Comparative Organellar Genomics of Pellidae: Insights into Codon Usage, Nucleotide Diversity, and Structural Evolution

Liverwort organellar genomes are generally highly conserved, but the subclass Pellidae (simple thalloids) shows unusual variation. This ancient yet unexplored lineage of simple thalloid liverworts provides an excellent model for investigating organellar genome evolution. In this study, we assembled four new plastid and four new mitochondrial Pellidae genomes using Oxford Nanopore sequencing, supplementing 86 plastomes and 82 mitogenomes from databases. We assessed nucleotide diversity and codon usage, and inferred phylogenies using IQ-TREE with fossil-calibrated dating. Plastomes ranged 120.6–126.5 kb, and mitogenomes 109–180 kb, with Apopellia endiviifolia featuring an exceptionally reduced mitogenome (~109 kb). Native RNA sequencing enabled a revised annotation of the mitochondrial atp1 gene in Apopellia, revealing two introns (previously thought absent) and reducing the intergenic region share to 36.26%, the lowest known among liverworts. Comparative analyses revealed contrasting evolutionary dynamics between organelles: Plastomes displayed higher nucleotide diversity and phylogenetically inconsistent codon usage patterns, likely influenced by compositional bias, whereas mitogenomes were more conserved and largely consistent with established phylogenetic relationships among the orders. Phylogenomic analyses yielded discordant topologies: Chloroplast data recovered Pellidae as a monophyletic clade, whereas mitochondrial data placed Pelliales (Pellia/Apopellia) as basal Jungermanniopsida, rendering Pellidae paraphyletic. Within Pellidae-relevant clades, several major divergences were dated to the Carboniferous–Permian, but with systematic chloroplast–mitochondrial offsets. These results highlight recurrent organellar incongruence and the dynamic evolutionary history of Pellidae organellar genomes.