Codon usage bias and selective constraints in Gentianales mitogenomes

Mitochondrial genome evolution(MGE) in flowering plants is quasi-intertwined-dynamic. MGE is driven via mutational pressures, translational selection, and functional constraints. However, unveiling the intra- and inter-genomic interplay governing evolutionary drives remains challenging. We investigate MGE-dynamic across twelve Gentianales species, revealing distinct codon usage patterns influenced by opposing evolutionary forces. While the first and second codon positions are highly conserved, the third codon positions show significant variability (27.7% - 45.8%), reflecting diverse selective pressures. Multi-dimensional analyses, including ENc-GC3s plots, neutrality plots, and PR2 bias, indicate that natural selection predominantly governs codon usage, outweighing mutational biases. Key findings include, non-significant correlations between GC12 and GC3 (R² ≤ 0.21), suggesting minimal mutational impact on genome composition; ENc-GC12 analysis showing codon optimization results from both selection and mutation; and PR2-plot analysis highlighting a preference for T- and G-ending codons, indicative of translational efficiency constraints. Gene-specific analyses of substitution rates (dN, dS, and dN/dS ) uncover heterogeneous selective landscapes, with genes such as atp, ccm, nad, and rps exhibiting signatures of positive selection. Substantial mutually offsetting dynamics between T3s and C3s (r = - 0.73), coupled with strong correlations between G3s and translational-efficiency indices (CAI: r = 0.69; CBI: r = 0.65), underscore that third-codon biases optimize translation. Evolutionary rates (dS and dN/dS ) show positive correlations with GC3 content (r = 0.45 and r = 0.33, respectively), indicating the influence of nucleotide composition on synonymous substitutions. Thus, these results reveal the interplay of the mutation-selection balance in non-recombining genomes and offer new perspectives on mitochondrial diversity in flowering plants.