Liverworts, with approximately 7300 species worldwide, exhibit remarkable morphological diversity in terms of growth form, ontogeny, and architecture. Their mitochondrial genome exhibits lower average substitution rates compared to their nuclear and plastid genomes, and shows less structural variation, suggesting its suitability for inferring relationships at higher taxonomic levels. In this study, we substantially expanded mitochondrial sampling in liverworts by adding complete mitochondrial gene sets from 97 species across 25 families, thereby increasing family-level coverage to 71%. Among these, we newly assembled 23 complete mitochondrial genomes. Although four species with structural variants were newly identified, the overall architecture of liverwort mitochondrial genomes remains highly conserved, with taxa that diverged over 470 million years ago still having collinearity. Phylogenetic inferences from mitochondrial genome sequences confirmed the monophyly of most suprafamilial taxa, with the exceptions of Porellales, Ptilidiales, and Pelliidae. Herzogianthus (Ptilidiales) was well-supported as a sister group to Jungermanniales sensu lato, rather than forming a monophyletic lineage with Ptilidium (Ptilidiales). This work provides an important resource for future genetic and phylogenetic studies of liverworts.
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