Complete Mitochondrial Genome Sequence of Ruffe Acerina cernua (Perciformes, Percidae): Mitogenomic Characteristics and Phylogenetic Implications

Yuping Liu, Sige Wang and Tianyan Yang*

Fishery College, Zhejiang Ocean University, Zhoushan, Zhejiang, China, 316022

ABSTRACT

Acerina cernua is mainly distributed in Europe and Asia, but has recently invaded in North America and new areas of Europe. In China, it is native to the Irtysh River and Ulungur Lake, and considered to be an indigenous fish endemic to Xinjiang. In this paper, the complete mitochondrial genome (16607 bp) of A.cernua was sequenced by high-throughput sequencing technology, including 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes, a control region (CR) and a L-strand replication origin (OL), and its gene composition and order were similar to those of most teleosts. The A + T content of the whole mtDNA was 55.39 %, suggesting an obvious anti-G bias (16.64 %). The positive AT-skew (0.01) and negative GC-skew (-0.25) were revealed. The analysis of codon usage showed that NNA-type codons were used most frequently, which was consistent with the A bias at the third codon positions in PCGs. Three base mismatches were detected in the secondary structures of tRNAs, namely A-C, U-U and A-A, which mainly occurred in the amino acid receptor arm and TψC arm. The CR contained three different domains: extended termination-associated sequences, central conserved region (CSB-F, CSB-E and CSB-D) and conserved sequence region (CSB1, CSB2 and CSB3). Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic analyses were performed based on 12 PCGs. The similar topological structures confirmed that the relationship between Acerina and Sander was the closest.

Article Information

Received 30 March 2023

Revised 25 April 2023

Accepted 16 May 2023

Available online 21 July 2023

(early access)

Published 02 May 2024

Authors’ Contribution

YL and TY presented the concept of the study and wrote the manuscript. TY, YL and SW designed research, conducted experiments, explained research results, and wrote and revised manuscripts. TY helped write and revise the manuscript.

Key words

Acerina cernua, Mitogenome, Sequence analysis, Phylogeny

DOI: https://dx.doi.org/10.17582/journal.pjz/20230330030335

* Corresponding author: [email protected]

0030-9923/2024/0003-1389 $ 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

Mitochondria (mt) are important functional organelles in eukaryotic cells and mitochondrial DNA (mtDNA) is a small closed circular molecule, which is independent of the nuclear genome with a length of 15-20 kb. It normally contains 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, a L-strand replication origin (OL), as well as a large non-coding region, namely control region (CR) or D-loop region (Boore, 1999). Because of the advantages of maternal inheritance, simple structure, independent replication, high mutation rate and relatively stable probability of variation, mtDNA has been widely applied in genetic structure, phylogeny and phylogeography at various taxonomic levels, and has become a powerful tool to solve both the interspecific genetic differentiation and intraspecific genetic variation (Inoue et al., 2001; Kawahara et al., 2008; Perea et al., 2010; Imoto et al., 2013). In recent years, the advancement of DNA sequencing technology has provided great convenience for the rapid and accurate acquisition of fish mitogenome data. More and more complete mitochondrial DNA sequences of teleosts have been reported, which intensively accelerate the taxonomic and phylogenetic researches of bony fishes.

The ruffe Acerina cernua (Linnaeus, 1758), also known as Gymnocephalus cernua (Linnaeus, 1758), belongs to the family Percidae and order Perciformes. This small spiny freshwater fish is originated in the Northern Europe and northwest Asia, but inadvertently introduced into North America and Western Europe in the 1980s. Nowadays, it has expanded its natural distribution areas and widely spreads in the Great Lakes and major rivers in Europe as an invasive species (Guo, 2012; Kottelat and Freyhof, 2007). In China, it is an aboriginal fish that naturally occurs in Irtysh River and Ulungur Lake of Xinjiang (Zhu et al., 2014). Enormous amounts of researches centering on physiology, ecology, biology and population dynamics of this fish have been carried out until now (Van and Van, 1994; Zhu et al., 2014). However, the investigations into its genetic background are very limited, which seriously hinder the rational utilization of the germplasm resources of exotic species (Nesbø et al., 1998; Zhang et al., 2017; Li et al., 2019). In this study, high-throughput sequencing (HTS) technology was used to obtain the complete mtDNA sequence of A. cernua collected from Irtysh River in Xinjiang, China. By comparing with the mitogenomes of 15 species of Percidae, the molecular phylogenetic relationship of Percidae was further discussed. The results of this study are expected to provide reference information for germplasm identification and genetic diversity evaluation of A. cernua.

MATERIALS AND METHODS

Experimental sample and genomic DNA extraction

In this study, the samples of A. cernua were collected from Irtysh River in Xinjiang, China in September 2017. About 2g fresh muscle tissue from the dorsal fin base was cut into 1.5 mL EP centrifuge tubes, immersed in 95% alcohol for 48 hours, and then frozen at −20 °C. Genomic DNA was extracted by traditional chloroform-Tris saturated phenol method (Maniatis et al., 1985). The concentration of genomic DNA was determined by NanoDrop ND-1000 ultramicro spectrophotometer, and DNA integrity was detected by 1 % agarose gel electrophoresis.

High-throughput sequencing and gene annotation

The qualified DNA samples were randomly broken into fragments with the length of about 300 bp by Covaris ultrasonic disruptor to construct a small fragment genomic DNA library of A. cernua. Sequencing was commissioned to Hengchuang Gene Technology Co., Ltd (Shenzhen, China) based on Illumina Hiseq 2500 high-throughput sequencing platform. The measured clean reads were assembled by SOAPdenovo 2.04 software (http://soap.genomics.org.cn/soapdenovo.html) (Li et al., 2010), and the local assembly results were optimized based on the paired-end and overlap relationships of reads. GapCloser 1.12 software (http://soap.genomics.org.cn/soapdenovo.html) (Zhao et al., 2011) was used to make up and repair the gaps introduced in the process of splicing scaffold. Ultimately, the redundant segment sequences were removed to obtain the final assembly result. The mitogenome was extracted and assembled using NOVO Plasty software (Dierckxsens et al., 2017). The complete sequence obtained by splicing was uploaded to the MITOS web server (http://mitofish.aori.u-tokyo.ac.jp/) for annotation of protein-coding genes (PCGs), RNAs and non-coding regions, and then supplemented by manual alignment correction to finally determine the location and length of each gene.

Data analysis

The secondary structure of transfer RNA (tRNA) gene was predicted by online software tRNAscan-SE (http://trna.ucsc.edu/tRNAscan-SE/) (Lowe and Chan, 2016). Ribosomal RNA (rRNA) secondary structure was mapped using the Visusalise RNA 2D Structure (R2DT) tool on the RNAcentral website (https://rnacentral.org/) (The RNA central Consortium, 2019), and the free energy of different rRNA gene was calculated by RNAfold software (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). The nucleotide composition and codon usage were analyzed by MEGA-X software (Kumar et al., 2008). The AT-skew and GC-skew values were calculated and analyzed by Microsoft Excel software. The complete mitogenome sequences of 15 Percidae species were downloaded from GenBank database (https://www.ncbi.nlm.nih.gov/) (Table I). Using Lateolabrax maculatus (GenBank accession number: KT852999) and Micropterus salmoides (GenBank accession number: DQ536425) as outgroups, the phylogenetic tree was constructed based on concatenated sequences of 12 PCGs (except ND6 gene encoded by L-strand) after removing stop codons by using maximum likelihood (ML) (Guindon and Gascuel, 2003) and Bayesian inference (BI) (Huelsenbeck and Ronquist, 2001) methods, respectively.

 

Table I. List of 15 Percidae species and two outgroups used in this paper.

Species	A+T (%)	Length (bp)	GenBank accession No.
Sander vitreus	55.23	16858	KP013098
Sander lucioperca	55.63	16542	KP125333
Percina macrolepida	53.25	16602	DQ536430
Percina kusha	53.65	16626	OP238461
Percina freemanorum	53.64	16578	OP326604
Percina copelandi	53.13	16607	MW856850
Percina brevicauda	53.02	16608	MK778456
Perca schrenkii	54.93	16536	KR905930
Perca fluviatilis	55.10	16537	KM410088
Perca flavescens	55.38	16537	JX629442
Gymnocephalus cernua	55.45	16614	KM978956
Etheostoma spectabile	52.42	16539	MK243404
Etheostoma jessiae	52.20	16600	KY965072
Etheostoma chuckwachatte	54.00	16603	KY965071
Etheostoma caeruleum	52.90	16588	KY660678
Acerina cernua	55.39	16607	This study
Micropterus salmoides (outgroup)	53.48	16484	DQ536425
Lateolabrax maculatus (outgroup)	53.13	16723	KT852999

 

RESULTS AND DISCUSSION

General features of the mt genome of A. cernua

The complete mitogenome of A. cernua was 16607 bp in length, which was in the length range (16484-16858 bp) of the mitogenome of 15 reported Percidae fish (Table I). The mtDNA structure of A. cernua was highly conserved and conforms to the typical mitogenome structure of vertebrates (Miya et al., 2003), which contained 13 PCGs, 22 tRNA genes, 2 rRNA genes, and 2 major non-coding regions (Fig. 1, Table II). Among them, ND6 gene and 8 tRNAs (tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-SerUGA, tRNA-Glu, and tRNA-Pro) were encoded on the light-strand (L-strand), and the remainders were encoded by the heavy-strand (H-strand). Two non-coding regions included a light-strand replication origin (OL) (38 bp) between tRNA-Asn and tRNA-Cys, and a control region (CR) (944 bp) between tRNA-Pro and tRNA-Phe, respectively.

 

Table II. Features of the mitogenome of A. cernua.

Gene	Coding strand	Start	End	Length/ bp	Space or overlap/bp	Amino acid (aa)	Star codon	Stop codon	Anticodon	Anticodon site
tRNA-Phe (F)	H	1	68	68	0				GAA	31-33
12S rRNA	H	69	1017	949	0
tRNA-Val (V)	H	1018	1089	72	0				TAC	34-36
16S rRNA	H	1090	2781	1692	0
tRNA-LeuUAA (L1)	H	2782	2855	74	0				TAA	36-38
ND1	H	2856	3830	975	5	324	ATG	TAA
tRNA-Ile (I)	H	3836	3905	70	-1				GAT	31-33
tRNA-Gln (Q)	L	3905	3975	71	-1				TTG	33-35
tRNA-Met (M)	H	3975	4043	69	0				CAT	31-33
ND2	H	4044	5089	1046	0	348	ATG	TA
tRNA-Trp (W)	H	5090	5160	71	1				TCA	33-35
tRNA-Ala (A)	L	5162	5230	69	1				TGC	31-33
tRNA-Asn (N)	L	5232	5304	73	0				GTT	34-36
OL	L	5305	5342	38	0
tRNA-Cys (C)	L	5343	5408	66	0				GCA	29-31
tRNA-Tyr (Y)	L	5409	5479	71	1				GTA	33-35
COI	H	5481	7031	1551	0	516	GTG	TAA
tRNA-SerUGA (S1)	L	7032	7102	71	3				TGA	33-35
tRNA-Asp (D)	H	7106	7177	72	6				GTC	34-36
COII	H	7184	7874	691	0	230	ATG	T
tRNA-Lys (K)	H	7875	7948	74	1				TTT	35-37
ATP8	H	7950	8117	168	-10	55	ATG	TAA
ATP6	H	8108	8790	683	0	227	ATG	TA
COIII	H	8791	9575	785	0	261	ATG	TA
tRNA-Gly (G)	H	9576	9646	71	0				TCC	33-35
ND3	H	9647	9995	349	0	116	ATG	T
tRNA-Arg (R)	H	9996	10064	69	0				TCG	31-33
ND4L	H	10065	10361	297	-7	98	ATG	TAA
ND4	H	10355	11735	1381	0	460	ATG	T
tRNA-His (H)	H	11736	11804	69	0				GTG	31-33
tRNA-SerGCU (S2)	H	11805	11872	68	5				GCT	28-30
tRNA-LeuUAG (L2)	H	11878	11950	73	0				TAG	34-36
ND5	H	11951	13789	1839	-4	612	ATG	TAA
ND6	L	13786	14307	522	0	173	ATG	TAG
tRNA-Glu (E)	L	14308	14376	69	5				TTC	31-33
Cyt b	H	14382	15522	1141	0	380	ATG	T
tRNA-Thr (T)	H	15523	15594	72	-1				TGT	33-35
tRNA-Pro (P)	L	15594	15663	70	0				TGG	32-34
D-loop	H	15664	16607	944	0

 

The mitogenome of A. cernua had 9 internal spacer regions and 6 overlapping regions (Table II). The total length of the former was 28 bp, accounting for 0.169 % of the whole mitogenome, and the interval spacer region between tRNA-Asp and COII was the largest (6 bp). The total length of the latter was 24 bp, with the largest overlapping region detected between ATP8 and ATP6 (10 bp). The overall base composition was listed as: 27.83 % A, 27.98 % C, 27.55 % T, 16.64 % G (Table III). The percentages of A, T and C were basically not much different from those of published Percidae fishes, but the content of guanine was the least (Zhang et al., 2017; Li et al., 2019). The mitogenome of A. cernua owned a higher proportion of A+T (55.39 %), showing the obvious AT bias. This phenomenon also existed in other Percidae fishes, but the content varied slightly from species to species. The reason for this base preference might be related to natural mutations and selection pressures during the process of replication and transcription (Zhong et al., 2002).

Protein-coding genes (PCGs) and the codon usage bias

Base composition analysis indicated that the total sequence length of 13 PCGs was 11428 bp, occupying 68.81 % of the whole mitogenome, apparently showing an AT bias (55.11 %). The number of encoded amino acid residues amounted to 3800. Among all the PCGs, ND5 had the longest base sequence (1839 bp), encoding 612 amino acids. While, ATP8 was the shortest (168 bp), encoding only 55 amino acids. Excepting for COI starting with GTG, the other genes all used ATG as the initiation codon. The incomplete codon phenomenon was common in metazoan mitogenomes. In the present study, five genes (ND1, COI, ATP8, ND4L and ND5) ended with TAA, ND6 was terminated by TAG, and the remaining genes were incomplete codons (TA- in ND2, ATP6 and COIII; T-- in COII, ND3, ND4 and Cyt b) (Table II). Metazoan mt genomes usually present a clear strand bias in nucleotide composition, which can be measured as AT- and GC-skews (Perna and Kocher, 1995; Hassanin et al., 2005). The AT-skew and GC-skew values are important yardsticks

 

Table III. Composition and skewness of A. cernua mitogenome.

Gene name/ Codon site	Length/ bp	Base composition/ %						AT-skew	GC-skew
		T	C	A	G	A+T	C+G
ND1	975	31.49	28.00	24.92	15.59	56.41	43.59	-0.12	-0.28
ND2	1046	27.72	33.56	26.20	12.52	53.92	46.08	-0.03	-0.46
COI	1551	30.75	27.27	23.40	18.57	54.16	45.84	-0.14	-0.19
COII	691	29.23	26.34	27.79	16.64	57.02	42.98	-0.03	-0.23
ATP8	168	25.00	33.33	30.95	10.71	55.95	44.05	0.11	-0.51
ATP6	683	30.60	32.50	23.13	13.76	53.73	46.27	-0.14	-0.41
COIII	785	29.04	29.04	25.99	15.92	55.03	44.97	-0.06	-0.29
ND3	349	32.38	31.23	19.77	16.62	52.15	47.85	-0.24	-0.31
ND4L	297	28.62	32.66	22.56	16.16	51.18	48.82	-0.12	-0.34
ND4	1381	28.02	30.56	26.57	14.84	54.60	45.40	-0.03	-0.35
ND5	1839	28.87	28.98	28.93	13.21	57.80	42.20	0.00	-0.37
ND6	522	37.93	14.37	16.48	31.23	54.41	45.59	-0.39	0.37
Cyt b	1141	29.54	30.50	24.98	14.99	54.51	45.49	-0.08	-0.34
1st codon	-	21.09	27.56	25.23	26.12	46.32	53.68	0.09	-0.03
2nd codon	-	40.35	27.78	18.22	13.65	58.57	41.43	-0.38	-0.34
3rd codon	-	27.98	31.79	32.48	7.75	60.46	39.54	0.07	-0.61
PCGs	11428	29.80	29.04	25.31	15.85	55.11	44.89	-0.08	-0.29
rRNAs	2641	22.38	24.95	31.81	20.86	54.18	45.82	0.17	-0.09
tRNAs	1552	26.68	21.52	28.48	23.32	55.15	44.85	0.03	0.04
OL	38	21.05	31.58	10.53	36.84	31.58	68.42	-0.33	0.08
D-loop	944	32.20	19.17	31.57	17.06	63.77	36.23	-0.01	-0.06
mtDNA	16607	27.55	27.98	27.83	16.64	55.39	44.61	0.01	-0.25

 

for nucleotide differences between the heavy chain and the light chain. The larger the absolute values, the more noteworthy differences between the two (Perna and Kocher, 1995). In PCGs, the AT-skews were from -0.39 (ND6) to 0.11 (ATP8), and all the GC-skews were negative except ND6 (Table III). As the conventional preference in most mitogenomes, AT-skews are positive and GC-skews are negative, besides, the former (absolute value) is generally lower in magnitude than the latter (Fonseca et al., 2014). However, the AT-skews and GC-skews of most PCGs were negative in this study, indicating that the chain asymmetry in the nucleotide composition and the excess of cytosine and thymine in the H-strand (Table III, Fig. 2).

 

The amino acid composition and the relative synonymous codon usage (RSCU) were preliminarily analyzed. The results showed that among the 3795 amino acids encoded, Leu2 was the most frequently used one (14.45 %). It was speculated that this result was related to the fact that the transmembrane protein encoded by the mitochondrial gene was mainly composed of hydrophobic amino acids (Zou, 2008). The percentage of Cys was the least, only accounting for 0.61 % (Fig. 3). RSCU means the preference for the use of synonymous codon, and it is defined as the ratio of the observed value of the synonymous codon number to the expected value of codon occurrence (Behura and Severson, 2013). There were 30 preferred codons (RSCU ≥ 1) in 13 PCGs, in which the highest RSCU value (2.18) was the codon CGA encoding Arg. The codons AGA and AGG were not used, so the codons with base A at the third site except UUA, CCA and AGA were all preferred codons, which coincided with A bias at the third codon position in the PCGs. UAA and UAG were stop codons and didn’t encode any amino acids at all (Table IV).

 

Transfer and ribosomal RNA genes

The typical 22 tRNA genes were identified in the mt genome of A.cernua ranging from 66 bp (tRNA-Cys) to 74 bp (tRNA-Lys). The base contents of tRNAs were 28.48% A, 21.52% C, 26.68% T, and 23.32% G, respectively (Table III). All 21 tRNAs could fold into typical clover secondary structure, but only tRNA-Ser GCU lacked dihydrouracil (DHU) stem (Fig. 4). Three base mismatches (A-C, U-U and A-A) were detected in tRNAs. Except for mismatches in the anticodon arms of tRNA-Trp and tRNA-Ser GCU as well as the DHU stem of tRNA-Arg, the others mainly occurred on the amino acid receptor arms and the TψC arms. However, partial mismatches of these tRNA genes can be corrected by later RNA editing without causing amino acid transport disorders, and these mismatches help eliminating deleterious mutations (Tomita et al., 1996; Lynch, 1997). The length of DHU stems was almost 4 bp excluding tRNA-Val, tRNA-Tyr and tRNA-Ser UGA (3 bp). Only the TψC stems of tRNA-Phe, tRNA-Lys and tRNA-Gln were 4 bp in length. The number of bases in the TψC loop and anticodon loop is mostly 7, but it varied greatly in DHU loop, ranging from 5 to10.

The full lengths of 12S rRNA and 16S rRNA genes of A. cernua mitogenome were 949 bp and 1692 bp, respectively. Both of them were located regularly between tRNA-Phe and tRNA-Leu UUA, and separated by tRNA-Val (Table II). The AT content (54.18%) was higher than GC content (45.82%), and the A base accounted for the largest proportion (31.81%) in the rRNAs (Table III). The secondary structures of rRNAs gene were relatively conserved, forming multiple stem-loop structures of different sizes. The predicted free energy of 12S rRNA gene was -260.32 kcal/ mol, and the free energy of 16S rRNA gene was -475.35 kcal/ mol.

 

Table IV. The number of codons and RSCU in A. cernua.

Codon	Count	RSCU	Codon	Count	RSCU	Codon	Count	RSCU	Codon	Count	RSCU
UUU(F)	128	1.09	UCU(S1)	44	1.13	UAU(Y)	52	0.92	UGU(C)	7	0.61
UUC(F)	107	0.91	UCC(S1)	70	1.79	UAC(Y)	61	1.08	UGC(C)	16	1.39
UUA(L1)	110	0.99	UCA(S1)	57	1.46	UAA(*)	0	0	UGA(W)	105	1.75
UUG(L1)	9	0.08	UCG(S1)	9	0.23	UAG(*)	0	0	UGG(W)	15	0.25
CUU(L2)	180	1.62	CCU(P)	85	1.55	CAU(H)	35	0.64	CGU(R)	15	0.78
CUC(L2)	139	1.25	CCC(P)	83	1.51	CAC(H)	74	1.36	CGC(R)	11	0.57
CUA(L2)	179	1.61	CCA(P)	42	0.76	CAA(Q)	82	1.71	CGA(R)	42	2.18
CUG(L2)	51	0.46	CCG(P)	10	0.18	CAG(Q)	14	0.29	CGG(R)	9	0.47
AUU(I)	181	1.36	ACU(T)	60	0.79	AAU(N)	39	0.67	AGU(S2)	14	0.36
AUC(I)	86	0.64	ACC(T)	126	1.66	AAC(N)	77	1.33	AGC(S2)	40	1.03
AUA(M)	91	1.24	ACA(T)	110	1.45	AAA(K)	66	1.76	AGA(*)	0	0
AUG(M)	56	0.76	ACG(T)	7	0.09	AAG(K)	9	0.24	AGG(*)	0	0
GUU(V)	80	1.45	GCU(A)	79	0.89	GAU(D)	28	0.74	GGU(G)	37	0.60
GUC(V)	49	0.89	GCC(A)	143	1.62	GAC(D)	48	1.26	GGC(G)	80	1.30
GUA(V)	71	1.29	GCA(A)	120	1.36	GAA(E)	71	1.42	GGA(G)	85	1.38
GUG(V)	20	0.36	GCG(A)	12	0.14	GAG(E)	29	0.58	GGG(G)	44	0.72

 

Commonly, the lower absolute value of the free energy means the more stable the molecular structure (Zuker and Stiegler, 1981). It indicated that the 12S rRNA was more conservative than 16S rRNA. Because the evolutionary rate of 12S rRNA was relatively low in the mitogenome, it was suitable for the study of the evolutionary relationship of the family and above classification system (Song et al., 2008).

The rRNA secondary structures were predicted refering to the Salmo salar mitochondrial 12S rRNA (b.16.m. S. salar) file provided by CRW (Cannone et al., 2002) and Homo sapiens mitochondrial 16S rRNA (mHS_LSU_3D) file provided by Ribo Vision (Bernier et al., 2014), respectively. The 12S rRNA secondary structure of A. cernua mitochondrial DNA contained four domains (Fig. 5A), of which domain I and domain II were variable regions, and domain III and domain IV were conserved regions. The secondary structure of 16S rRNA comprised six domains (Fig. 5B), of which the I − III and VI domains were variable regions, and IV and V domains were conserved regions. In contrast with S. salar, there were base substitutions in both four regions of 12S rRNA, but base insertion and reposition were just observed in domain I and domain IV. Compared with H. sapiens, due to the tremendous differences between two species, the base substitution, insertion and reposition of A. cernua 16S rRNA appeared more frequently. The reposition and insertion were only discovered in the loop region. Therefore, the sequence of the rRNA stem region was considered to be relatively conservative than the sequence of the loop region.

Non-coding regions

The OL was located inside the WANCY tRNA cluster (tRNA-Trp, tRNA-Ala, tRNA-Asn, tRNA-Cys and tRNA-Tyr) with a length of 38 bp. The AT content of OL was 31.58 %, versus 68.42 % of the GC content, showing obvious GC preference. Just like most vertebrates, this small DNA fragment could be folded into a stable stem-loop structure with the stem and loop lengths of 24 bp and 14 bp, respectively (Macey et al., 1997; Zhang et al., 2014; Shi et al., 2015). The highly conserved block (5 ‘ -CCCCGG-3 ‘) was also found in tRNA-Cys gene after this sequence, suggesting that it was involved in regulating genome replication and transcription (Boore, 1999; Hixson et al., 1986).

Another non-coding region CR is proved to be the fastest evolutionary sequence in mtDNA and has been widely used in population genetics and molecular systematics researches (Xiao and Zhang, 2000; Liu et al., 2008). The CR was located between tRNA-Pro and tRNA-Phe, with a length of 931 bp and the AT content of 63.77 %. The mitochondrial CR sequence can be divided into three distinct domains: extended termination-associated sequences (ETAS), central conserved domain (CD), and conserved sequence block (CSB). Among them, the central conserved domain can be further organized into CSB-F, CSB-E, CSB-D, CSB-C and CSB-B regions, and the conserved sequence block also can fall into CSB1, CSB2 and CSB3 blocks (Lee et al., 1995; Li et al., 1996; Liu et al., 2008).

Reference to a variety of CR sequences in the order Perciformes, the length of ETAS was identified to be 364 bp in A. cernua. It contained two repetitive motif “TACAT”

 

and an inverse complementary sequence “ATGTA”, which could form a hairpin structure. A tandem repeat sequence “CAAGT ATTTG” was found in ETAS, and it also appeared in sturgeons (Zhang et al., 2000), Coilia (Tang et al., 2007), pikeperches (Faber and Stepien, 1998) and other fishes. The similar conserved sequences, corresponding to CSB-F, CSB-E and CSB-D were defined in CD, in which CSB-E could be identified by “GTGGG”-box sequence. It is reported that CSB-E and CSB-D are very similar and highly conserved among various Perciformes fishes (Wang, 2008; Zhao et al., 2016). Here, a highly conserved sequence “TCTTT TTTTT TTTTT TTCCT TTC” was also recognized, which was found in many fish control regions and was similar to a conserved fragment (CSB-B) in mammalian control regions (Southern et al., 1988). The sequence length of CSB (including CSB1, CSB2 and CSB3) was 220 bp. Among them, CSB1 is related to the initiation of mitochondrial DNA replication, so the conserved sequence region is considered to be the most critical part of the entire CR (Su et al., 2012) (Fig. 6).

 

 

Phylogenetic analysis

Considering that only the ND6 gene was located on the L-strand and the heterogeneity of base composition was likely to lead to poor phylogenetic analysis (Miya et al., 2003), therefore, 12 PCGs sequences encoded by H-strand were adopted to explore the phylogenetic relationships among Perciformes species. The topological trees were separately constructed by ML and BI methods (Fig. 7). The results showed that two trees possessed the similar topological structures, and species from the same genus clustered into the one evolutionary branch. Because G. cernua was regarded as synonyms of A. cernua, they were clustered into a separate branch both in the ML tree and BI tree. The genera Acerina and Sander first gathered into a small branch, and then converged together with genus Perca. Therefore, Acerina and Sander had the closest genetic relationship, followed by Perca. By contrast, Acerina and Percina, Acerina and Etheostama were distantly related. Our result was consistent with the phylogenetic relationship constructed by Li et al. (2019) using the Neighbour-joining (NJ) method.

CONCLUSION

In summary, the complete mitogenome of A. cernua was determined by using High-throughput sequencing. The mitogenome (16607 bp) had similar gene order and orientation to those of other Percidae fishes. Based on 12 PCGs sequences, two kinds of molecular evolutionary trees were constructed and the phylogenetic status of A. cernua were also discussed, which provided new clues and references for the study of species evolution, classification and genetic diversity of this fish group.

ACKNOWLEDGEMENTS

The authors would like to express their sincere thanks to Peng Chen in Xinjiang Fisheries Research Institute for his help in sample collecting.

Funding

This study was supported by the National Innovation and Entrepreneurship Training Program for College Students (No. 202210340023); Science and Technology Innovation Project of College Students in Zhejiang Province (No. 2023R411006).

IRB approval and ethical statement

The animal study protocol was approved by the Ethics Committee of Zhejiang Ocean University (protocol code: ZJOU-ECAE20211876, date of approval: 16 December 2021).

Statement of conflict of interest

The authors have declared no conflict of interest.

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