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Mitochondrial genomic variation and validation of cytochrome b as a species-level marker in anemonefish (Amphiprioninae).

K. Somkham,C. Boonphakdee,J. Thamnawasolos,T. Boonphakdee,T. Mataroepayotorn

Published 2025 in Brazilian journal of biology = Revista brasleira de biologia

ABSTRACT

Accurate species identification within the anemonefish subfamily (Amphiprioninae) is essential for ecological research, biodiversity conservation, and regulation of the ornamental fish trade. This study presents a comparative analysis of 19 publicly available complete mitochondrial genomes from 11 Amphiprioninae species, including multiple strains of Amphiprion ocellaris and Premnas biaculeatus. Although gene order was conserved across species, we identified lineage- and strain-specific polymorphisms, such as a 3-bp deletion in the nd5 gene, codon variation in atp6 and cox1, and variability in tandem repeats within the 12S/16S rRNA genes and the control region (D-loop). Phylogenetic analyses based on concatenated mitochondrial genes confirmed the monophyly of Amphiprioninae and supported the taxonomic reclassification of P. biaculeatus within Amphiprion. Among all loci examined, cytochrome b (cytb) showed the highest phylogenetic resolution. We validated its diagnostic potential by amplifying and sequencing a 927-bp cytb fragment from juveniles of A. ocellaris, A. percula, and A. perideraion, achieving >99% sequence identity with species-specific GenBank references. These findings establish cytb as a reliable and cost-effective molecular marker for distinguishing morphologically similar species, with applications in taxonomy, aquaculture, conservation genetics, and trade monitoring.

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