The complete mitochondrial genome of Sardinella zunasi (Clupeiformes: Clupeidae)

Abstract The complete mitogenome of Sardinella zunasi was determined by next-generation sequencing. The S. zunasi mitogenome was a circular 16,307 bp molecule that contained 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and one control region (D-loop). The gene arrangement was consistent with other Sardinella mitogenomes. The phylogenetic relationships of 29 Clupeoidei species based on 13 protein-coding genes from the available mitogenomes were analyzed. Sardinella zunasi clustered with Sardinella among Clupeidae, suggesting a closer relationship with this genus. These results will be useful for understanding the phylogenetic relationships, taxonomic classification, and phylogeography of the genus Sardinella relative to other genera of Clupeoidei.

Complete mitochondrial genome; Clupeoidei; Sardinella zunasi; phylogenetic analysis Sardinella zunasi (Japanese sardinella) is a ray-finned fish in the family Clupeidae, which includes herrings and sardines. It is native to the northwestern Pacific, where it occurs near shore along the Asian coastline from southwestern Korea and southern Japan to Taiwan. It is commercially important in Korea, Japan, and China (Wang et al. 2008) and has been heavily fished and is considered overexploited. Numerous ecological and morphological studies have examined the species; however, the complete mitochondrial DNA (mtDNA) sequence of this species has not been reported (Kim et al. 2001).
A specimen was bought at a Korean fish market and stored at Soonchunhyang University, Republic of Korea, under accession no. SUC-30000. The species identification of the specimen was confirmed morphologically.
Total genomic DNA was extracted from muscle tissue using a NucleoSpin Tissue Kit (Macherey-Nagel, D€ uren, Germany) and a genomic library for next-generation sequencing (NGS) was constructed using the MGIEasy DNA Library Prep Kit (MGI Tech, Shenzhen, China). After obtaining raw NGS data on an MGISEQ-2000 (MGI Tech), the mtDNA sequence of a circular 16,307 bp molecule was assembled using CLC Assembly Cell 5.1.1 (Qiagen, Hilden, Germany), and the assembled sequence was annotated using the web-based automatic MITOS annotation server (Bernt et al. 2013).
An NCBI BlastN (Johnson et al. 2008) search of the complete mtDNA sequence showed 98.37% identity to sequence NC_039553 of the Sardinella lemuru mitochondrion (Jiang et al. 2017). Therefore, the full mitochondrial sequence of S. zunasi has not been registered in the GenBank database, and this sequence is presumed to have high similarity with the mitochondrial sequence of S. lemuru. We performed a detailed NCBI BlastN search using the sequences of the COX1, 12S rRNA, and 16S rRNA regions of S. zunasi, which showed sequence identities of 99.64-99.69%, 99.42-100%, and 99.64-100%, respectively, with reported S. zunasi sequences and 99.54%, 99.90%, and 94.45% with S. lemuru sequences. The comparison of these regions confirmed that our mtDNA sequence was more similar to S. zunasi than to S. lemuru. Therefore, the mtDNA sequence completed here was for S. zunasi. This sequence was registered in GenBank under accession no. MW118114.
The complete mitogenome of S. zunasi (MW118114) was 16,307 bp in length and consisted of 13 protein-coding genes (PCGs), 22 tRNAs, two rRNAs (12S and 16S), and a non-coding region including the putative control region (D-loop). The S. zunasi gene arrangement was identical to those of its relatives: S. lemuru (Jiang et al. 2017 (Sebastian et al. 2017). Twelve PCGs were on the heavy strand, while ND6 was on the light strand. All of the tRNAs were predicted to form typical stem-loop secondary structures, as in S. lemuru. The noncoding D-loop region was located between tRNA-Pro and tRNA-Phe. All PCGs had the typical ATG start codon, except for COX1 (ATC), COX2 (GTG), and ND6 (GTG). Incomplete stop codons were identified in ND2, COX2, COX3, ND3, ND4, and CYTB, as in S. melanura.
A maximum-likelihood phylogenetic tree of S. zunasi and 28 other Clupeoidei species was constructed based on the 13 protein-coding genes using MEGA-X (Kumar et al. 2018). Interestingly, this showed that S. zunasi clustered with S. lemuru, suggesting a very close relationship, confirming that the current sequence belongs to Sardinella (Figure 1). To confirm that the species identification of S. lemuru NC_039553 was correct, a p-distance analysis using the complete mitogenome sequences of Sardinella species gave values of 0.196-0.214 with other species in Sardinella, while the distance between S. zunasi and S. lemuru was 0.008. In a more detailed analysis, the p-distances were determined for the other cox1 genes of S. zunasi and S. lemuru in GenBank and ranged from 0.0031 to 0.0053 between the cox1 genes of S. zunasi; NC_039553 S. lemuru had a value of 0.0044, while other S. lemuru sequences had values of 0.1799-0.1928 and Sardinella maderensis had a value of 0.1920. Therefore, NC_039553 was verified to be the S. zunasi mitochondrial sequence. The complete mtDNA sequence of S. zunasi should improve the taxonomic classification of Sardinella species with similar mtDNA sequences.

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

Funding
This study was a part of the project titled 'Development of Discrimination Method and On-site Kit for Geographical Origin of Fishery Product' (Project no. 20200425-04)