI) and maximum likelihood (ML) revealed all Sinocyclocheilus species in this research formed a solid monophyletic group and grouped into two major clades with strong support excluded S. jii. Additionally, S. cyphotergous in this study was closely related to S. multipunctatus and S. punctatus. In summary, this study provided novel insights into the phylogeny of the Sinocyclocheilus fishes, conducive to the conservation genetics and cave adaptation for S. cyphotergous.

Article Information

Received 13 February 2023

Revised 20 March 2023

Accepted 09 April 2023

Available online 09 October 2023

(early access)

Published 26 July 2024

Authors’ Contribution

XG, YL and RZ conceived and designed the experiments, XG, YL, RZ, and YLv analyzed the data. YL wrote the original draft; YW, JS, JX, KC, YLv and LL reviewed and revised the manuscript. All authors have read and approved the publication of this manuscript.

Key words

Sinocyclocheilus cyphotergous, Mitogenome assembly, Annotation, Phylogenetic relationship, Evolution analysis

DOI: https://dx.doi.org/10.17582/journal.pjz/20230213080243

* Corresponding author: liyanping@njtc.edu.cn

0030-9923/2024/0005-2245 $ 9.00/0

Copyright 2024 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

The genus Sinocyclocheilus (Cypriniformes: Cyprinidae) is monophyletic and endemic to the Karst area of the Yunnan-Guizhou Plateau and its surrounding region Guangxi, southwestern China, being the most diverse genus of cyprinid fishes in China (Zhao and Zhang, 2009). There are more than 60 valid species that have been recognized in this genus, and most of them distribute in the tributaries of Xijiang River, while few are distributed in the Jinsha River and Honghe River. Also, most of the Sinocyclocheilus have various cave-dwelling behaviors or underground streams (Zhao and Zhang, 2009).

At present, although there are some studies referring to the phylogenetic relationship of the genus Sinocyclocheilus (Li and He, 2009; Liang et al., 2011; Luo and Zhang, 2021; Xiao et al., 2005; Zhang and Wang, 2018), the phylogenetic relationship among the species of this genus has not been completely solved due to the continuous discovery of new species, and the inconsistency of the phylogenetic relationship between morphological characteristics and molecular data. Additionally, the lack of genetic markers in molecular phylogenetic investigations further complicates determining this genus’ evolutionary affiliation. Mitochondrial DNA has many valuable features including relatively conserved gene content and organization, lack of genetic recombination, maternal inheritance, and relatively fast evolutionary rate. Hence, partial or complete mitochondrial genes have been used to determine molecular phylogenetic and evolutionary relationships (Boore, 1999; Lv et al., 2018; Zhang et al., 2022; Zhang and Wang, 2018). However, the study of mitogenomes is still scarce in the genus of Sinocyclocheilus compared to its rich species.

S. cyphotergous is the most representative cave fish in Guizhou province and is named for its unique horn protrusion on its back. It bears vulnerable status on the International Union for the Conservation of Nature (IUCN) Red List, and is listed as national second-grade key protected fish. However, the available genetic data for this species remains scarce. The purposes of this study is shown as: (1) Assemble and annotate the mitochondrial genome of S. cyphotergous to determine its genomic organization and structure of the complete mitochondrial sequence; (2) reconstruct the phylogeny of the genus Sinocyclocheilus using the mitochondrial genome obtained here and NCBI; (3) determine the substitution rates of protein coding genes based on the complete mitochondrial genome of the genus Sinocyclocheilus and to identify signals of positive selection. This study not only improves understanding of genomic and phylogenetic information of Sinocyclocheilus, but also provides reference for the conservation genetics of S. cyphotergous.

MATERIALS AND METHODS

Sample collection

Samples were collected from Luodian County, Guizhou Province, China (25°34′31″N, 106°50′18″E). Muscle samples were preserved in 95% ethanol, and voucher specimens were deposited at the School of Life Sciences, Guizhou Normal University, Guiyang, Guizhou Province, China. (Voucher no. GZNUSLS202009042). Sampling was performed according to Chinese animal protection laws.

DNA extraction and sequencing

Genomic DNA was extracted from muscle tissue using a DNeasy blood and tissue kit (QIAGEN, Hilden, Germany). DNA integrity, purity and concentration were assessed with an Agilent 5400 fragment analyzer (Agilent Technologies, Santa Clara, CA, U.S.A.). After the DNA sample was qualified, and the template size was 100 ng/ul, it was randomly sheared with a Covaris ultrasonicator (Covaris Inc., Woburn, MA, U.S.A.), and then the library was constructed through several steps: End repair and phosphorylation, adding A-tailing, ligating index adapter, purification, denaturing and PCR amplification. After the library was constructed, a Qubit 2.0 (Life Technologies, Singapore) was used to quantify and dilute the library. We then employed an Agilent 2100 Bioanalyzer (Agilent) to detect inserted fragments in the library. Finally, the effective concentration of the library was accurately quantified by q-PCR to ensure the library quality. After that, different libraries were pooled into the flow cell according to the effective concentration and target drop-off data. Illumina paired-end sequencing was conducted with Illumina Hiseq X Ten sequencer (Illumina, San Diego, CA, U.S.A.).

Mitochondrial genome assembly and annotation

The raw data contained adapter information, low-quality bases, and undetected bases (indicated by N), which would interfere with subsequent analysis. We therefore filtered the raw data using the following criteria: (1) filtered out reads containing adapter sequences; (2) removed paired reads, when the content of N in a single-ended sequence exceeded 10%; (3) base with quality no more than 5 was regarded as low-quality base based on phred+33. If more than half were low-quality bases in a sequence, this sequence, along with the paired one was discarded. The reads belonging to the mitochondrial genome of Sinocyclocheilus cyphotergous were identified by alignment with the other nine released mitogenomes of congeneric species within Sinocyclocheilus (Accession number: MG026730.1; MZ781221.1; NC_058003.1; MT361975.1; KX528071.1; MK387704.1; NC_057312.1; NC_056194.1; NC_056143.1), which was performed by minimap2 version 2.24. Then, the reads within the alignments were extracted and assembled to a complete circle by ABYSS version 2.1.5 with a k-mer length of 27 bp. The assembled mitogenome was annotated using the MitoAnnotator on the homepage (Iwasaki et al., 2013).

Mitogenome characteristic analyses

All putative tRNA genes were identified using tRNAscan-SE search server (Lowe and Chan, 2016) and MITOS (Bernt et al., 2013). The secondary structures of tRNAs were drawn by MITOS. The base composition, amino acid composition, codon usage and relative synonymous codon usage (RSCU) of 13 protein coding genes (PCGs) were analyzed using MEGA version 7.0 (Kumar et al., 2016). Nucleotide compositional skew was calculated using the formula: AT-skew = (A − T) / (A + T) and GC-skew = (G − C) / (G + C) (Perna and Kocher, 1995).

 

Table I. The information of the mitochondrial genomes used in this study.

No.	Taxonomic position/ Species	Size (bp)	Accession no.
Cypriniformes, Cyprinidae
1	Sinocyclocheilus punctatus	16582	NC_058003
2	S. ronganensis	16587	NC_032385
3	S. grahami	16585	NC_013189
4	S. microphthalmus	16589	MN145877
5	S. yishanensis	16573	MK387704
6	S. huizeensis	16585	NC_044072
7	S. qujingensis	16588	NC_043910
8	S. wumengshanensis	16585	NC_039769
9	S. tingi	16584	NC_039594
10	S. oxycephalus	16585	NC_037858
11	S. jii	16577	NC_037197
12	S. multipunctatus	16586	MG026730
13	S. bicornutus	17426	NC_031382
14	S. anophthalmus	16574	NC_023472
15	S. furcodorsalis	16581	NC_019995
16	S. altishoulderus	16589	NC_013186
17	S. longibarbatus	16787	MW345239
18	S. lingyunensis	16572	NC_056143
19	S. angularis	16586	MW362289
20	S. anshuiensis	16618	NC_027169
21	S. rhinocerous	16588	NC_027168
22	S. cyphotergous	16611	this study
23	Cyprinus carpio	16581	KF856965
24	Barbus barbus	16600	NC_008654

 

Phylogenetic analysis

To investigate the phylogenetic relationships between S. cyphotergous and other species of Sinocyclocheilus fishes, the phylogenetic analyses were conducted based on the mitochondrial genome sequence of S. cyphotergous assembled in this study and other 21 Sinocyclocheilus fishes that were downloaded from NCBI GenBank (Table I). We used two species of Cyprinus carpio and Barbus barbus as outgroups. Therefore, the complete datasets used for phylogenetic analysis included 24 complete mitochondrial genomes (Table I).

All sequences were aligned with MAFFT version 5.0 (Katoh et al., 2005), and the phylogenetic tree was inferred by IQ-TREE version 1.6.12 (Lam-Tung et al., 2015) with the “-spp” option to allow partition-specific evolution rates. Twenty-four mitochondrial genomes were partitioned to six partitions, and the best model for each partition was detected with IQ-tree Model Finder (Kalyaanamoorthy et al., 2017) based on Bayesian information criterion. Maximum likelihood (ML) tree and Bayesian inference (BI) was performed in IQ-tree using fixed models as defined by the best-fit partitioning schemes. Node support of the trees were inferred by conducting non-parametric bootstrap (1000 replicates) and ultrafast bootstrap (5000 replicates) (Hoang et al., 2018), respectively. Trees were graphically visualized with FigTree version 1.4.2.

Substitution rate estimation and comparison

The rates of nonsynonymous (Ka) and synonymous (Ks) substitutions may differ during the evolution process among mitochondrial coding genes. To investigate the evolutionary patterns under different selective pressures among 13 PCGs in Sinocyclocheilus fishes, we further calculated the average Ka and Ka/ Ks values among all 13 PCGs of 21 Sinocyclocheilus fishes.

Based on the annotations of the 21 Sinocyclocheilus mitochondrial genomes, each of the 13 protein coding genes were extracted, and each protein coding gene was aligned separately with the codon-based model in the Muscle module of Mega version 7 (Kumar et al., 2016). The pairwise Ka and Ka/ Ks values between each pair of single-gene datasets were calculated in DnaSP version 5.0 (Librado and Rozas, 2009). The average values of Ka and Ka/ Ks were calculated in R, and were used to represent changes of each coding gene’s substitution rates.

RESULTS and DISCUSSION

Mitogenome features and nucleotide composition of S. cyphotergous

A total of 27,099,642,600 raw reads were generated and it has been deposited to NCBI database (see additional details in Data availability statement). After assembly, the complete mitogenome of S. cyphotergous was obtained (accession number: OQ319607), with a total length of 16,611 bp. The mitogenome of S. cyphotergous also consists of 13 protein-coding genes (ND1, ND2, COI, COII, ATP8, ATP6, COIII, ND3, ND4L, ND4, ND5, ND6, and Cyt b), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA genes, and one non-coding control region (D-loop region) (Fig. 1, Table II). The arrangement of these genes was identical to other previously published Sinocyclocheilus mitogenomes (Li and Yang, 2021; Luo and Zhang, 2021; Zhang and Wang, 2018). Most genes were transcribed from the heavy strand (2 rRNAs, 12 protein-coding genes and 14 tRNAs); only nine genes, including one protein coding gene (ND6) and eight tRNAs (tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser (UCN), tRNA-Glu and tRNA-Pro), were encoded on the light strand.

There were 6 overlapping regions totaling 22 bp and varied in size from 1 to 7 bp; the longest overlapping region was located between ATP8 and ATP6, ND4L and ND4. Likewise, a total of 68 intergenic nucleotides were dispersed in 13 intergenic spacer regions and ranged in length from 1 to 32 bp; the longest gap was located between tRNA-Asn and tRNA-Cys. The overlapping and intergenic regions of S. cyphotergous is similar to the most other fish mitochondrial genomes that without gene rearrangement occurred (He et al., 2012, 2016; Luo and Zhang, 2021; Wu et al., 2009; Zhang and Wang, 2018). The 12S and 16S rRNA genes were 955 and 1679 bp, respectively. They were located between tRNA-Phe and tRNA-Leu (UUR) and were separated by tRNA-Val (Table II). The D-loop region was located between tRNA-Pro and tRNA-Phe.

As is the case with other Sinocyclocheilus mitogenomes, the overall base composition of S. cyphotergous was 31.33% A, 25.89% T, 26.49% C, and 16.29% G, with a slight AT bias of 57.22%, which was in accordance with most other fish mitogenomes. The A+T contents of PCGs, rRNAs, tRNAs, and D-loop region were all above 50% (Table III). The D-loop region, known as A+T rich region, has the highest A+T content (68.83%), which is typical for animal mitochondrial genomes (Guo et al., 2003; Sbisa et al., 1997; Zhang and Wang, 2018; Zou et al., 2017). The skew statistics showed a similar pattern of base composition in S. cyphotergous mitogenome, except for the tRNA, which was also similar to other Sinocyclocheilus mitogenomes (Zhang and Wang, 2018).

 

Table II. Mitochondrial genome organization of Sinocyclocheilus cyphotergous.

Gene/ Element	Codon					Intergenic nucleotides*	Stra-nd†
	From	To	Length (bp)	Start	Stop
tRNA-Phe	1	69	69			0	H
12S rRNA	70	1024	955			0	H
tRNA-Val	1025	1096	72			0	H
16S rRNA	1097	2775	1679			0	H
tRNA-Leu (UUR)	2776	2851	76			+1	H
ND1	2853	3827	975	ATG	TAA	+4	H
tRNA- Ile	3832	3903	72			-2	H
RNA- Gln	3902	3972	71			+1	L
tRNA-Met	3974	4042	69			0	H
ND2	4043	5087	1045	ATG	T--	0	H
tRNA-Trp	5088	5160	73			+2	H
tRNA-Ala	5163	5231	69			+1	L
tRNA-Asn	5233	5305	73			+32	L
tRNA-Cys	5338	5404	67			-1	L
Tyr	5404	5474	71			+1	L
COI	5476	7026	1551	GTG	TAA	0	H
tRNA-Ser (UCN)	7027	7097	71			+3	L
tRNA-Asp	7101	7172	72			+13	H
COII	7186	7876	691	ATG	T--	0	H
tRNA-Lys	7877	7952	76			+1	H
ATP8	7954	8118	165	ATG	TAA	-7	H
ATP6	8112	8794	683	ATG	TA-	0	H
COIII	8795	9579	785	ATG	TA-	0	H
tRNA-Gly	9580	9651	72			0	H
ND3	9652	10000	349	ATG	T--	0	H
tRNA-Arg	10001	10070	70			0	H
ND4L	10071	10367	297	ATG	TAA	-7	H
ND4	10361	11741	1381	ATG	T--	0	H
tRNA-His	11742	11811	70			0	H
tRNA-Ser (AGY)	11812	11880	69			+1	H
RNA-Leu (CUN)	11882	11954	73			+3	H
ND5	11958	13781	1824	ATG	TAA	-4	H
ND6	13778	14299	522	ATG	TAA	0	L
tRNA-Glu	14300	14368	69			+5	L
Cyt b	14374	15514	1141	ATG	T--	0	H
RNA-Thr	15515	15586	72			-1	H
tRNA-Pro	15586	15655	70			0	L
D-loop	15656	16611	956			0	H

 

*Numbers correspond to the nucleotides separating different genes. Negative numbers indicate overlapping nucleotides between adjacent genes.

 

Protein coding genes and codon usage patterns

Almost all PCGs started with the typical ATG initiation codons whereas COI started with GTG. Among these 13 PCGs, six PCGs including ND1, COI, ATP8, ND4L, ND5 and ND6 were terminated with the typical TAA codons, while other seven PCGs (ND2, COII, ATP6, COIII, ND3, ND4 and Cyt b) were characterized by incomplete stop codons (T or TA) (Table II). The base compositions of the 13 PCGs were 28.10% for T, 27.01% for C, 29.14% for A and 15.75% for G (Table III).

Excluding the termination codons, a total of 3803 amino acids from 13 PCGs were encoded in the mitogenome of S. cyphotergous. The relative synonymous codon usage (RSCU) of the 13 PCGs in S. cyphotergous was shown in Figure 2. The most frequently used codon in S. cyphotergous was GCC-Ala (1.69%), followed by AAA-Lys (1.51%) and AGG-Arg (1.48%) (Fig. 2).

 

Transfer RNAs and ribosomal RNAs

The mitogenome of S. cyphotergous was predicted to encode 22 tRNA genes, which were interspersed along the genome, with the length varying from 67 bp (tRNA-Cys) to 76 bp (tRNA-Leu (UUR) and tRNA-Lys) (Table II). All the tRNAs of S. cyphotergous were capable of folding into a typical clover-leaf secondary structure except for tRNA-Ser (AGY) with an incomplete dihydrouridine arm (Fig. 3), which is similar to other Sinocyclocheilus fishes (Wu et al., 2009; Zhang and Wang, 2018). 15 of 22 tRNA genes, including tRNA-Leu, tRNA-Gln, tRNA-Met, tRNA-Trp,

 

Table III. Nucleotide composition of the S. cyphotergous mitochondrial genome.

	Length (bp)	T%	C%	A%	G%	A+ T%	AT-skew	GC-skew
Genome	16611	25.89	26.49	31.33	16.29	57.22	0.09	-0.24
PCGs	11409	28.10	27.01	29.14	15.75	57.24	0.02	-0.26
1st codon position	3803	28.95	27.77	29.95	13.33	59.00	0.02	-0.35
2nd codon position	3803	27.24	27.03	29.03	16.70	56.27	0.03	-0.24
3rd codon position	3803	28.11	26.24	28.43	17.22	56.54	0.01	-0.21
rRNA	2634	19.70	24.72	34.51	21.07	54.21	0.27	-0.08
tRNA	1566	26.69	21.39	28.87	23.05	55.56	0.04	0.04
D-loop region	956	34.00	18.20	34.83	12.97	68.83	0.01	-0.17

 

 

tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Asp, tRNA-Lys, tRNA-Gly, tRNA-His, tRNA-Ser, tRNA-Glu, and tRNA-Pro, were found to have 29 G-U mismatches in their secondary structures, which formed a weak bond. Six tRNA including tRNA-Ile, tRNA-Gly, tRNA-Arg, tRNA-His, tRNA-Asp, and tRNA-Thr, were found to have A-C mismatches. Three C-C mismatches of tRNA-Thr, tRNA-Met, and tRNA-Ser were found in their amino acid acceptor arm, TΨC stem and anticodon arm, respectively. Two U-U mismatches of tRNA-Gln and tRNA-Asn were found in their TΨC stem, and one U-U mismatch of tRNA-Cys was found in the anticodon arm. In addition, an A-A mismatch of tRNA-Trp was found in the dihydrouridine arm (Fig. 3).

As in other fish mitogenome sequences, there were two rRNA genes including 12S rRNA and 16S rRNA in S. cyphotergous. The size of 12S rRNA was 955 bp and located between tRNA-Phe and tRNA-Val, and the size of 16S rRNA was 1679 bp and located between tRNA-Val and tRNA-Leu (UUR), respectively. The base composition of the two rRNAs was 19.70% for T, 24.72% for C, 34.51% for A, and 21.07 for G. The A + T content was 54.21%, thus slightly higher than the G + C content (Table III). The length and A + T content of these two rRNAs are similar to the other Sinocyclocheilus species mitogenomes (Hao et al., 2016; He et al., 2012, 2016; Wu et al., 2009; Zhang and Wang, 2018).

Phylogenetic analysis within Sinocyclocheilus fishes

To date, there are 21 other Sinocyclocheilus mitogenomes available on GenBank. Our multiple sequence alignment of 24 complete mitochondrial genomes, including two outgroup species (Cyprinus carpio and Barbus barbu) and 22 Sinocyclocheilus species. The total alignment for phylogenetic analysis was 11,385 bp. Model selection using Partition Finder 2 indicated an optimal partitioning scheme with six partitions, and the first partition was ND4+ATP6+CYTB+ND5 with GTR+F+I+G4 model, the second partition was ND1+ND2 with TIM3+F+I+G4 model, the third partition was ND6 with HKY+F+G4 model, the fourth partition was COIII+COI+COII+ND4L with GTR+F+I+G4 model, the fifth partition was ATP8 with K3Pu+F+I model, and the sixth partition was ND3 with GTR+F+G4 model. The phylogeny trees resulting from BI and ML analyses showed identical topologies, and only slight differences were occurred between the Bayesian posterior probabilities and ML bootstrap values (Fig. 4). Phylogenetic analysis revealed all Sinocyclocheilus species formed a solid monophyletic group and grouped into two major clades with strong support. S. cyphotergous in this study was clustered together and closely related to S. multipunctatus and S. punctatus. Additionally, S. jii is the sister species to a clade of all other Sinocyclocheilus species based on the phylogenetic reconstruction (Fig. 4), which is also supported by the previous research (Li and Yang, 2021). In summary, our study provides a new resource for understanding the whole mitochondrial genome of S. cyphotergous, which will promote the molecular study of this genus.

 

The nucleotide substitution rate in the Sinocyclocheilus fishes

Of all average values of Ka and Ka/Ks across the 13 PCGs of the Sinocyclocheilus fishes, ATP8 had the largest average Ka and Ka/Ks, while COI had the lowest (Fig. 5), which implies that ATP8 might evolve more quickly than other mitochondrial protein coding genes. The evolutionary patterns under different selective pressures among 13 PCGs in the Sinocyclocheilus fishes was similar to the Bagridae catfishes (Zhang et al., 2022) and Glyptosternoid fishes (Lv et al., 2018). In addition, the average Ka/Ks ratios for all PCGs were far lower than one, which indicates that they were all under strong purifying selection.

 

CONCLUSION

In the present study, we first collected a rare cavefish S. cyphotergous, and then determined the complete mitochondrial genome using Illumina sequencing. The mitogenome was 16,611 bp in length, which contained 37 genes and one control region, as is typical of teleost mitogenomes. All tRNAs could fold into the typical cloverleaf secondary structures, except for tRNA-Ser (AGY) that lacked the dihydrouracil arm. The ratio of non-synonymous and synonymous substitutions (Ka/Ks) of all the 13 PCGs were less than 1, indicating negative or purifying selection evolved in these genes. The evolutionary rate of ATP8 was the fastest and COI was the slowest. The reconstructed phylogenetic tree based on the 13 PCGs from the mitochondrial genome of 24 species supported that S. cyphotergous in this study was closely related to S. multipunctatus and S. punctatus, and all Sinocyclocheilus species in this research formed a solid monophyletic group and grouped into two major clades with strong support excluded S. jii. Finally, this study provides novel insights into the phylogeny of the Sinocyclocheilus fishes, and offers genetic basis for the conservation of S. cyphotergous.

ACKNOWLEDGMENTS

We thank Dr. Yunhai Yi for her valuable and constructive suggestions in revising the manuscript. We also thank three anonymous reviewers for their valuable comments that improved the quality of this article.

Funding

This work was supported by the Opening Foundation of the Key Laboratory of Sichuan Province for Fishes Conservation and Utilization in the Upper Reaches of the Yangtze River (NJTCCJSYSYS14), and the Neijiang Normal University Key Program Fund (2021ZD09).

IRB approval

This study was reviewed and approved by the IRB of Neijiang Normal University for being conducted in accordance with ethical guidelines for animal research.

Ethical statement

The experimental samples used for this study were collected and processed in accordance with the approval of Animal Experiment Ethics Committee in Neijiang Normal University.

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. OQ319607. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA916069, SRR22894312, and SAMN32411344, respectively.

Statement of conflict of interest

The authors have declared no conflict of interest.

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