Comparative phylogenetic analyses of RNA editing in fern plastomes suggest possible adaptive innovations.

RNA editing in plant organelles is widely regarded as a neutral corrective mechanism, yet it persists as a complex, energetically costly process, requiring numerous nuclear-encoded pentatricopeptide repeat proteins. Ferns are the most diverse lineage of land plants that uniquely retain both cytidine-to-uridine (C-to-U) and uridine-to-cytidine (U-to-C) RNA editing in their plastomes, offering a powerful system to investigate the evolutionary forces shaping both editing types. Two distantly related fern lineages - Hymenophyllaceae and Vittarioideae (Pteridaceae) - each containing sister sublineages with contrasting evolutionary rates, were selected for comparative analysis. Genomic and transcriptomic data were combined with bioinformatic and phylogenetic methods to identify RNA editing sites and investigate the evolutionary dynamics of both C-to-U and U-to-C editing in fern plastomes. Nonsynonymous edits were frequently lost, consistent with neutral evolution. By contrast, C-to-U edits at start codons and U-to-C edits at internal stop codons were evolutionarily conserved, displaying lower and more variable editing efficiencies that suggest these edits are regulated. C-to-U edits at start codons and U-to-C edits at internal stop codons are evolutionarily conserved and exhibit signatures of selective regulation, suggesting that they function as molecular checkpoints. These findings provide the strongest evidence to date that RNA editing in plants plays an adaptive role in modulating plastid gene expression.