Abstract

Mitochondrial genome is a powerful molecule marker to provide information for phylogenetic relationships and revealing molecular evolution in ichthyological studies. Sebastiscus species, a marine rockfish, are of essential economic value. However, the taxonomic status and phylogenetic relationships of Sebastidae have been controversial so far. Here, the mitochondrial genomes (mitogenomes) of three species, S. tertius, S. albofasciatus, and S. marmoratus, were systemically investigated. The lengths of the mitogenomes’ sequences of S. tertius, S. albofasciatus, and S. marmoratus were 16910, 17056, and 17580 bp, respectively. It contained 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNA (tRNA) genes, and one identical control region (D-loop) among the three species. The genetic distance and Ka/Ks ratio analyses indicated 13 PCGs were suffering purifying selection and the selection pressures were different from certain deep-sea fishes, which were most likely due to the difference in their living environment. The phylogenetic tree was constructed by Bayesian Inference (BI) and Maximum Likelihood (ML). Most interestingly, the results indicated that Sebastidae and Scorpaenidae were grouped into a separate branch, so the taxonomic status of Sebastidae should be classified into subfamily Sebastinae. Our results may lead to a taxonomic revision of Scorpaenoidei.

Introduction

The family Sebastidae belongs to Scorpaeniformes, commonly known as rockfishes or false kelpfishes, comprises 7 genera and approximately 133 species, and it is a diverse and economically important group distributed in Atlantic, Indian, and Pacific Oceans [1]. Sebastidae has become one of the most important marine fishes due to its huge economic value and high biodiversity. However, there is still no consensus about the taxonomic status of Sebastidae up to now. Some authors considered Sebastidae as a family of the order Scorpaeniformes [2–6]. By contrast, some authorities recognized it as a subfamily of Scorpaenidae [7–12]. Besides, the databases that are widely referenced also showed different results, like FishBase (https://www.fishbase.se) acknowledged the validity of Sebastidae, but Integrated Taxonomic Information System (ITIS) (https://www.itis.gov) is opposed to this opinion. Before the conclusion, it will be unified use of the appellation ‘Sebastidae’ in the present study.

The genus Sebastiscus is mainly distributed in the western Pacific [13,14]. Despite their diversity, abundance, and economic importance, our understanding of the relationships within the genus remains limited. It was classified as part of the genus Sebastes until 1904 when Jordan and Starks identified it as a separate genus by recognizing the development of the air bladders [13]. Besides, Matsubara used the structure of the suborbital bone and the number of vertebrae as the diagnosis traits of Sebastiscus [15,16]. Although Barsukov and Chen classified Sebastiscus as a subgenus of Sebastes, it is commonly regarded as an independent genus at present [17–21]. Three valid species and a newly reported species are included in genus Sebastiscus: S. albofasciatus [22], S. marmoratus [23], S. tertius [17], and S. vibrantus [6]. It is worth noting that S. marmoratus is widely distributed in the northwestern Pacific Ocean and the remaining species are likely to be confined to the warm waters of East Asia and Indonesia [6,24].

Mitochondrial genomes (mitogenomes) have become a powerful molecule marker of species classification, population genetics, molecular systematic geography, molecular ecology, and other fields [25–29]. Small size, maternal inheritance, compact gene arrangement, high conservatism, and simple structure are the main features of mitochondrial genomes [30–32]. The structure, characteristics, and properties of the mitochondrial genomes of fish have been studied more and more widely since Johansen et al. completed the complete sequence determination of the mitochondrial genome of Gadus morhua [33].

From the above, there have been so many meaningful achievements about phylogenetic analyses and fish taxa through research of mtDNA. However, there exists some limitations in short mitochondrial gene fragments in discussing and resolving more complicated phylogenetic relationships in many fish lineages [34]. For these limitations, the longer DNA sequences liked complete mitochondrial genomes which have additional informative sites will have better ways to solve these higher level relationships and deeper branches thoroughly [35]. Therefore, the mitochondrial genomes applied in this study may help recognize the evolutionary and relationships of the family Sebastidae and verify the accuracy of traditional taxonomy.

In the present study, we sequenced and annotated three complete mitochondrial genomes of Sebastiscus species (S. tertius, S. albofasciatus, and S. marmoratus) and compared them with each other. The characteristics were described to evaluate the variation and conservation in the mitochondrial genome among Sebastiscus species. The relative synonymous codon usage (RSCU) and AT skew value of protein-coding genes (PCGs) were analyzed to better understand the functional inference of related genes. Moreover, the phylogenetic analyses among Sebastiscus species were performed and related species in Scorpaenoidei to preferably discuss the taxonomic status of Sebastidae. All information reported in this article will help supplement and enhance the limited molecular data available for Sebastiscus species, and provide the essential evidence for the taxonomic status of Sebastidae.

Materials and methods

Sampling and DNA extraction

The sample of rockfishes were collected using hook-and-line fishing from the coastal waters of Zhoushan in China (coordinates: 30.0607°N, 122.3546°E) during April 2018, coastal waters of Qingdao in China (coordinates: 35.7607°N, 120.2016°E) during May 2019, and coastal waters of Kozagawa in Japan (coordinates: 33.4145°N, 135.7524°E) during June 2019, respectively. All samples were identified based on morphological characteristics (Katoh and Tokimura (2001) [10]; Morishita et al. (2018) [6]) and stored at −20°C for further study. Muscle tissue was taken from the tails of each sample and digested using proteinase K (Merck, Germany). Genomic DNA was isolated following a standard phenol–chloroform method and detected by 2.0% agarose electrophoresis. DNA precipitation was dissolved in double-distilled water and stored at 4°C after concentration quantification. The study protocol was approved by the Experimental Animal Ethics Committee of the Ocean University of China.

PCR amplification and sequencing

Three Sebastiscus mitogenomes were amplified with 34 pairs of Sebastiscus-specific universal primer sets (Supplementary Table S1) and 4 pairs of specific primer sets (Supplementary Table S2) which were designed based upon the mitochondrial genome sequence of S. marmoratus (accession no. KF667491) by Primer Premier 6.0 [36]. We used the normal PCR method with Takara Taq DNA polymerase (Takara, China) in a 25-µl reaction volume including 17.5 µl of ultrapure water, 2.5 µl of 10× PCR buffer, 2 µl of dNTPs, 1 µl of each primer (5 µmol/l), 0.15 µl of Taq polymerase, and 1 µl of DNA template. PCR amplification was carried out in a Biometra thermal cycler (Göttingen, Germany) under the following conditions: 5 min initial denaturation at 95°C, and 35 cycles of 45 s at 94°C for denaturation, 45 s at 53°C for annealing, and 45 s at 72°C for an extension, and a final extension at 72°C for 10 min. All PCR products were sequenced in both directions using the primer-walking method by Qingdao Qingke Biotechnology Co. Ltd. (Qingdao, China).

Mitogenome annotation and sequence analyses

SeqMan (DNAStar, U.S.A.) software was used to manually correct and compare the contiguous sequence fragments, then splice the complete mitogenome sequence and calculate the full length of mitogenome. The starting and ending positions of each gene were determined by comparing the full mitogenome sequence of S. marmoratus (Accession no. KF667491) published in GenBank (https://www.ncbi.nlm.nih.gov/genbank/). Online software tRNAscan-SE2.0 (https://lowelab.ucsc.edu/tRNAs can-SE) was used to identify the transfer RNA (tRNA) gene and predict the secondary structure diagram of tRNA [37]. The online software Tandem Repeats Finder [38] was used to search and analyze the tandem repeats in the control region. The OL and tandem repeat structures were simulated and drawn by RNAstructure6.2 software. All complete mitogenomes were preliminarily annotated and drawn the mitochondrial genome map by Mitofish (https://mitofish.aori.u-tokyo.ac.jp) [39,40].

The composition of complete mitochondrial genome sequence and each segment (including the non-coding region, ribosomal RNA (rRNA), tRNA, and the PCGs) were calculated by MEGA5.0 software [41], and the PCG codon and base content were determined. Composition skewness of each segment was calculated by the following formulas: AT-skew = (A − T)/(A + T); GC-skew = (G − C)/(G + C).

Phylogenetic analyses

In order to discuss the phylogenetic relationships of Sebastidae and explore the taxonomic status of Sebastiscus in Scorpaenoidei, mitogenomes of 33 previously sequenced Scorpaenoidei and 2 previously sequenced Gobiidae species (with the latter as the outgroup taxon; Table 1) were used in the phylogenetic analyses. We used the nucleotide sequences of the 13 PCGs as the dataset to construct the phylogenetic tree. Sequences were aligned using SeqMan from DNAStar software (U.S.A.). The optimal model for nucleotide sequences was estimated by MEGA 5.0 (Tamura et al. 2011). TN93 + G + I captured the minimum values of Bayesian Information Criterion (BIC) and it was considered to be the best model for phylogenetic tree construction. The Maximum Likelihood (ML) phylogenetic tree was constructed by MEGA 5.0 software with 1000 replicates of bootstrap and the Bayesian Inference (BI) analysis was inferred by the software of MrBayes 3.2.6 based on 10000000 generations [42]. The divergence time was predicted by MEGA 10 [43] with the ML method of RelTime. The calibration of divergence times was obtained from online Time Tree database (http://www.timetree.org/) [44].

Table 1
Information of the complete mitogenome sequences cited in this study
Family (Latin name)Species (Latin name)GenBank numberLength (bp)
Sebastidae Sebastes oblongus KF836441 16396 
 Sebastes fasciatus KX897946 16400 
 Sebastes thompsoni KJ834064 16405 
 Sebastes trivittatus KJ834062 16409 
 Sebastes pachycephalus KF836442 16415 
 Sebastes longispinis KJ834061 16445 
 Sebastes taczanowskii KJ525744 16452 
 Sebastes hubbsi KJ525745 16453 
 Sebastes vulpes KJ525743 16462 
 Sebastes owstoni KJ834063 16465 
 Sebastes inermis KF725093 16504 
 Sebates schlegelii AY491978 16525 
 Sebastes minor MH378782 16850 
 Sebastes rubrivinctus MH378777 16896 
 Sebastes nigrocinctus MH378778 16922 
 Sebastes aleutianus MH378781 17038 
 Sebastes steindachneri MH378779 17109 
 Sebastes koreanus KJ775792 17606 
 Helicolenus avius NC020349 16651 
 Helicolenus hilgendorfi AP002948 16728 
Scorpaenidae Pterois miles LK022697 16497 
 Pterois volitans KJ739816 16500 
 Parapterois heterura LC493917 16475 
 Scorpaenopsis ramaraoi LC493915 16972 
 Scorpaenopsis cirrosa KR701907 16966 
Peristediidae Satyrichthys amiscus AP004441 16526 
Triglidae Chelidonichthys kumu KY379222 16495 
 Pterygotrigla hemisticta LC493913 16499 
 Pterygotrigla ryukyuensis LC495487 16508 
 Lepidotrigla kanagashira MK784116 16504 
 Lepidotrigla guentheri LC493914 16509 
 Lepidotrigla hime MN104592 16606 
 Lepidotrigla microptera KY012348 16610 
Gobiidae Parapocryptes serperaster KT965855 17243 
 Boleophthalmus pectinirostris NC016195 17111 
Family (Latin name)Species (Latin name)GenBank numberLength (bp)
Sebastidae Sebastes oblongus KF836441 16396 
 Sebastes fasciatus KX897946 16400 
 Sebastes thompsoni KJ834064 16405 
 Sebastes trivittatus KJ834062 16409 
 Sebastes pachycephalus KF836442 16415 
 Sebastes longispinis KJ834061 16445 
 Sebastes taczanowskii KJ525744 16452 
 Sebastes hubbsi KJ525745 16453 
 Sebastes vulpes KJ525743 16462 
 Sebastes owstoni KJ834063 16465 
 Sebastes inermis KF725093 16504 
 Sebates schlegelii AY491978 16525 
 Sebastes minor MH378782 16850 
 Sebastes rubrivinctus MH378777 16896 
 Sebastes nigrocinctus MH378778 16922 
 Sebastes aleutianus MH378781 17038 
 Sebastes steindachneri MH378779 17109 
 Sebastes koreanus KJ775792 17606 
 Helicolenus avius NC020349 16651 
 Helicolenus hilgendorfi AP002948 16728 
Scorpaenidae Pterois miles LK022697 16497 
 Pterois volitans KJ739816 16500 
 Parapterois heterura LC493917 16475 
 Scorpaenopsis ramaraoi LC493915 16972 
 Scorpaenopsis cirrosa KR701907 16966 
Peristediidae Satyrichthys amiscus AP004441 16526 
Triglidae Chelidonichthys kumu KY379222 16495 
 Pterygotrigla hemisticta LC493913 16499 
 Pterygotrigla ryukyuensis LC495487 16508 
 Lepidotrigla kanagashira MK784116 16504 
 Lepidotrigla guentheri LC493914 16509 
 Lepidotrigla hime MN104592 16606 
 Lepidotrigla microptera KY012348 16610 
Gobiidae Parapocryptes serperaster KT965855 17243 
 Boleophthalmus pectinirostris NC016195 17111 

Results and discussion

Mitogenome organization and composition

The complete mitochondrial genomes of S. albofasciatus (Accession no. MT117230), S. tertius (Accession no. MT117231), and S. marmoratus (Accession no. MT789709) in GenBank were 17056, 16910, and 17580 bp in length, respectively (Figure 1). The size variation of the three mitogenomes was mainly caused by the differences in the lengths of the non-coding regions. Of all 36 sequenced Scorpaenoidei mitogenomes, the length of the mitogenome of Sebastes koreanus (17606 bp) was the longest, whereas that of Sebastes oblongus (16396 bp) was the shortest. The mitogenome lengths of five Scorpaenoidei species were longer (>17000 bp) because of a longer control region (>1300 bp). The mitogenome of S. albofasciatus contained the typical 37 genes (13 PCGs, 22 tRNAs, and 2 rRNAs), 1 control region, and 1 OL. The mitogenome of S. tertius and S. marmoratus had the same composition (Table 2). Most mitochondrial genes were encoded on the H-strand, except for ND6 and eight tRNA (Glu, Ala, Asn, Cys, Tyr, Ser-UCN, Gln, and Pro) genes that were encoded on the L-strand. The nucleotide composition of S. albofasciatus, S. tertius, and S. marmoratus mitogenomes had a higher A+T bias of 55.15, 55.04, and 55.30%, respectively, and both showed positive AT-skew and negative GC-skew (Figure 2 and Supplementary Table S3).

Mitochondrial genome maps of S. tertius (A) S. albofasciatus (B) and S. marmoratus (C)

Figure 1
Mitochondrial genome maps of S. tertius (A) S. albofasciatus (B) and S. marmoratus (C)

The innermost circle of the images represents GC% per every 5 bp of the mitogenome; the darker lines are, the higher their GC% are.

Figure 1
Mitochondrial genome maps of S. tertius (A) S. albofasciatus (B) and S. marmoratus (C)

The innermost circle of the images represents GC% per every 5 bp of the mitogenome; the darker lines are, the higher their GC% are.

The nucleotide skewness of three species of Sebastisucs

Figure 2
The nucleotide skewness of three species of Sebastisucs

(A) S. tertius; (B) S. albofasciatus; (C) S. marmoratus. The incomplete T-/TA- of the stop codon is not included.

Figure 2
The nucleotide skewness of three species of Sebastisucs

(A) S. tertius; (B) S. albofasciatus; (C) S. marmoratus. The incomplete T-/TA- of the stop codon is not included.

Table 2
Summary of gene/element feature of S. tertius (ST), S. albofasciatus (SA) and S. marmoratus (SM)
Gene/RegionPosition startPosition endSize (bp)Intervening spacer (bp)*Amino acidInitial codonTerminal codonStrandLetter code
STSASMSTSASMSTSASMSTSASMSTSASMSTSASMSTSASM
tRNAPhe 68 68 68 68 68 68          
12S rRNA 69 69 69 1015 1014 1014 947 946 946           
tRNAVal 1016 1015 1015 1087 1086 1086 72 72 72          
16S rRNA 1088 1087 1087 2779 2778 2778 1692 1692 1692           
tRNALeu(UUR) 2780 2779 2779 2853 2852 2852 74 74 74          L1 
ND1 2854 2853 2853 3828 3827 3827 975 975 975 324 324 324 ATG ATG ATG TAG TAA TAA  
tRNAIle 3832 3833 3831 3903 3902 3902 72 70 72 −2 −1 −2          
tRNAGln 3902 3902 3901 3972 3972 3972 71 71 72 −1 −1 −2          
tRNAMet 3972 3972 3971 4042 4042 4041 71 71 71          
ND2 4043 4043 4042 5088 5088 5087 1046 1046 1046 348 348 348 ATG ATG ATG TA TA TA  
tRNATrp 5089 5089 5088 5159 5160 5158 71 71 71          
tRNAAla 5161 5162 5160 5229 5230 5228 69 69 69          
tRNAAsn 5231 5232 5230 5303 5304 5302 73 73 73          
OL 5305 5306 5303 5341 5342 5340 37 37 38 −3 −3 −4           
tRNACys 5339 5340 5337 5405 5406 5403 67 65 67          
tRNATyr 5408 5409 5406 5478 5479 5476 69 71 71          
COI 5480 5481 5478 7030 7031 7028 1551 1551 1551 516 516 516 GTG GTG GTG TAA TAA TAA  
tRNASer(UCN) 7031 7032 7029 7101 7102 7099 71 69 71          
tRNAAsp 7105 7106 7103 7177 7178 7175 73 73 73          
COII 7184 7185 7182 7874 7875 7872 691 691 691 230 230 230 ATG ATG ATG  
tRNALys 7875 7876 7873 7948 7949 7946 74 74 74          
ATPase8 7950 7951 7948 8117 8118 8115 168 168 168 −10 −10 −10 55 55 55 ATG ATG ATG TAA TAA TAA  
ATPase6 8108 8109 8106 8790 8791 8788 683 683 683 227 227 227 ATG ATG ATG TA TA TA  
COIII 8791 8792 8789 9575 9576 9573 785 785 785 261 261 261 ATG ATG ATG TA TA TA  
tRNAGly 9576 9577 9574 9647 9648 9645 72 72 72          
ND3 9648 9649 9646 9996 9997 9994 349 349 349 116 116 116 ATG ATG ATG  
tRNAArg 9997 9998 9995 10065 10066 10063 69 69 69          
ND4L 10066 10067 10064 10362 10363 10360 297 297 297 −7 −7 −7 98 98 98 ATG ATG ATG TAA TAA TAA  
ND4 10356 10357 10354 11736 11737 11734 1381 1381 1381 460 460 460 ATG ATG ATG  
tRNAHis 11737 11738 11735 11805 11806 11803 69 69 69          
tRNASer(AGY) 11806 11807 11804 11873 11874 11871 68 72 68          
tRNALeu(CUN) 11878 11879 11876 11950 11951 11948 73 73 73          L2 
ND5 11951 11952 11949 13789 13790 13787 1839 1839 1839 −4 −4 −4 612 612 612 ATG ATG ATG TAA TAA TAA  
ND6 13786 13787 13784 14307 14308 14305 522 522 522 173 173 173 ATG ATG ATG TAG TAG TAG  
tRNAGlu 14308 14309 14306 14376 14377 14374 69 69 69          
Cyt b 14383 14384 14381 15523 15524 15521 1141 1141 1141 380 380 380 ATG ATG ATG  
tRNAThr 15524 15525 15522 15595 15596 15593 72 73 72 −1 −1 −1          
tRNAPro 15595 15596 15593 15664 15665 15662 70 70 70          
D-loop 15665 15666 15663 16910 17056 17580 1246 1391 1918              
Gene/RegionPosition startPosition endSize (bp)Intervening spacer (bp)*Amino acidInitial codonTerminal codonStrandLetter code
STSASMSTSASMSTSASMSTSASMSTSASMSTSASMSTSASM
tRNAPhe 68 68 68 68 68 68          
12S rRNA 69 69 69 1015 1014 1014 947 946 946           
tRNAVal 1016 1015 1015 1087 1086 1086 72 72 72          
16S rRNA 1088 1087 1087 2779 2778 2778 1692 1692 1692           
tRNALeu(UUR) 2780 2779 2779 2853 2852 2852 74 74 74          L1 
ND1 2854 2853 2853 3828 3827 3827 975 975 975 324 324 324 ATG ATG ATG TAG TAA TAA  
tRNAIle 3832 3833 3831 3903 3902 3902 72 70 72 −2 −1 −2          
tRNAGln 3902 3902 3901 3972 3972 3972 71 71 72 −1 −1 −2          
tRNAMet 3972 3972 3971 4042 4042 4041 71 71 71          
ND2 4043 4043 4042 5088 5088 5087 1046 1046 1046 348 348 348 ATG ATG ATG TA TA TA  
tRNATrp 5089 5089 5088 5159 5160 5158 71 71 71          
tRNAAla 5161 5162 5160 5229 5230 5228 69 69 69          
tRNAAsn 5231 5232 5230 5303 5304 5302 73 73 73          
OL 5305 5306 5303 5341 5342 5340 37 37 38 −3 −3 −4           
tRNACys 5339 5340 5337 5405 5406 5403 67 65 67          
tRNATyr 5408 5409 5406 5478 5479 5476 69 71 71          
COI 5480 5481 5478 7030 7031 7028 1551 1551 1551 516 516 516 GTG GTG GTG TAA TAA TAA  
tRNASer(UCN) 7031 7032 7029 7101 7102 7099 71 69 71          
tRNAAsp 7105 7106 7103 7177 7178 7175 73 73 73          
COII 7184 7185 7182 7874 7875 7872 691 691 691 230 230 230 ATG ATG ATG  
tRNALys 7875 7876 7873 7948 7949 7946 74 74 74          
ATPase8 7950 7951 7948 8117 8118 8115 168 168 168 −10 −10 −10 55 55 55 ATG ATG ATG TAA TAA TAA  
ATPase6 8108 8109 8106 8790 8791 8788 683 683 683 227 227 227 ATG ATG ATG TA TA TA  
COIII 8791 8792 8789 9575 9576 9573 785 785 785 261 261 261 ATG ATG ATG TA TA TA  
tRNAGly 9576 9577 9574 9647 9648 9645 72 72 72          
ND3 9648 9649 9646 9996 9997 9994 349 349 349 116 116 116 ATG ATG ATG  
tRNAArg 9997 9998 9995 10065 10066 10063 69 69 69          
ND4L 10066 10067 10064 10362 10363 10360 297 297 297 −7 −7 −7 98 98 98 ATG ATG ATG TAA TAA TAA  
ND4 10356 10357 10354 11736 11737 11734 1381 1381 1381 460 460 460 ATG ATG ATG  
tRNAHis 11737 11738 11735 11805 11806 11803 69 69 69          
tRNASer(AGY) 11806 11807 11804 11873 11874 11871 68 72 68          
tRNALeu(CUN) 11878 11879 11876 11950 11951 11948 73 73 73          L2 
ND5 11951 11952 11949 13789 13790 13787 1839 1839 1839 −4 −4 −4 612 612 612 ATG ATG ATG TAA TAA TAA  
ND6 13786 13787 13784 14307 14308 14305 522 522 522 173 173 173 ATG ATG ATG TAG TAG TAG  
tRNAGlu 14308 14309 14306 14376 14377 14374 69 69 69          
Cyt b 14383 14384 14381 15523 15524 15521 1141 1141 1141 380 380 380 ATG ATG ATG  
tRNAThr 15524 15525 15522 15595 15596 15593 72 73 72 −1 −1 −1          
tRNAPro 15595 15596 15593 15664 15665 15662 70 70 70          
D-loop 15665 15666 15663 16910 17056 17580 1246 1391 1918              

Intervening spacer* (bp): positive values indicate the interval sequence of adjacent genes, and negative values indicate the overlapping of adjacent genes. H represents heavy strand and L represents light strand.

PCGs and codon usages

All the 13 PCGs in the mitogenomes of the three rockfishes were similar to those of other vertebrates. Twelve PCGs (ND1, ND2, COI, COII, ATP8, ATP6, COIII, ND3, ND4L, ND4, ND5, and CYTB) were coded on the heavy strand (H-strand) and the remaining one (ND6) was coded on the light strand (L-strand). The length, codon usage, and A+T content of PCGs in the S. albofasciatus, S. tertius, and S. marmoratus mitogenomes were nearly identical. Among the three mitogenomes, all the 13 PCGs of Sebastiscus species encoded 3800 amino acids in total. All the PCGs used the initiation codon ATG except COI used GTG. ATG is an accepted conventional initiation codon for many Osteichthyes mitogenomes including among Scorpaeniformes fishes [45,46]. At the same time, GTG was commonly used as the initiation codon of COI in many other Osteichthyes mitogenomes [47–51]. All three mitogenomes have the same termination codon for 11 PCGs (ND2, COII, ATP8, ATP6, COIII, ND3, ND4L, ND4, ND5, ND6, and CYTB). TAA were commonly used as the termination codons, although the incomplete termination codons T or TA were found in ND2, COII, ATP6, COIII, ND3, ND4, and CYTB in all three mitogenomes. The incomplete termination codon has also been found in all other sequenced Osteichthyes species [48–52]. It has been confirmed that the incomplete termination codons could act as the complete functional termination codons in polyadenylation processes and polycistronic transcription cleavage [29,51,53]. TAG was used as the termination codon of ND6 in the three Sebastiscus mitogenomes. Additionally, in the S. tertius mitogenome, ND1 used TAG as the termination codon, too. Although TAG is the typical termination codon in many mitogenomes, it is not used frequently, due to the high percentage of AT nucleotide use by the PCGs [32].

The average AT contents of the 13 PCGs in S. albofasciatus and S. tertius were 54.50 and 54.40%, respectively, and both were similar values to that of S. marmoratus (54.54%). The PCGs encoded by the H-strand displayed T-skews (T > A) and C-skews (C > G) whereas the L-strand displayed T-skews and G-skews (G >C). We calculated the RSCU of the three Sebastiscus species mitogenomes (Figure 3 and Supplementary Table S4) and the result showed that the frequency of using NNA and NNC (N represents A, T, C, G) were higher than NNT and NNG in S. albofasciatus, S. tertius, and S. marmoratus. The most frequent amino acids in the coding sequences of S. albofasciatus, S. tertius, and S. marmoratus mitochondrial proteins were Leu (CUN), Ala, and Thr (>290) (Supplementary Figure S1). These three amino acids were also frequently used in other Osteichthyes mitogenomes [48–51]. Moreover, the minimally used amino acid in the three mitogenomes was Cys (<30).

The RSCU in three Sebastiscus mitogenomes

Figure 3
The RSCU in three Sebastiscus mitogenomes

The RSCU of the mitogenome in S. tertius (A), S. albofasciatus (B), and S. marmoratus (C). Codon families are labeled on the x-axis. The termination codon is not given.

Figure 3
The RSCU in three Sebastiscus mitogenomes

The RSCU of the mitogenome in S. tertius (A), S. albofasciatus (B), and S. marmoratus (C). Codon families are labeled on the x-axis. The termination codon is not given.

Genetic distance and evolution rates of PCGs

The genetic distance could be used to evaluate different mutation pressures among genes [54]. The pairwise genetic distances (p-distance) were calculated to reveal the sequence conservation and divergence of the PCGs among the Sebastiscus species (Figure 4). The genetic distance at the third nucleotide position was obviously higher than the first and second nucleotide position, indicating that the evolution of the third position was faster than the first and the second. The highest p-distance was found in ND1 (0.289) and ND4 (0.243, 0.161) at the third nucleotide of codons, while explored in ND2 (0.039, 0.034) and ND3 (0.013) base on the first and second nucleotide position. The COI-III and ATP8 genes had the low genetic distance in both first + second and third analysis. ND1, ND2, ND3, and ND4 genes might have high evolutionary rates among the three species, while COI-III and ATP8 were low.

The pair genetic distances of 13 PCGs among S. tertius (ST), S. albofasciatus (SA), and S. marmoratus (SM)

Figure 4
The pair genetic distances of 13 PCGs among S. tertius (ST), S. albofasciatus (SA), and S. marmoratus (SM)

The values were calculated based on the first and second nucleotide position, and on the third nucleotide position, respectively.

Figure 4
The pair genetic distances of 13 PCGs among S. tertius (ST), S. albofasciatus (SA), and S. marmoratus (SM)

The values were calculated based on the first and second nucleotide position, and on the third nucleotide position, respectively.

The value of nonsynonymous substitution (Ka)/synonymous substitution (Ks) is a common indicator to assess selective pressure and evolutionary relationships of species in molecular studies [55]. Ka/Ks < 1, Ka/Ks = 1, and Ka/Ks > 1 were represented purifying selection, neutral mutation, and positive selection, respectively [56]. All 13 PCG genes were under strong purifying selection with Ka/Ks values below 1 (Figure 5). The result was different from deep-sea fishes, where most genes exhibited positive selection or convergent/parallel signals with the exception of ND4L and ND5 [57]. One of the reasons might be the different living environment between them. Environmental difference leads to the different expression levels of protein-coding, which involved in energy regulation, reproduction, and immune behaviors [26]. The basic characteristics of genome evolution depended on random genetic drift and mutation pressure that closely connected with the environment [58]. The deep-sea fishes inhabited in the condition of oxygen deficiency, lacked food, no sunlight, and extreme cold, while the Sebastiscus species survived in the warm coastal waters [57,59,60]. Positive selection usually related to the adaptation of new environments and the development of the new function, and most nonsynonymous mutations were disadvantage [61,62]. The Ka/Ks values in Sebastiscus species showed they were under purifying selection, indicating that the environment variation was not great enough to change their genetic function.

The rates of nonsynonymous substitutions and synonymous substitutions for each PCG in pairwise mitochondrial genome of S. tertius (ST), S. albofasciatus (SA), and S. marmoratus (SM)

Figure 5
The rates of nonsynonymous substitutions and synonymous substitutions for each PCG in pairwise mitochondrial genome of S. tertius (ST), S. albofasciatus (SA), and S. marmoratus (SM)
Figure 5
The rates of nonsynonymous substitutions and synonymous substitutions for each PCG in pairwise mitochondrial genome of S. tertius (ST), S. albofasciatus (SA), and S. marmoratus (SM)

ND2 and ATP8 genes had high Ka/Ks (mean: 0.135, 0.188) values across three Sebastiscus mitogenomes compared with other genes, while ND3, ATP6, and Cytb genes were low (mean: 0.013, 0.014, 0.014). Low mutation rates tended to occur on highly expressed genes due to DNA repair mechanisms [63]. Compared with other genes, the ND3, ATP6, and Cytb showed low Ka/Ks representing a low mutation rate, indicating that they may have higher expression level.

rRNAs and tRNAs

Similar to S. marmoratus, the mitogenomes of S. albofasciatus and S. tertius each had one 12S rRNA and one 16SrRNA gene. The 12S rRNA gene was located between tRNAPhe and tRNAVal, and the 16S rRNA gene was located between tRNAVal and tRNALeu(UUR) as also occurs in some other Scorpaeniformes fishes [64,65]. The size of the 12S rRNA in S. albofasciatus and S. marmoratus were 946 bp, both a little shorter than in S. tertius (947 bp). The size of the 16S rRNA was 1692 bp in S. albofasciatus and S. tertius, which was consistent with S. marmoratus. In the S. tertius mitogenome, the A+T content of the rRNA genes was the minimum (52.25%) whereas the A+T content of rRNA in the S. albofasciatus and S. marmoratus mitogenomes were approximately 52.31 and 52.35%, lower than the control region. In three Sebastiscus species, the AT-skew of rRNA was strongly positive whereas the GC-skew was slightly negative indicating that the contents of A and C were higher than those of T and G in the rRNA, respectively.

Like the typical set of tRNA genes in Osteichthyes mitogenomes, there were 22 tRNA genes predicted in the three species. All of them had two kinds of tRNALeu and tRNASer (Table 2). The secondary clover-leaf structures of tRNA genes identified in the mitogenome of S. tertius, S. albofasciatus, and S. marmoratus are shown in Figure 6. These tRNA genes varied in length from 65 to 74 bp. All the predicted tRNAs displayed the typical clover-leaf secondary structure, except for tRNASer(AGY), which can not form a stable secondary structure because of lack of the DHU arm [66,67], this structure was common among fish mitogenomes [68].

Inferred secondary structures of the 22 tRNA genes of S. marmoratus, S. tertius, and S. albofasciatus mitogenomes

Figure 6
Inferred secondary structures of the 22 tRNA genes of S. marmoratus, S. tertius, and S. albofasciatus mitogenomes
Figure 6
Inferred secondary structures of the 22 tRNA genes of S. marmoratus, S. tertius, and S. albofasciatus mitogenomes

Control region

The control region is the largest non-coding region of the fish mitogenome which is equivalent to the A+T-rich region of the insect mitogenome [29,69]. The control regions of Sebastiscus mitogenomes were located between tRNAPro and tRNAPhe with lengths of 1246 and 1391 bp, respectively (Table 2), much shorter than that of S. marmoratus (1918 bp). The length of the control region is variable, which is the main reason for the differences in mitochondrial DNA lengths in fishes [70–72]. In the mitogenomes of S. tertius and S. albofasciatus, the contents of A+T were 69.34 and 68.01%, respectively, similar to that of S. marmoratus (68.81%). The control regions of all three species genomes showed positive AT-skew values while all control regions of the three Sebastiscus species displayed negative GC-skew values. Additionally, we found tandem repetitive sequences in all three Sebastiscus species by Tandem Repeat Finder V 4.07 (http://tandem.bu.edu/trf/trf.html) [38]. The tandem repetitive sequence units of S. tertius, S. albofasciatus and S. marmoratus were 22, 275, and 269 bp, respectively. The secondary structures of tandem repetitive sequence for three species showed that the two kinds of structures can form multiple stem ring structures, respectively (Figure 7). As a non-coding region, the control region of mitochondrial DNA has a low evolutionary pressure. Therefore, it is frequency of base insertion and deletion is high, and the tandem repetitive sequence has often occurred, too. With the accumulation of data, the phenomenon of tandem repetition is becoming more and more frequent in the mitochondrial genome of fish [73]. The formation mechanism has been made some progress, of which slipped-strand mispairing is the most likely mechanism for tandem repeats [74,75]. In this study, the number of the core sequence repetitions in S. tertius and S. albofasciatus were 6 and 2, respectively, and that of S. marmoratus was 4. The difference between tandem repetitive sequence is the main reason the mitogenome lengths of the three species are different. Besides, the control region is responsible for the regulatory functions of DNA replication and transcription, which length variation will inevitably affect its functions and the metabolic frequency of the whole organism, thus resulting in interspecific differences.

Predicted secondary structure and free energy of tandem repeat sequences about S. tertius (A), S. albofasciatus (B), and S. marmoratus (C)

Figure 7
Predicted secondary structure and free energy of tandem repeat sequences about S. tertius (A), S. albofasciatus (B), and S. marmoratus (C)

On the left is Maximum Expected Accuracy (MEA) and the right is Minimum Free Energy (MFE).

Figure 7
Predicted secondary structure and free energy of tandem repeat sequences about S. tertius (A), S. albofasciatus (B), and S. marmoratus (C)

On the left is Maximum Expected Accuracy (MEA) and the right is Minimum Free Energy (MFE).

L-strand origin of replication

The L-strand origins of replication (OL) of S. tertius and S. albofasciatus mitogenomes were located between tRNAAsn and tRNACys with the same lengths of 37 bp, 1 bp shorter than S. marmoratus (38 bp). Secondary structures of OL were displayed in Figure 8. In these OL structures, the use of stem codons showed significant asymmetry, with more pyrimidines at the 5′ end of the sequence. Consistent of other studies in fish, all OL regions had an identical conserved sequence region at the end of the stem (5′-GCCGG-3′) [45,76–78]. This may be related to the mechanism of RNA transformation to DNA [79]. Like found in many fishes, the use frequency of T and C bases in the ring region was higher [76,80].

Inferred secondary structures of the OL of Sebastiscus mitogenome, S. tertius (A), S. albofasciatus (B), and S. marmoratus (C)

Figure 8
Inferred secondary structures of the OL of Sebastiscus mitogenome, S. tertius (A), S. albofasciatus (B), and S. marmoratus (C)
Figure 8
Inferred secondary structures of the OL of Sebastiscus mitogenome, S. tertius (A), S. albofasciatus (B), and S. marmoratus (C)

Phylogenetic analyses

Phylogenetic relationships were reconstructed based on the sequences of 13 PCGs of 38 mitogenomes using BI and ML methods. The phylogenetic trees constructed by two methods were consistent with high intermediate bootstrap values, post probabilities, and the topological structure of the two phylogenetic trees were basically the same (Figure 9). We found three Sebastiscus species (S. marmoratus, S. tertius, and S. albofasciatus) formed a monophyletic group in both the BI and ML analyses. S. tertius and S. albofasciatus formed a sister group, which together had a sister relationship with S. marmoratus. Barsukov and Chen (1978) regarded Sebastiscus as a subgenus of Sebastes [17]. However, the result of our phylogenetic inference showed that Helicolenus are more closely related to Sebastes than to Sebastiscus. Our results supported the view that Sebastiscus should be treated as a genus independent from Sebastes which also was supported by some previous studies [11,81–83]. It was the strong evidence that supports the Sebastiscus as an independent species.

Phylogenetic tree of 36 Scorpaenoidei species constructed by BI and ML methods based on concatenated sequences of 13 PCGs

Figure 9
Phylogenetic tree of 36 Scorpaenoidei species constructed by BI and ML methods based on concatenated sequences of 13 PCGs

Boleophthalmus pectinirostris and Parapocryptes serperaster were used as the outgroup. The species in red (Latin name) indicated the sequences generated in the present study. The numbers at codes showed the Bayesian posterior probabilities.

Figure 9
Phylogenetic tree of 36 Scorpaenoidei species constructed by BI and ML methods based on concatenated sequences of 13 PCGs

Boleophthalmus pectinirostris and Parapocryptes serperaster were used as the outgroup. The species in red (Latin name) indicated the sequences generated in the present study. The numbers at codes showed the Bayesian posterior probabilities.

Sebastidae and Scorpaenidae have been considered as two families by several authors [2–6]. Based on this study, however, if Sebastidae is valid, the family Scorpaenidae was regarded as paraphyletic with its subfamily Pteroinae in a sister relationship with Sebastidae (Figure 10). A number of authors thought that Sebastidae was considered as a subfamily of Scorpaenidae, and had an equal level with Pteroinae and Scorpaeninae [10,81,84]. Such taxonomic division was consistent with our results of phylogenetic relationships.

Phylogenetic relationships about Scorpaenoidei based on BI and ML methods by the concatenated sequences of 13 PCGs

Figure 10
Phylogenetic relationships about Scorpaenoidei based on BI and ML methods by the concatenated sequences of 13 PCGs

Each red frame represented a separate family.

Figure 10
Phylogenetic relationships about Scorpaenoidei based on BI and ML methods by the concatenated sequences of 13 PCGs

Each red frame represented a separate family.

We estimated the species divergence time estimated using the ML method of RelTime by MEGA 10 (Figure 11). The divergence time calculations indicated that the Scorpaenidae had differentiated from other species ∼45.45 million years ago and all the families of Scorpaenoidei began to diverge more than 40 million years ago, except for Sebastidae. The differentiation time of Sebastidae from other species of Scorpaenidae was the lastest, which began ∼17.96 million years ago and the evolutionary rate of Sebastidae fishes were similar to the average rate of other subfamilies of Scorpaenidae. In summary, the taxonomic status of Sebastidae maybe coincides with the subfamilies of Scorpaenidae according to the result.

Divergence time analysis of 36 Scorpaenoidei fish species base on ML topology using concatenated sequences of 13 PCGs

Figure 11
Divergence time analysis of 36 Scorpaenoidei fish species base on ML topology using concatenated sequences of 13 PCGs

Numbers near the nodes indicated the estimated divergence time (Mya).

Figure 11
Divergence time analysis of 36 Scorpaenoidei fish species base on ML topology using concatenated sequences of 13 PCGs

Numbers near the nodes indicated the estimated divergence time (Mya).

Conclusions

In the present study, the complete mitogenomes of S. tertius, S. albofasciatus, and S. marmoratus were successfully determined. Their mitogenomes were with a total length of 16910 bp in S. tertius, 17056 bp in S. albofasciatus, and 17580 bp in S. marmoratus, respectively. A total of 22, 275, and 269 bp tandem repetitive sequences were respectively detected in three mitogenomes, which may be a typical characteristic of Sebastiscus fish. The ratio of Ka and Ks indicated that three species were suffering a purifying selection, while the ND2 and ATP8 showed the highest Ka/Ks values. Both ML and BI analyses indicated that Sebastiscus was monophyletic and S. marmoratus was a sister clade to S. tertius and S. albofasciatus. That is to say, the taxonomic status of Sebastidae, well supported by results of a phylogenetic tree, should be subfamily Sebastinae. We believe the present study will greatly improve our understanding of the evolution profile and phylogenetic position in Scorpaeniformes, which could benefit resource management and species protection in fishery and aquaculture.

Data Availability

The data used in the manuscript can be found on NCBI website (https://www.ncbi.nlm.nih.gov/) by accession number.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported by the National Key R&D Program of China [grant number 2019YFD0901301]; and the National Natural Science Foundation of China [grant number 41776171].

Author Contribution

Chenghao Jia: Methodology, Validation, Formal analysis, Writing original draft. Xiumei Zhang: Validation, Supervision, Writing review and editing. Shengyong Xu, Tianyan Yang and Takashi Yanagimoto: Resources, Writing review and editing. Tianxiang Gao: Conceptualization, Validation, Supervision, Writing review and editing, Funding acquisition. All authors have read and agreed to the published version of the manuscript.

Acknowledgements

We sincerely thank the reviewers for their critique and suggestions.

Abbreviations

     
  • BI

    Bayesian Inference

  •  
  • Ka

    nonsynonymous substitution

  •  
  • Ks

    synonymous substitution

  •  
  • ML

    maximum likelihood

  •  
  • PCG

    protein-coding gene

  •  
  • rRNA

    ribosomal RNA

  •  
  • RSCU

    relative synonymous codon usage

  •  
  • tRNA

    transfer RNA

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