Complete mitochondrial genome characterization and exploratory mitochondrial DNA modification signals in square-head catfish Chaca chaca.

Complete mitochondrial genomes provide powerful insights into evolutionary relationships, phylogeography, and emerging epigenetic regulation, yet remain poorly characterized for many fishes. Here, we report the first complete mitogenome of the square-head catfish Chaca chaca and integrate mitogenomic evolution with mitochondrial DNA (mtDNA) methylation profiling. High-molecular-weight DNA was sequenced using Oxford Nanopore adaptive sampling, followed by de novo assembly, annotation, and comparative analyses. The assembled mitogenome is 16,591 bp long and contains the standard complement of 13 protein-coding genes, 22 tRNAs, two rRNAs, and a complete control region. Phylogenetic reconstruction based on concatenated alignments of the 13 mitochondrial protein-coding genes (PCGs) recovered C. chaca within Chacidae and adjacent to Pimelodidae + Pseudopimelodidae. Genome-wide methylation analysis detected signals consistent with 5-methylcytosine (5mC) and N6-methyladenine (6mA) across the mitogenome, with differences in modification frequency observed between strands after normalization for base composition within this dataset. The separation was more pronounced for 5mC compared to 6mA and is consistent with strand-associated asymmetry observed in this dataset. In addition to this, two cytosine positions within an intergenic spacer between tRNA-Asp and COXII showed detectable 5mC-associated signal. This mitogenome provides a foundational genomic resource for C. chaca, enabling refined phylogenetic resolution, conservation genetics, and future investigations into the potential relevance of mtDNA modification signals, pending further validation in fishes.