Complete Mitochondrial Genome and Phylogenetic Position of Chirolophis wui (Perciformes: Stichaeidae)

: The complete mitochondrial genome of Chirolophis wui (Wang and Wang, 1935) was sequenced using the Illumina platform. The genome sequence is 16,522 bp in length with 54% A+T content and contains 13 protein coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and 1 control region (D-loop). The H-strand contains 28 genes (12 PCGs, 14 tRNAs, and 2 rRNAs), whereas the L-strand accommodates 9 genes ( ND6 and 8 tRNAs). The nucleotide composition of the mitochondrial genome of C. wui is AT-biased, accounting for 54.0%, with an AT skew value of − 0.0556 and a GC skew value of − 0.2043. The majority of PCGs utilized the start codon, ATG, while only one gene, COI, utilized the alternative start codon, GTG. Of the 13 PCGs, 6 genes used the termination codon (TAA or TGA), whereas 7 genes used the incomplete termination codon (T or TA). Among the 22 tRNA genes, the tRNA-Leu and tRNA-Ser were found in duplicates. A phylogenetic tree was constructed using 10 complete mitochondrial genome sequences and indicated that C. wui has a very close relationship with C. japonicus and other species in the family Stichaeidae, with a high supporting bootstrap value. This study can provide valuable information for future evolutionary studies on C. wui and Stichaeidae.


Introduction
The mitochondrion is a type of organelle that is capable of directly converting organic materials into energy, which is then used to sustain many biological processes that occur within a cell [1][2][3].Mitochondria have semiconservatively self-replicating, closed circular, double-stranded mitochondrial DNA [4,5].The mitochondrial genomes of vertebrates are small, at around 14-26 kb on average, and the order of encoded genes is highly conserved [6][7][8].The mitochondrial genome of vertebrates typically consists of 13 protein-coding genes (PCGs), 2 ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and 2 noncoding regions: the control regions and the origin of L-strand replication [9].The complete mitochondrial genomes have been shown to be useful genetic markers in detecting and differentiating distinct or similar, and even concealed, species within closely related taxa [10,11].The sequencing of fish mitogenomes has been made possible by recent advancements in molecular biology analytical techniques, resulting in a clear comprehension of the structure of fish mitogenomes (which are 16-18 kb in size) [1,12].Fish mitogenomes also have highly conserved protein-coding genes, transfer RNAs, ribosomal RNAs, and noncoding regions, although the gene spacing and length vary between species [9,13].The mitogenome has been a prominent molecular guide in the study of the phylogeny and evolution of fish since the determination of the mtDNA sequences of several fish species [1,14,15].
The family Stichaeidae comprises six different subfamilies: Azygopterinae, Chirolophinae, Lumpeninae, Opisthocentrinae, Stichaeinae, and Xiphisterinae.The family Stichaeidae inhabits the North Pacific, North Atlantic, and Arctic oceans, with the majority of species inhabiting the North Pacific.They are coastal fishes found in the intertidal zone and shallow bays beneath rocks and in algae.They can be found on the outer continental shelf at depths of more than 250 m [16][17][18].The subfamily Chirolophinae contains four genera: Bryozoichthys, Chirolophis, Gymnoclinus, and Sodatovia [16,19].The body is moderately elongate and relatively robust.The head is uncovered, and the body is coated with tiny scales.The head, anterior part of the body, and first few dorsal fins have spines with dermal appendages.The anal fin has one weakly developed spine.The pectoral fins are large, and the pelvic fins have one spine and 2-4 soft rays [16,19].There are eight species in the genus Chirolophis, including C. ascanii, C. decorates, C. japonicus, C. nuagtor, C. saitone, C. snyderi, C. tarsodes, and C. wui (http://www.fishbase.se/search.php,accessed on 10 January 2022) [20].Chirolophis wui (Wang and Wang, 1935) has only been found in the Republic of Korea, China, and Japan [21,22].However, molecular studies are limited in comparison to morphological and environmental investigations.
To date, the mitochondrial genome of C. wui has not been studied.As a result, the complete mitochondrial genome of C. wui was sequenced in this study, and their phylogenetic relationship with other Stichaeidae species was investigated.This study can help with future evolutionary studies on C. wui and Stichaeidae.

Sample and DNA Extraction
An individual sample of C. wui was captured from the coast of Taean in the Republic of Korea (36 • 34 26.27 N; 126 • 1 86.3822E) (Figure 1), and deposited at the Marine Fish Resources Bank of Korea (MFRBK) in Pukyong National University (PKNU), Busan, Republic of Korea (Dr.Jin-Koo Kim, taengko@pknu.ac.kr) under the voucher number PKU-21087.
Using the DNeasy Blood and Tissue Kit (Qiagen, Germany), total genomic DNA was isolated from the muscle tissue according to the manufacturer's recommendations.The quality and purity of gDNA were evaluated using a NanoDrop 3300 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).The gDNA was stored at −20 °C for further analysis.

Next-Generation Sequencing and Mitogenome Assembly
The TrueSeq Nano DNA Kit was used to create the DNA library, which was then sequenced on the Illumina platform using 150 bp paired-end reads (Illumina, HiSeq 2500, San Diego, CA, USA) at Macrogen (Daejeon, Republic of Korea).For the library, a total of 67,988,441 clean reads of each direction were produced.Using Trimmomatic [23], adapter sequences and low-quality reads were deleted to reduce analytical bias.The overall quality of the produced sequencing reads was verified with FastQC v0.11.5 (Babraham Institute, Bioinformatics) [24].Mitogenome assembly was accomplished de novo using various κ-mers and the SPAdes v3.13.0 tool [25].The filtered Illumina reads and reconstructed mitogenome were submitted to BioProject, the Sequence Read Archive (SRA), and GenBank under the corresponding accession numbers PRJNA855310, SRR19965989, and OP388414, respectively.Using the DNeasy Blood and Tissue Kit (Qiagen, Germany), total genomic D isolated from the muscle tissue according to the manufacturer's recommendatio quality and purity of gDNA were evaluated using a NanoDrop 3300 spectropho (Thermo Scientific, Wilmington, DE, USA).The gDNA was stored at −20 ℃ for analysis.

Next-Generation Sequencing and Mitogenome Assembly
The TrueSeq Nano DNA Kit was used to create the DNA library, which w sequenced on the Illumina platform using 150 bp paired-end reads (Illumina, HiS San Diego, CA, USA) at Macrogen (Daejeon, Republic of Korea).For the library, a 67,988,441 clean reads of each direction were produced.Using Trimmomatic [23], sequences and low-quality reads were deleted to reduce analytical bias.The quality of the produced sequencing reads was verified with FastQC v0.11.5 (Ba Institute, Bioinformatics) [24].Mitogenome assembly was accomplished de nov various κ-mers and the SPAdes v3.13.0 tool [25].The filtered Illumina rea reconstructed mitogenome were submitted to BioProject, the Sequence Read (SRA), and GenBank under the corresponding accession numbers PRJNA SRR19965989, and OP388414, respectively.

Mitogenome Annotation, Sequence Analysis, and Phylogenetic Analysis
The mitogenome of C. wui was analyzed for annotation using the MITOS server (http://mitos.bioinf.uni-leipzig.de/index.py,accessed on 10 January 2022) [26].The PCGs were validated using the ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/,accessed on 10 January 2022) following translation into the predicted amino acids according to the vertebrate mitochondrial genetic code.Based on the mitochondrial code of vertebrates, the codon usage in PCGs was predicted via the Codon Usage web server [27].The Tandem Repeat Finder v4.09 web server was used to look into the number of repetitions in the region [28].MEGA-X v10.2.4 was used to determine the nucleotide composition of the D-loop, rRNAs, tRNAs, PCGs, and mitogenome of C. wui [29].The following formulae were used to determine the asymmetry in the mitogenome base composition.In terms of the four nucleotides (A, T, G, and C), the skews were calculated as follows: AT skew = (A − T)/(A + T) and GC skew = (G − C)/(G + C) [30].The MITOS server [26] and ARWEN server [31] were used to estimate the secondary structures of tRNA and rRNA genes.
To determine the phylogenetic position of C. wui within Stichaeidae, 9 mitogenomes of Stichaeidae species were downloaded from GenBank, and complete sequences of Cottus szanaga species were used as an outgroup (Table 1).The complete mitochondrial genomes of these species were aligned using ClustalW [32], maximum-likelihood (Tamaru-Nei model) [33] analysis was conducted using MEGA XI v11.0.8 [34], and the tree topology was evaluated with 1000 bootstrap replicates.

Mitochondrial Genome Assembly, Annotation, and Sequence Analysis
A total of 135.9 million reads (Table S1) were produced with the next-generation sequencing of C. wui, and after eliminating the adapter sequences and low-quality reads, the remaining 113.1 million reads (Table S2) were appropriate for the genomic assembly methods.
The mitogenome of C. wui consisted of 13 PCGs, 2 rRNA genes, 22 tRNA genes, and 1 control region (D-loop) (Figure 3, Table 2).Only ND6 and 8 tRNA genes (Gln, Ala, Asn, Cys, Tyr, Ser, Glu, and Pro) were encoded on the L-strand of the 37 mitochondrial genes; all of the other genes were encoded on the H-strand.As in the typical vertebrate mitochondrial genome, the 12S and 16S rRNA genes of C. wui are located between the tRNA-Phe and tRNA-Leu genes, with the tRNA-Val gene in between.Regarding the results of comparative mitochondrial genome analysis with C. japonicus and C. ascanii, they were of the same composition and order as C. wui.

Ribosomal RNA, Transfer RNA Genes, and Control Region
In C. wui, the length of the ribosomal RNA genes was 2641 bp (15.98% of the total mitogenome).The sizes of the 12S rRNA and 16s rRNA genes were 948 bp and 1693 bp, respectively.The overall base composition of the 12S rRNA was T = 21.7%,C = 25.5%,A = 30.1%,G = 2.7%, and AT = 51.8%.The 12S rRNA gene showed a weakly positive AT skew (0.1622) and GC skew (−0.0581) compared with the AT skew (0.1554) and GC skew (−0.0419) of the 16s RNA gene.
The mitochondrial genome of C. wui was analyzed using the MITOS website, and 22 tRNA-encoding genes were found (Figure 4).The length of 22 tRNAs ranged from 66 bp (tRNA-Cys) to 74 bp (tRNA-Leu-1 and tRNA-Lys).The tRNAs of leucine (Leu) and serine (Ser) existed in two copies with different anticodons.The existence of these tRNAs in the mitochondrial genome as numerous copies has been proven in various vertebrates [11,35,40,41].The sequences of all tRNA genes of C. wui were folded into a canonical cloverleaf secondary structure consisting of an amino acid accepter (AA) arm, a dihydrouridine (DHU) arm, an anticodon (AC) arm, a TΨC arm, and variable (V) arms [10,11,42].The tRNA-Ser-1 (AGT) and tRNA-Cys (GCA) had an incorrect DHU arm, which have been reported in several fish [11,43].All tRNAs of C. wui had AT skew values of 0.1073, and GC skew values of −0.0746.
Located between the tRNA-Pro and tRNA-Phe, the C. wui CR (D-loop) was 851 bp in length (Table 2).The D-loop had a total of T = 28.6%,C = 21.7%,A = 30.6%,and G = 19.2% by base composition.In addition, the AT skew value was 0.0338, whereas the GC skew value was −0.0611 (Table 3, Figure 3).As a result of frequent insertions/deletions and substitutions of nucleotides, it is known that this region exhibits substantial length variation [44].The tandem repeat sequences were not detected in the D-loop region in C. wui using the Tandem Repeats Finder web server [28].Although the exact purpose of the control region (D-loop) is unknown, it is anticipated that it will be crucial to the replication and transcription of the genome [11].

Phylogenetic Analysis
The phylogenetic tree was constructed using the mitogenome sequences from ten species, including C. wui and eight additional Stichaeidae species, and one Cottage species, Cottus szanaga (KX762050) [45], as an outgroup (Figure 5).Table 1 lists the mitogenomes that were analyzed in this study and their corresponding accession numbers.
The taxa of the nine Stichaeidae species were well clustered, and C. szanaga was distinct from the Stichaeidae.Within Stichaeidae, the phylogeny was shown by the Chirolophinae, Stichaeinae, and Opisthocentrinae.Within each of the three subfamilies, there was only one genus (Chirolophis, Stichaeus, and Opisthocentrus).The phylogenetic analysis showed that C. wui is most closely related to C. japonicus.C. wui and C. japonicus are sisters of C. ascanii.
The results presented in this study would play an important role in the investigation of the phylogenetic relationships and taxonomy of the family Stichaeidae.[11,35,40,41].The sequences of all tRNA genes of C. wui were folded into a canonical cloverleaf secondary structure consisting of an amino acid accepter (AA) arm, a dihydrouridine (DHU) arm, an anticodon (AC) arm, a TΨC arm, and variable (V) arms [10,11,42].The tRNA-Ser-1 (AGT) and tRNA-Cys (GCA) had an incorrect DHU arm, which have been reported in several fish [11,43].All tRNAs of C. wui had AT skew values of 0.1073, and GC skew values of −0.0746.

Phylogenetic Analysis
The phylogenetic tree was constructed using the mitogenome sequences from ten species, including C. wui and eight additional Stichaeidae species, and one Cottidae species, Cottus szanaga (KX762050) [45], as an outgroup (Figure 5).Table 1 lists the mitogenomes that were analyzed in this study and their corresponding accession numbers.The results presented in this study would play an important role in the investigation of the phylogenetic relationships and taxonomy of the family Stichaeidae.

Conclusions
In conclusion, the current study presented the first complement mitogenome assembly and annotation of C. wui.We described the characterization of the mitochondrial genome of C. wui using various genetic and phylogenetic research approaches.These

Conclusions
In conclusion, the current study presented the first complement mitogenome assembly and annotation of C. wui.We described the characterization of the mitochondrial genome of C. wui using various genetic and phylogenetic research approaches.These results can help to advance understanding and collect fundamental genetic data for the Stichaeidae family.
Author Contributions: Y.-S.L., M.P.P., K.R.M. and J.-O.K. performed the experiments, analyzed the data, were involved in certain tools for analysis and drafting of the paper, and approved the final draft.Y.B.S., Y.-J.L., K.R.M. and J.-K.K. were involved in certain tools for analysis, organizing the results, and preparing figures.G.-D.K. was involved in the conception and design of the work, funding acquisition, revising it critically for intellectual content, and the final approval of the version to be published.All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement:
The sample used for this study was the dead body of a fish and, as per the animal experimental ethics in the Republic of Korea (Standard operating guideline; IACUC-Institutional Animal Care and Use Committee, Book no.11-1543061-000457-01, effective from December 2020), we did not require approval from the Ethics Committee.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The mitogenome sequence data that support the findings of this study are openly available in GenBank of NCBI at https://www.ncbi.nlm.nih.gov/under accession number OP388414.The associated BioProject, SRA, and the complement mitogenome numbers are PRJNA855310, SRR19965989, and OP388414, respectively.

Fishes 2023, 8 , 13 Figure 2 .
Figure 2. Sample image and the mitochondrial genome map of Chirolophis wui.(a) Specimen reference image; (b) the mitochondrial genome of C. wui.Genes outside the circle are transcribed in a clockwise direction and those inside in a counterclockwise direction.

Figure 2 .
Figure 2. Sample image and the mitochondrial genome map of Chirolophis wui.(a) Specimen reference image; (b) the mitochondrial genome of C. wui.Genes outside the circle are transcribed in a clockwise direction and those inside in a counterclockwise direction.

Figure 3 .
Figure 3. Graphical representation of nucleotide composition contents and AT and GC skew.They should be listed as: (a) nucleotide composition AT% and GC%; (b) AT and GC skew.

Figure 5 .
Figure 5.The phylogenetic tree was obtained using the complete mitochondrial genome sequences from ten species (nine from the Stichaeidae family and Cottus szanaga from the Cottidae family as an outgroup) and 1000 bootstrap repetitions using the maximum-likelihood approach.The numbers on the branches represent the bootstrap values, and the star next to Chirolophis wui denotes the species used in this research.The taxa of the nine Stichaeidae species were well clustered, and C. szanaga was distinct from the Stichaeidae.Within Stichaeidae, the phylogeny was shown by the Chirolophinae, Stichaeinae, and Opisthocentrinae.Within each of the three subfamilies, there was only one genus (Chirolophis, Stichaeus, and Opisthocentrus).The phylogenetic analysis showed that C. wui is most closely related to C. japonicus.C. wui and C. japonicus are sisters of C. ascanii.The results presented in this study would play an important role in the investigation of the phylogenetic relationships and taxonomy of the family Stichaeidae.

Figure 5 .
Figure 5.The phylogenetic tree was obtained using the complete mitochondrial genome sequences from ten species (nine from the Stichaeidae family and Cottus szanaga from the Cottidae family as an outgroup) and 1000 bootstrap repetitions using the maximum-likelihood approach.The numbers on the branches represent the bootstrap values, and the star next to Chirolophis wui denotes the species used in this research.

Funding:
This research was part of the project titled 'Development of Discrimination Method and On-Site Kit for Geographical Origin of Fishery Product (Project No. 20200425)', funded by the Ministry of Ocean and Fisheries, Republic of Korea.

Table 1 .
The list of mitogenomes used in this study.

Table 2 .
Gene annotations of the complete mitochondrial genome of C. wui.

Table 2 .
Gene annotations of the complete mitochondrial genome of C. wui.

Table 3 .
General metrics of nucleotide composition of C. wui.Note: The triplet codon of amino acids is denoted by the numbers 1st, 2nd, and 3rd, which indicate the relative abundance of specific nucleotide bases at each position.

Table 4 .
Codon usage analysis of PCGs in the mitochondrial genome of C. wui.