Ancient reticulation, incomplete lineage sorting and the evolution of the pyrenoid at the dawn of hornwort diversification

Abstract Background and Aims Resolving the phylogeny of hornworts is critical in understanding the evolution of key morphological characters that are unique to the group, including the pyrenoid. Extensive phylogenomic analyses have revealed unexpected complexities in the placement of Leiosporoceros, the previously identified sister taxon to other hornworts. We explore the role of incomplete lineage sorting (ILS) and ancient reticulation in resolving interrelationships and comprehending the diversification and evolutionary processes within hornworts. Methods Using the GoFlag probe set, we sequenced 405 exons representing 234 nuclear genes, sampling 79 hornwort specimens, including representatives of all hornwort genera. We inferred the species phylogeny from gene tree analyses using concatenated and coalescence approaches, assessed ancient reticulation, ILS, and estimated the timing of divergences based on fossil calibrations. Key Results Extreme levels of gene tree incongruence challenge the sister relationship of Leiosporoceros to other hornworts. This phylogenetic discordance is due to incomplete lineage sorting and ancient reticulation, the latter revealed using a network approach to identify evidence of gene flow among hornwort lineages. Hornworts diversification began in the Carboniferous with widespread family-level divergences during the mid-Cretaceous and Paleogene. Conclusions Incomplete lineage sorting and ancient reticulation are identified as important in hornwort evolution. Patterns of hornwort diversification parallel those in other plants groups (e.g., liverworts, mosses, ferns, and gymnosperms). Two scenarios on pyrenoid evolution are plausible based on the variable position of the pyrenoid-free Leiosporoceros. Pyrenoids were retained from a green algal ancestor and are plesiomorphic, or they evolved in response to the substantial drop in atmospheric CO2 levels during the Carboniferous as has been hypothesized in other photosynthetic organisms. Both hypotheses require losses and gains during hornwort speciation.