Comprehensive phylogenomic time tree of bryophytes reveals deep relationships and uncovers gene incongruences in the last 500 million years of diversification.

PREMISE: Bryophytes form a major component of terrestrial plant biomass, structuring ecological communities in all biomes. Our understanding of the evolutionary history of hornworts, liverworts, and mosses has been significantly reshaped by inferences from molecular data, which have highlighted extensive homoplasy in various traits and repeated bursts of diversification. However, the timing of key events in the phylogeny, patterns, and processes of diversification across bryophytes remain unclear. METHODS: Using the GoFlag probe set, we sequenced 405 exons representing 228 nuclear genes for 531 species from 52 of the 54 orders of bryophytes. We inferred the species phylogeny from gene tree analyses using concatenated and coalescence approaches, assessed gene conflict, and estimated the timing of divergences based on 29 fossil calibrations. RESULTS: The phylogeny resolves many relationships across the bryophytes, enabling us to resurrect five liverwort orders and recognize three more and propose 10 new orders of mosses. Most orders originated in the Jurassic and diversified in the Cretaceous or later. The phylogenomic data also highlight topological conflict in parts of the tree, suggesting complex processes of diversification that cannot be adequately captured in a single gene-tree topology. CONCLUSIONS: We sampled hundreds of loci across a broad phylogenetic spectrum spanning at least 450 Ma of evolution; these data resolved many of the critical nodes of the diversification of bryophytes. The data also highlight the need to explore the mechanisms underlying the phylogenetic ambiguity at specific nodes. The phylogenomic data provide an expandable framework toward reconstructing a comprehensive phylogeny of this important group of plants.

A novel thylakoid-less isolate fills a billion-year gap in the evolution of Cyanobacteria.

Cyanobacteria have played pivotal roles in Earth's geological history, especially during the rise of atmospheric oxygen. However, our ability to infer the early transitions in Cyanobacteria evolution has been limited by their extremely lopsided tree of life-the vast majority of extant diversity belongs to Phycobacteria (or "crown Cyanobacteria"), while its sister lineage, Gloeobacteria, is depauperate and contains only two closely related species of Gloeobacter and a metagenome-assembled genome. Here, we describe a new cultured member of Gloeobacteria, Anthocerotibacter panamensis, isolated from a tropical hornwort. Anthocerotibacter diverged from Gloeobacter over 1.4 Ga ago and has low 16S rDNA identities with environmental samples. Our ultrastructural, physiological, and genomic analyses revealed that this species possesses a unique combination of traits that are exclusively shared with either Gloeobacteria or Phycobacteria. For example, similar to Gloeobacter, it lacks thylakoids and circadian clock genes, but the carotenoid biosynthesis pathway is typical of Phycobacteria. Furthermore, Anthocerotibacter has one of the most reduced gene sets for photosystems and phycobilisomes among Cyanobacteria. Despite this, Anthocerotibacter is capable of oxygenic photosynthesis under a wide range of light intensities, albeit with much less efficiency. Given its key phylogenetic position, distinct trait combination, and availability as a culture, Anthocerotibacter opens a new window to further illuminate the dawn of oxygenic photosynthesis.

Population genomics of a reindeer lichen species from North American lichen woodlands.

PREMISE: Lichens are one of the main structural components of plant communities in the North American boreal biome. They play a pivotal role in lichen woodlands, a large ecosystem situated north of the closed-crown forest zone, and south of the forest-tundra zone. In Eastern Canada (Quebec), there is a remnant LW found 500 km south of its usual distribution range, in the Parc National des Grands-Jardins, originated mainly because of wildfires. We inferred the origin of the lichen Cladonia stellaris from this LW and assessed its genetic diversity in a postfire succession. METHODS: We genotyped 122 individuals collected across a latitudinal gradient in Quebec. Using the software Stacks, we compared four different approaches of locus selection and single-nucleotide polymorphism calling. We identified the best fitting approach to investigate population structure and estimate genetic diversity of C. stellaris. RESULTS: Populations in southern Quebec are not genetically different from those of northern LWs. The species consists of at least four phylogenetic lineages with elevated levels of genetic diversity and low co-ancestry. In Parc National des Grands-Jardins, we reported high values of genetic diversity not related with time since fire disturbance and low genetic differentiation among populations with different fire histories. CONCLUSIONS: This first population genomic study of C. stellaris is an important step forward to understand the origin and biogeographic patterns of lichen woodlands in North America. Our findings also contribute to the understanding of the effect of postfire succession on the genetic structure of the species.

Population Genomics and Phylogeography of a Clonal Bryophyte With Spatially Separated Sexes and Extreme Sex Ratios.

The southern Appalachian (SA) is one of the most biodiversity-rich areas in North America and has been considered a refugium for many disjunct plant species, from the last glacial period to the present. Our study focuses on the SA clonal hornwort, Nothoceros aenigmaticus J. C. Villarreal & K. D. McFarland. This hornwort was described from North Carolina and is widespread in the SA, growing on rocks near or submerged in streams in six and one watersheds of the Tennessee (TR) and Alabama (AR) Rivers, respectively. Males and female populations occur in different watersheds, except in the Little Tennessee (TN) River where an isolated male population exists ca. 48 km upstream from the female populations. The sex ratio of 1:0 seems extreme in each population. In this study, we use nuclear and organellar microsatellites from 250 individuals from six watersheds (seven populations) in the SA region and two populations from Mexico (23 individuals). We, then, selected 86 individuals from seven populations and used genotyping by sequencing to sample over 600 bi-allelic markers. Our results suggest that the SA N. aenigmaticus and Mexican plants are a nested within a clade of sexual tropical populations. In the US populations, we confirm an extreme sex ratio and only contiguous US watersheds share genotypes. The phylogenetic analysis of SNP data resolves four clusters: Mexican populations, male plants (Little Pigeon and Pigeon river watersheds) and two clusters of female plants; one from the Little Tennessee and Hiwassee Rivers (TR) and the other from the Ocoee (TR) and Coosa (AR) Rivers. All clusters are highly differentiated (Fst values over 0.9). In addition, our individual assignment analyses and PCAs reflect the phylogenetic results grouping the SA samples in three clades and recovering males and female plants with high genetic differentiation (Fst values between 0.5 and 0.9 using microsatellites and bi-allelic markers). Our results point to Pleistocene events shaping the biogeographical pattern seen in US populations. The extreme sex ratio reflects isolation and highlights the high vulnerability of the populations in the SA.

Genome-wide organellar analyses from the hornwort Leiosporoceros dussii show low frequency of RNA editing.

Because hornworts occupy a pivotal position in early land colonization as sister to other bryophytes, sister to tracheophytes, or sister to all other land plants, a renewed interest has arisen in their phylogenetic diversity, morphology, and genomes. To date, only five organellar genome sequences are available for hornworts. We sequenced the plastome (155,956 bp) and mitogenome (212,153 bp) of the hornwort Leiosporoceros dussii, the sister taxon to all hornworts. The Leiosporoceros organellar genomes show conserved gene structure and order with respect to the other hornworts and other bryophytes. Additionally, using RNA-seq data we quantified the frequency of RNA-editing events (the canonical C-to-U and the reverse editing U-to-C) in both organellar genomes. In total, 109 sites were found in the plastome and 108 in the mitogenome, respectively. The proportion of edited sites corresponds to 0.06% of the plastome and 0.05% of the mitogenome (in reference to the total genome size), in contrast to 0.58% of edited sites in the plastome of Anthoceros angustus (161,162 bp). All edited sites in the plastome and 88 of 108 sites in the mitogenome are C-to-U conversions. Twenty reverse edited sites (U-to-C conversions) were found in the mitogenome (17.8%) and none in the plastome. The low frequency of RNA editing in Leiosporoceros, which is nearly 88% less than in the plastome of Anthoceros and the mitogenome of Nothoceros, indicates that the frequency of RNA editing has fluctuated during hornwort diversification. Hornworts are a pivotal land plant group to unravel the genomic implications of RNA editing and its maintenance despite the evident evolutionary disadvantages.

Divergence times and the evolution of morphological complexity in an early land plant lineage (Marchantiopsida) with a slow molecular rate.

We present a complete generic-level phylogeny of the complex thalloid liverworts, a lineage that includes the model system Marchantia polymorpha. The complex thalloids are remarkable for their slow rate of molecular evolution and for being the only extant plant lineage to differentiate gas exchange tissues in the gametophyte generation. We estimated the divergence times and analyzed the evolutionary trends of morphological traits, including air chambers, rhizoids and specialized reproductive structures. A multilocus dataset was analyzed using maximum likelihood and Bayesian approaches. Relative rates were estimated using local clocks. Our phylogeny cements the early branching in complex thalloids. Marchantia is supported in one of the earliest divergent lineages. The rate of evolution in organellar loci is slower than for other liverwort lineages, except for two annual lineages. Most genera diverged in the Cretaceous. Marchantia polymorpha diversified in the Late Miocene, giving a minimum age estimate for the evolution of its sex chromosomes. The complex thalloid ancestor, excluding Blasiales, is reconstructed as a plant with a carpocephalum, with filament-less air chambers opening via compound pores, and without pegged rhizoids. Our comprehensive study of the group provides a temporal framework for the analysis of the evolution of critical traits essential for plants during land colonization.

Structure and development of Nostoc strands in Leiosporoceros dussii (Anthocerotophyta): a novel symbiosis in land plants.

The presence of Nostoc in longitudinally oriented schizogenous canals is a feature that separates Leiosporoceros from all other hornworts and represents a novel symbiotic arrangement in land plants. In surface view, Nostoc canals are visible as elongated, dichotomously branched blue-green strands. All other hornworts develop numerous discrete globose colonies through continuous production of mucilage clefts as avenues for multiple invasions within a single thallus. To elucidate the anatomy and development of the unusual Nostoc strands in Leiosporoceros, we examined sporeling development in culture and the structure of strands in field-collected plants using light and electron microscopy. Rosette-like sporelings have mucilage clefts scattered along swollen apices. All field specimens were strap-shaped, contained Nostoc, and lacked mucilage clefts. Nostoc strands are located in the center of the thallus and develop behind the apical cell by separation of the middle lamella between apical derivatives. Strands elongate and branch in synchrony with apical growth, and thus only a single invasion is required for strand production. Two distinct ultrastructural morphotypes in the collections suggest nonspecificity of Nostoc. We speculate that Nostoc enters the thallus in the sporeling stage through mucilage clefts, and once colonies are established, cleft production ceases.