Intron-Associated Mitogenome Size Variation and Gene-Specific Stop Codon Absence in the Lichen-Forming Genus Ramalina

Mitochondrial genomes of lichen-forming fungi provide a useful system for examining genome evolution, yet the fruticose lichen genus Ramalina has not been studied comparatively. We newly sequenced three Ramalina mitogenomes and reconstructed one from published data, then compared these with six additional public mitogenomes to obtain a 10-species dataset. Nine other Ramalinaceae mitogenomes were used as a reference panel. Within Ramalina, core gene content and gene order are highly conserved, whereas genome size varies 1.83-fold and is strongly associated with intron content and homing endonuclease abundance. Introns repeatedly occupy homologous insertion sites across the genus. Four genes, cox1, nad3, nad4, and nad5, recurrently lack canonical stop codons at conserved terminal positions, while cob consistently lacks a stop immediately after the conserved ENSLIELA motif. These states are supported by multiple assembly approaches and reanalysis of public reads, while the affected genes show patterns consistent with purifying selection. These results show that Ramalina mitogenomes combine conserved core architecture with intron-associated size variation and recurrent changes in mitochondrial gene-terminal signals.