Complete mitochondrial genome of 10 anemonefishes belonging to Amphiprion and Premnas

Abstract

Anemonefish are widely distributed in tropical areas with phenotypic color variation often observed in the same species. Complete mitochondrial genomes (mitogenomes) of 10 anemonefishes belonging to Amphiprion and Premnas were determined to support taxonomic status. Average mitogenome sequence was 16,838 ± 19.69 bp, containing 37 genes with identical gene order to most teleost mitogenomes. The percula complex comprised A. percula and A. ocellaris and was phylogenetically clustered with P. biaculeatus. Color morphs of A. ocellaris and P. biaculeatus were identified, suggesting large phenotypic variation at species level. Results will facilitate further genetic studies of mitochondrial variation and species diversity in anemonefish.

Keywords:

• Anemonefish
• Amphiprion
• mitochondrial genome
• Premnas

Anemonefish (Pomacentridae) are found along coral reefs. They have a mutualistic symbiotic relationship with tropical sea anemones (Elliott et al. 1999) and are indicators to understand the geographical distribution of high ecological speciation in the marine environment (Timm et al. 2008). Most are distinguished by their color patterns; however, several species show variable morphs (Allen 1975). Recent molecular analyses reported color patterns as not always sufficient indicators of species diversity (Li et al. 2015; Thongtam na Ayudhaya et al. 2017). Genetic diversity studies of more molecular markers will provide important information for systematic conservation anemonefish management. Here, we determined complete mitochondrial genomes (mitogenomes) of 10 anemonefishes in Amphiprion ocellaris (common morph) collected from Rayong (12.6814° N, 101.2816° E), A. ocellaris (black morph), A. percula and Premnas biaculeatus (yellow striped morph) from Chonburi, Thailand (13.3611° N, 100.9847° E), P. biaculeatus (white striped morph) from Phetchaburi, Thailand (12.9649° N, 99.6426° E), and A. clarkii, A. polymnus, A. akallopisos, A. ephippium and A. frenatus from Krabi, Thailand (8.0863° N, 98.9063° E). All specimens were stored at Thailand Natural History Museum (THNHM). Whole genomic DNA was extracted following standard salting-out protocol (Supikamolseni et al. 2015). Polymerase chain reaction (PCR) amplifications were performed using universal mitogenome primers (Mauro et al. 2004) and specific primers were developed to amplify remaining parts of the genome. All PCR products were sequenced by DNA sequencing service, First Base Laboratories Sdn Bhd (Selangor, Malaysia), with annotation performed following Srikulnath et al. (2012) and Prakhongcheep et al. (2018). Mitogenome sequence size of 10 anemonefishes (16,762–16,980 bp) was similar to previous reports (Mabuchi et al. 2007; Li et al. 2015; Hu et al. 2016; Tao et al. 2016). All mitogenomes comprised 37 genes and a control region (CR). Gene organization patterns were identical to teleosts (Miya et al. 2013). GAT deletion was found in ND5 of A. percula and A. ocellaris differed from previous reports with CCT deletion (Li et al. 2015), suggesting the possibility of intra-specific variation. Nucleotide diversity among anemonefish mitogenomes was 8.36 ± 0.27%, with sequence divergence between Amphiprion and Premnas at 10.98%. Two morphs of A. ocellaris and P. biaculeatus gave nucleotide divergence at 1.30% and 2.48%, respectively, similar to analysis of a single mitochondrial gene (COI, Cytb, or 16S rRNA) at species level (Thongtam na Ayudhaya et al. 2017), suggesting intra-specific sequence diversity. At least one tandem repeat was identified in the CR of most anemonefishes, except for A. ocellaris. The first tandem repeat was identified in Amphiprion spp., while the second in two different morphs of P. biaculeatus. A phylogenetic tree of 18 anemonefishes was constructed based on concatenated 12 protein-coding genes without ND6, with Abudefduf vaigiensis and Paratilapia polleni as the outgroup using Bayesian inference, MrBayes version 3.2.6 (Huelsenbeck and Ronquist 2001). All GenBank sequences were grouped within the same species, supporting Amphiprioninae as a monophyletic group (Litsios et al. 2014). The percula complex containing A. percula and two morphs of A. ocellaris was clustered with two morphs of P. biaculeatus (Figure 1). Both morphs of A. ocellaris and P. biaculeatus were identified, suggesting large phenotypic variation at species level. Availability of complete mitogenomes will increase understanding of evolutionary processes and diversification of anemonefish and facilitate comprehensive analyses.

Figure 1 Phylogenetic relationships among concatenated mitochondrial twelve protein-coding genes, without ND6 sequences of 20 mitochondrial genomes, including Abudefduf vaigiensis and Paratilapia polleni as the outgroup using Bayesian inference analysis. The complete mitochondrial genome sequence was downloaded from GenBank. Accession and voucher numbers are indicated in parentheses after the scientific name of each species. Bold letter indicates specimens collected in this study and stored at Thailand Natural History Museum (THNHM). An asterisk (*) indicates specimens collected from Chonburi, Thailand (13.3611° N, 100.9847° E). A plus (+) indicates specimens collected from Phetchaburi, Thailand (12.9649° N, 99.6426° E). A hash (#) indicates specimens collected from Rayong, Thailand (12.6814° N, 101.2816° E). A section sign (§) indicates specimens collected from Krabi, Thailand (8.0863° N, 98.9063° E). Support values at each node are Bayesian posterior probabilities. Branch-lengths represent the number of nucleotide substitutions per site.

Acknowledgements

The authors would like to thank Sahabhop Dokkaew (Kasetsart University) for advice on sample preparation.

Disclosure statement

The authors report no conflict of interest and are entirely responsible for the content and writing of this article. Animal care and all experimental procedures were approved by the Animal Experiment Committee, Kasetsart University, Thailand (approval no. ACKU01257) and conducted in accordance with the Regulations on Animal Experiments at Kasetsart University.

Additional information

Funding

This study was financially supported by grants from the Kasetsart University Doctoral Scholarship, the Center for Advanced Studies in Tropical Natural Resources, National Research University-Kasetsart University (CASTNAR, NRU-KU, Thailand), the Center of Excellence on Agricultural Biotechnology, Science and Technology Postgraduate Education and Research Development Office, Office of Higher Education Commission, Ministry of Education (AG-BIO/PERDO-CHE), the Fellowship of Capacity Building for Kasetsart University on Internationalization at Kasetsart University [No. 0513.10109/8384], Faculty of Medicine, Ramathibodi Hospital Scholarship (No. 678/2558), and the Thailand Research Fund [TRF; nos. RSA6180075 and PHD60I0014]