Comparative mitochondrial genomics of Ophiocordycipitaceae fungi reveals AT bias and site-specific positive selection in the nad4 gene.

The fungal family Ophiocordycipitaceae (Hypocreales, Ascomycota) includes the medicinally important Ophiocordyceps sinensis. However, comparative mitogenomic analyses remain limited. Here, we analyzed mitochondrial genomes from 18 species spanning Ophiocordyceps, Cordyceps sensu stricto, Beauveria, Tolypocladium, and three outgroups, using maximum-likelihood phylogenetics (GTR + F + R3, 1000 ultrafast bootstraps), codon-usage analysis, and site-specific selection pressure estimation. Genome sizes ranged 20.7-272.5 kb (~ 13-fold), with low GC content (mean GC = 27.7%). Codon usage showed moderate-to-strong bias (ENC = 30.0-35.5), with neutrality plots supporting AT mutation pressure as the dominant force. Site-specific dN/dS analysis revealed heterogeneous selection: cob had the strongest purifying selection (mean ω = 0.80), while nad4 showed the highest mean ω (2.40). Two positively selected sites in nad4 were identified (Site 245: ω = 6.79, P = 0.045; Site 384: ω = 7.21, P = 0.036), confirmed by pairwise Nei-Gojobori analysis (mean ω = 1.70; 80.8% of comparisons ω > 1) and sliding window tests. PAML codeml M7/M8 LRT validated significant positive selection (2ΔlnL = 14.88, P = 0.00058), with BEB identifying the same two sites (posterior probability > 0.95). These results advance our understanding of mitochondrial evolution in entomopathogenic fungi and provide candidate sites for functional validation.