Mitochondrial genome evolution across marine–freshwater divergence in the flatfish genus Brachirus

Abstract Freshwater adaptation from marine ancestors represents a major ecological transition in teleost evolution, yet its genomic basis remains poorly understood. The genus Brachirus is predominantly marine, although a small subset of species has secondarily adapted to freshwater habitats, providing a rare framework for studying ecological transitions. We sequenced and assembled complete mitochondrial genomes of two freshwater species, B. panoides and B. villosus, generating circular genomes of 16,629 bp and 16,609 bp encoding 13 protein-coding genes, 22 transfer RNAs, and two ribosomal RNAs. Phylogenetic analyses using maximum likelihood (ML) and Bayesian inference (BI) placed Brachirus within Soleidae and dated their divergence to 19–24.3 million years ago (MYA). Selection test detected no widespread adaptive signals in mitochondrial genes associated with freshwater lineages; only two genes showed episodic selection under branch models. These findings suggest that mitochondrial genomes alone may provide limited insight into freshwater adaptation and highlight the need for nuclear genomic data to fully resolve the evolutionary mechanisms underlying habitat transitions in Brachirus.