Complete mitochondrial genome and phylogenetic analysis of the copper shark Carcharhinus brachyurus (Günther, 1870)

Abstract Copper shark (Carcharhinus brachyurus Günther, 1870) is one of the most widely distributed but least known species in the family Carcharhinidae. Herein, we report the first complete mitogenome of C. brachyurus. The overall structure of the 16,704 bp C. brachyurus mitogenome was similar to that of other Carcharhinus species and showed the highest average nucleotide identity (97.1%) with the spinner shark (Carcharhinus brevipinna). Multigene phylogeny using 13 protein-coding genes (PCGs) in the mitogenome resolved C. brachyurus clustered with other species within the genus; the overall tree topology was congruent with recent phylogenetic studies of this species. These results provide important information for conservation genetics and further evolutionary studies of sharks.

Copper shark (Carcharhinus brachyurus G€ unther, 1870), in the family Carcharhinidae, is distributed in the equatorial temperate region of the eastern Atlantic, northwestern and eastern Pacific, southern Africa, Australia, and New Zealand (Compagno et al. 2005;Kim et al. 2019). In the IUNC Red List of threatened species, C. brachyurus is classified as 'vulnerable' (Huveneers et al. 2020). Various traits, including slow growth and late maturity render them vulnerable to anthropogenic activities (Drew et al. 2017). In the mitogenome of C. brachyurus, only the mitochondrial control region has been partially sequenced and used for phylogeographic analysis (Benavides et al. 2011). In this study, we analyzed the complete mitogenome sequence of C. brachyurus.
The specimens of C. brachyurus used in this study were collected from the Moseulpo fish markets in Jeju, Korea, in October 2017, as previously described (Kim et al. 2019 Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen Korea Ltd., Seoul, Korea) according to the manufacturer's instructions and subjected to direct PCRbased sequencing. Fifteen primer pairs, designed from the mitogenome of a related species in the family Carcharhinidae, the spinner shark (Carcharhinu brevipinna, NC_027081.1), were used to amplify the C. brachyurus mitogenome. All PCR primers and amplification conditions are listed in Supplementary Table 1. The obtained partial sequences of C. brachyurus were assembled using Geneious R11.1.5 (Kearse et al. 2012), and the final complete mitogenome was annotated as previously described (Kim et al. 2017).
The complete C. brachyurus mitogenome (MT995631) was 16,704 bp long, with a 61.7% A þ T content. It consisted of a typical set of 37 genes (2 rRNAs, 22 tRNAs, and 13 proteincoding genes [PCGs]), and the overall structure was similar to the mitogenomes of other sharks in Carcharhinidae available in MitoFish (http://mitofish.aori.u-tokyo.ac.jp/). A putative Dloop (1061 bp, 66.4% A þ T content) was located between tRNA Pro and tRNA Phe , and the replication origin (35 bp) was located on the H-strand between tRNA Asn and tRNA Cys (Supplementary Table 2). The two rRNAs were 955 bp (12S rRNA) and 1676 bp (16S rRNA) long and separated by tRNA Val . The size of the 22 tRNAs varied from 67 bp (tRNA Ser and tRNA Cys ) to 75 bp (tRNA Leu ), with a total length of 1550 bp.
Orthologous average nucleotide identity (OrthoANI) values were analyzed with other related sharks in the family Carcharhinidae as previously described (Kim et al. 2017). The overall OrthoANI values between C. brachyurus and other sharks in the genus Carcharhinus were >93%, with the highest value (97.1%) obtained for spinner shark (C. brevipinna, KM244770); other genera in the family showed <92% identity (Supplementary Table 3). Thirty-three complete mitogenomes of other related species in Carcharhinidae were obtained from GenBank and used for multigene phylogenetic analysis. The concatenated sequences of the 13 PCGs were aligned as previously described (Kim et al. 2017), and the maximum likelihood tree was reconstructed using MEGAX version 10.0 (Kumar et al. 2018). In the resultant tree, copper shark was clustered with other Carcharhinus species (Figure 1). Our tree topology was congruent with previous phylogenetic analyses based on nuclear or mitochondrial genes, which clustered blue shark (Prionace glauca) and whitetip reef shark (Triaenodon obesus) with other sharks in the genus Carcharhinus (Dosay-Akbulut 2008; Li et al. 2016), indicating that taxonomic assignment of the two species warrants further reevaluation. The mitogenome and associated genomic data of C. brachyurus provide important insights into biodiversity and address phylogenetic relationships within the genus Carcharhinus.

Data availability statement
The data that support the findings of this study are openly available in the NCBI GenBank database at https://www.ncbi.nlm.nih.gov, reference number MT995631.