Relative performance of three phylogenetic methods based on complete mitochondrial genomes of barnacle

Mitochondrial Genome analysis is essential for understanding phylogenetic relationships. However, few studies have compared the performance of phylogenetic approaches for marine invertebrates, which have a complex evolutionary history. This study compared three phylogenetic trees based on 34 complete mitochondrial genomes, including Amphibalanus eburneus, Fistulobalanus kondakovi, and Megabalanus rosa, in terms of (1) gene order, (2) concatenated protein-coding genes, and (3) universal cytochrome c oxidase subunit I (COX1) marker regions. Each phylogenetic tree exhibited significant topological differences (Robinson-Foulds distance of 0.55-0.92). The protein-coding genes (78.8%) performed significantly better in terms of monophyletic preservation than the COX1 marker region (61.3%) and gene order (50.0%). Gene order analysis identified two genomic regions (I and II) as hotspots (regions with concentrated rearrangement activity) with significantly elevated breakpoint densities (319 and 100 breakpoints, respectively; p < 0.001), indicating concentrated genome rearrangement activity. Although all three methods consistently preserved some families, they strongly suggested that taxonomic re-evaluation is necessary for Balanidae. In conclusion, gene order provides insights into genome evolution patterns, concatenated protein-coding genes are the most suitable for phylogenetic studies, and COX1 markers are useful for rapid species identification rather than phylogenic classification. This comparative analysis provides important insights into the effects of method selection on mitochondrial phylogeny, especially when addressing complex phylogenetic problems in marine invertebrates.