Characteristics and phylogenetic analysis of the complete mitochondrial genome of Microstomus achne

Abstract Microstomus achne (Jordan and Starks, 1904) is an economically valuable flatfish belonging to the family Pleuronectidae and the only flatfish that inhabits Korea. Here, we report on the complete mitochondrial genome of M. achne and the phylogenetic relationship between close species. The mitogenome is 16,971 bp long and encodes 13 protein-coding genes (PCGs), 22 transfer RNAs, and two ribosomal RNAs. The phylogenetic analysis showed that M. achne clustered with Glyptocephalus stelleri, which supports the conclusion that M. achne belongs to the family Pleuronectidae. The results of this study provide a better understanding of M. achne.


Introduction
Microstomus achne (Jordan and Starks, 1904) is a benthic fish belonging to the order Pleuronectiformes and family Pleuronectidae, and its most striking morphological characteristics is the white spots on the body (Cooper and Chapleau 1998). The genus Microstomus was reported to comprise 10 nominal species worldwide, five of which have recently been excluded from the genus, and four of which are currently accepted as valid (Norman 1934;Cooper and Chapleau 1998;Froese and Pauly 2022). Of this genus, only M. achne is known to inhabit Korea (Kim et al. 2005). However, genetic information on members of these genera is remarkably limited, and in particular, no complete mitochondrial DNA sequences have yet been reported for this genus. We describe the completed mitochondrial genome of M. achne, which was acquired using next-generation sequencing, and we anticipate that this information will help to understand the phylogenetic status of M. achne.

Materials
We obtained the fin of a flatfish fin that was collected from Boryeong South Korea (36 22 0 N, 126 34 0 E) and deposited in the Pukyong National University storage facility (Figure 1

Methods
The genomic DNA was extracted using the PureHelix TM Genomic DNA Prep Kit [Animal], Solution Type (NANOHELIX, Daejeon). For species identification using the cox1 sequence, the cox1 gene was amplified by PCR using the fish universal primer set (Ward et al. 2005) and sequenced by Macrogen (South Korea). The partial cox1 sequence was compared using a BLASTN (Johnson et al. 2008) search.
Raw data from next-generation sequencing were obtained using the method described in Chae et al. (2022) and then deposited in the Sequence Read Archive (SRA) database (SRR21722070). The raw data were trimmed with Cutadapt ver. 4.1 (Martin 2011), and a contig sequence was produced with the default option in the de novo assembler of the CLC Genomics Workbench (ver. 20.04; QIAGEN). The circular form of the mitogenome was confirmed by using the "Map to Reference" tool in Geneious software (ver. 2021.2.2; https:// www.geneious.com) to map the filtered data onto the contig sequence. Annotation of this final sequence was performed in the MITOS Webserver (Bernt et al. 2013), after which the detailed annotation was manually corrected using SnapGene software (ver. 5.3.2; GSL Biotech LLC; snapgene.com). Finally, the completed circular form of the mitogenome sequence was registered at the NCBI GenBank (OP066370). The mitogenome map was prepared using ORDRWA (Greiner et al. 2019  ; Cynoglossus sinicus, JQ348998 ; Cynoglossus roulei, MK574671 ). Each mtDNA sequence was retrieved from GenBank. These nucleotide sequences of protein-coding genes (PCGs) were aligned and analyzed using a GTR substitution model and 1,100,000 chain length.
The total length of the final mitogenome was 16,971 bp, and it comprised 13 PCGs, 22 tRNA genes, and two rRNA genes. Its gene order matched that of Glyptocephalus stelleri (MT258402). Among the PCGs, only nad6 was transcribed on the negative strand, and all others were transcribed on the positive strand (Figure 2). The ATG codon was used as a start codon in 12 PCGs (nad1, nad2, cox2, atp8, atp6, cox3, nad3, nad4l, nad4, nad5, nad6, and cob), while the GTG codon was used as a start codon in cox1. nad1, atp8, atp6, nad4l, and nad5 used the TAA stop codon. While the truncated T-codon terminated translation in nad2, cox2, nad3, nad4, and cob, and the truncated TA-stopped translation in cox3. The AGA codon and the TAG codon were utilized as stop codons in cox1 and nad6, respectively.
The mitogenome of M. achne contained 22 tRNA genes, including two tRNA-L and two tRNA-S. Of these tRNA genes, 14 (tRNA-F, tRNA-V, tRNA-L2, tRNA-I, tRNA-M, tRNA-W, tRNA-D, tRNA-K, tRNA-G, tRNA-R, tRNA-H, tRNA-S1, tRNA-L1, and tRNA-T) were transcribed on the positive strand; the remaining tRNA genes (tRNA-Q, tRNA-A, tRNA-N, tRNA-C, tRNA-Y, tRNA-S2, tRNA-E, and tRNA-P) were transcribed on the negative strand ( Figure 2). In the predicted secondary structure, there were standard and abnormal structures in the tRNA loop or stem. tRNA-C and tRNA-S1 contained an abnormality in the D-loop. Imperfect base pairing in the T-loop was observed in tRNA-V, tRNA-W, tRNA-M, tRNA-N, and tRNA-E. In addition, incomplete base pairing in the acceptor stem was found in tRNA-F, tRNA-V, tRNA-I, tRNA-R, tRNA-H, tRNA-L1, and tRNA-T. Uniquely, tRNA-S1 had partial base pairing in every stem. The other genes possessed standard tRNA structure.
The two rRNA genes were located close to the border of tRNA-V (Figure 2). Between tRNA-F and tRNA-V, a small rRNA was placed, while a large rRNA was placed between tRNA-V and tRNA-L2 (Figure 2). The small and large rRNA were 951 bp and 1713 bp in length, respectively. The putative control region was located between tRNA-P and tRNA-F and had a length of 1261 bp (Figure 2).
Each species was identified as belonging to either family Pleuronectidae, family Cynoglossidae, or the outgroup (A. fulvescens). M. achne clustered with G. stelleri of the family Pleuronectidae and was separated from other nodes in the phylogenetic analysis (Figure 3).

Discussion and conclusion
Microstomus achne is the only flatfish belonging to the genus Microstomus that can be observed in South Korea. In addition, this genus lacks any complete mitogenomes. Here, the complete mitochondrial genome of M. achne was identified, and its genetic characteristics were elucidated. Significantly, the gene composition was the same as the general mitogenome composition of vertebrates (Pereira, 2000). In addition, gene composition and order had no significant features compared with the family Pleuronectidae. This is the first study to report the complete mitogenome of flatfish of the genus Microstomus. These data can be utilized to reveal the phylogenetic relationships between members of the genus Microstomus, especially M. achne.

Acknowledgment
This article was reviewed by Dr. Khawaja Muhammad Imran Bashir for English, the authors are thankful for his suggestions and support.

Authors contributions
Jun Young Chae, Moo-Sang Kim, Jinkoo Kim, and Hyung-Ho Lee conceived the idea for this study. Jun Young Chae conducted the experiments. Jun Young Chae wrote the manuscript with support from Moo-Sang Kim and Hyung-Ho Lee. Tae-Wook Kang and Jin Ho Kim performed the data analysis, and Jinkoo Kim supplied the specimen. All authors agree to be accountable for all aspects of the work.

Ethical approval
No ethical approval was required for this study. We used a flatfish fin from a specimen that was previously collected by the MBRIS, outside this study. This specimen was dead, and the sample was provided with permission by the MBRIS (permission no. 2022-130).

Disclosure statement
No potential conflict of interest was reported by the author(s).

Data availability statement
The genome sequence data supporting this study's findings are available in the GenBank of the NCBI at (https://www.ncbi.nlm.nih.gov/) under accession no. OP066370. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA882383, SRR21722070, and SAMN30933649, respectively.