A review of molecular-clock calibrations and substitution rates in liverworts, mosses, and hornworts, and a timeframe for a taxonomically cleaned-up genus Nothoceros

Absolute times from calibrated DNA phylogenies can be used to infer lineage diversification, the origin of new ecological niches, or the role of long distance dispersal in shaping current distribution patterns. Molecular-clock dating of non-vascular plants, however, has lagged behind flowering plant and animal dating. Here, we review dating studies that have focused on bryophytes with several goals in mind, (i) to facilitate cross-validation by comparing rates and times obtained so far; (ii) to summarize rates that have yielded plausible results and that could be used in future studies; and (iii) to calibrate a species-level phylogeny for Nothoceros, a model for plastid genome evolution in hornworts. Including the present work, there have been 18 molecular clock studies of liverworts, mosses, or hornworts, the majority with fossil calibrations, a few with geological calibrations or dated with previously published plastid substitution rates. Over half the studies cross-validated inferred divergence times by using alternative calibration approaches. Plastid substitution rates inferred for "bryophytes" are in line with those found in angiosperm studies, implying that bryophyte clock models can be calibrated either with published substitution rates or with fossils, with the two approaches testing and cross-validating each other. Our phylogeny of Nothoceros is based on 44 accessions representing all suspected species and a matrix of six markers of nuclear, plastid, and mitochondrial DNA. The results show that Nothoceros comprises 10 species, nine in the Americas and one in New Zealand (N. giganteus), with the divergence between the New Zealand species and its Chilean sister species dated to the Miocene and therefore due to long-distance dispersal. Based on the new tree, we formally transfer two species of Megaceros into Nothoceros, resulting in the new combinations N. minarum (Nees) J.C. Villarreal and N. schizophyllus (Gottsche ex Steph.) J.C. Villarreal, and we also newly synonymize eight names described in Megaceros.