Evolution and classification of hornworts: new insights from the first plastome-based phylogeny

Hornworts (Anthocerotophyta) represent a key lineage for understanding fundamental questions in land plant evolution, but their phylogeny and evolutionary history are still not well understood, primarily due to limited genomic resources and insufficient taxon sampling. We conducted comparative genomic analyses of 106 hornwort plastid genomes, including 91 newly generated ones. RNA editing sites were identified by integrating transcriptome data and in silico predictions. Additionally, a new method inspired by marker-capture strategies was proposed to estimate the total number of U-to-C editing sites. Hornwort plastomes are larger than those of liverworts and mosses, with rare gene loss or pseudogenization. Both C-to-U and U-to-C RNA editing occur across all lineages except Leiosporoceros. Diversification rate analyses indicate a major shift between c. 100 and 50 million years ago, possibly linked to the Cretaceous-Paleogene extinction event. Both morphological and molecular evidence support the merging of Folioceros into Anthoceros, and the recognition of two new species in the small genera Paraphymatoceros and Phymatoceros, respectively. This study presents the first large-scale plastome phylogeny of hornworts, introduces a DNA-only method for estimating the number of U-to-C RNA editing sites, and updates the classification. These results contribute broadly to our understanding of early land plant evolution.