Land plants (embryophytes) are characterized by a remarkable variety of sexual systems, so much so that Linnaeus used this diversity as the basis for his classification of flowering plants. Since at least Darwin, plant mating systems have been employed to evaluate theories regarding the evolutionary processes that shape the diversity of life. Over the last few decades, at first in a handful of model systems but increasingly in species specifically targeted for their life history, investigators have generated a preliminary catalogue of genes and pathways that govern transitions in sexual system across the phylogeny of land plants, albeit biased towards angiosperms. Remarkably, while some sexual system shifts are concurrent with the recruitment of new genes or pathways, the relatively conserved embryophyte gene family content indicates that aspects of the genetic control of sexual reproduction may be widely shared. Here we examine the underlying genetic control of gametogenesis, in particular sex determination and sexual differentiation of the gametophyte, potentially highly conserved traits that nonetheless are central to sexual system transitions across the embryophytes.

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 understanding 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 and 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 ILS and ancient reticulation, the latter revealed using a network approach to identify evidence of gene flow among hornwort lineages. Hornwort diversification began in the Carboniferous with widespread family-level divergences during the mid-Cretaceous and Palaeogene. CONCLUSIONS:ILS 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.

The mating system strongly influences genetic variation, reproductive isolation, speciation, and invasiveness. In haplodiploid species, inbreeding is expected to produce high homozygosity and purge deleterious mutations, potentially reducing inbreeding depression but increasing outbreeding depression at shallow genetic distances. In bark, ambrosia, and seed beetles (Scolytinae), haplodiploidy and inbreeding have been linked to small population persistence and invasiveness. However, observed heterozygosity and outbreeding in natural populations challenge these assumptions. To examine the relationship between genetic distance and fitness, we established F4 inbred lines of the date stone beetle, Coccotrypes dactyliperda, from populations in Israel and the United States. Genetic distances were estimated using mitochondrial genes and nuclear markers, and 814 experimental crosses were conducted to assess fecundity and hatch rates. Contrary to previous reports, we observed both inbreeding and outbreeding depression, especially in sibling matings and in extremely distant crosses. A non-linear model revealed reduced fitness at both ends of the relatedness spectrum, with highest fecundity at intermediate distances. Mitochondrial lineages showed the strongest inbreeding depression, although lineage assignments varied between mitochondrial and nuclear markers. Our results emphasize the importance of quantifying genetic distance continuously rather than categorically. The discordance between mitochondrial and nuclear markers suggests that incompatibility may involve unmeasured loci. These findings highlight the complexity of genetic structure and the need for further studies on the fitness consequences of genetic distance in haplodiploid species.

The maintenance of genetic variation by balancing selection is of considerable interest to evolutionary biologists. An important but understudied potential driver of balancing selection is antagonistic pleiotropy between diploid and haploid stages of the plant life cycle. Despite sharing a common genome, sporophytes (2n) and gametophytes (n) may undergo differential or even opposing selection. Theoretical work suggests antagonistic pleiotropy between life stages can generate balancing selection and maintain genetic variation. Despite the potential for far-reaching consequences of gametophytic selection, empirical tests of its pleiotropic effects (neutral, synergistic, or antagonistic) on sporophytes are generally lacking. Here, we examined the population genomic signals of selection across life stages in the angiosperm Rumex hastatulus and the moss Ceratodon purpureus. We compared gene expression between life stages and sexes, combined with neutral diversity statistics and the analysis of the distribution of fitness effects. In contrast to what would be predicted under balancing selection due to antagonistic pleiotropy, we found that unbiased genes between life stages were under stronger purifying selection, likely explained by a predominance of synergistic pleiotropy between life stages and strong purifying selection on broadly expressed genes. In addition, we found that 30% of candidate genes under balancing selection in R. hastatulus were located within inversion polymorphisms. Our findings provide novel insights into the genome-wide characteristics and consequences of plant gametophytic selection.

The development of CRISPR technologies provides a powerful tool for understanding the evolution and functionality of essential biological processes. Here we demonstrate successful CRISPR-Cas9 genome editing in the dioecious moss species, Ceratodon purpureus. Using an existing selection system from the distantly related hermaphroditic moss, Physcomitrium patens, we generated knock-outs of the APT reporter gene by employing CRISPR-targeted mutagenesis under expression of native U6 snRNA promoters. Next, we used the native homology-directed repair (HDR) pathway, combined with CRISPR-Cas9, to knock in two reporter genes under expression of an endogenous RPS5A promoter in a newly developed landing site in C. purpureus. Our results show that the molecular tools developed in P. patens can be extended to other mosses across this ecologically important and developmentally variable group. These findings pave the way for precise and powerful experiments aimed at identifying the genetic basis of key functional variation within the bryophytes and between the bryophytes and other land plants.

— The diverse and spectacular Hibisceae tribe comprises over 750 species. No studies, however, have broadly sampled across the dozens of genera in the tribe, leading to uncertainty in the relationships among genera. The non-monophyly of the genus Hibiscus is infamous and challenging, whereas the monophyly of most other genera in the tribe has yet to be assessed, including the large genus Pavonia. Here we significantly increase taxon sampling in the most complete phylogenetic study of the tribe to date. We assess monophyly of most currently recognized genera in the tribe and include three and thirteen newly sampled sections of Hibiscus and Pavonia, respectively. We also include five rarely sampled genera and 137 species previously unsampled. Our phylogenetic trees demonstrate that Hibiscus , as traditionally defined, encompasses at least 20 additional genera. The status of Pavonia emerges as comparable in complexity to Hibiscus . We offer clarity in the phylogenetic placement of several taxa of uncertain affinity (e.g. Helicteropsis , Hibiscadelphus, Jumelleanthus, and Wercklea ). We also identify two new clades and elevate them to the generic rank with the recognition of two new monospecific genera: 1) Blanchardia M.M.Hanes & R.L.Barrett is a surprising Caribbean lineage that is sister to the entire tribe, and 2) Astrohibiscus McLay & R.L.Barrett represents former members of Hibiscus caesius s.l. Cravenia McLay & R.L.Barrett is also described as a new genus for the Hibiscus panduriformis clade, which is allied to Abelmoschus . Finally, we introduce a new classification for the tribe and clarify the boundaries of Hibiscus and Pavonia .

Terrestrial plants from the very limits of life are likely to harbor genes that confer an advantage in human space exploration. These plants are seemingly capable of performing mission critical functions in spaceflight and on extraterrestrial farms while informing directed gene manipulation in target plant species. However, their adaptations to physiologically extreme habitats may hinder the efficacy of routine laboratory techniques for model plants. We here present the development of Antarctic moss Ceratodon purpureus payload and flight operations for the ANT1 Radiation Tolerance Experiment with Moss in Orbit on the Space Station (ARTEMOSS) experiment to the International Space Station (ISS) given limited physical space and crew time. We demonstrate that the hydrophobic surface of Antarctic moss impedes chemical tissue fixation and precludes the use of RNAlater coupled with payload hardware deployed in standard plant spaceflight experiments. We show that deep-freezing the moss tissue on Petri plates provides adequate tissue fixation and allows the extraction of high-quality RNA suitable for gene expression profiling. We replaced hardware with stacks of Petri plates housing Antarctic moss and chemical fixation with deep-freezing in a cryogenic GLACIER freezer. Our design can be translated to other plant species to expand current experimentation techniques with plants from extreme terrestrial environments in order to advance human space exploration.

Ice retreat following the last glacial maximum, and the associated changes in landscape, climate, and hydrology, altered nutrient fluxes off the landscape and fostered the development of new biological communities. The role of feedbacks between the new plant communities and geological processes, such as mineral weathering, are not well understood. Differences in water chemistry between catchments in southwestern Greenland that were deglaciated ~ 7 and 10 kya suggests that nutrient differences cannot be explained solely by abiotic factors alone. One hypothesis to explain the variation in water chemistry is that plant communities mobilize different nutrients, either by releasing them directly or by differential weathering of the underlying substrates. To test this hypothesis, we first surveyed plant communities using a combination of Floristic Habitat Sampling and modified Point Quarter method in forty-five 1.25 m2 plots across five watersheds in 2022. The watersheds were ~ 150 km apart with one near the ice edge exposed ~6.8 ky ago and the other near the coastexposed ~ 10 ky. ago Using NMDS and ANOSIM (α = 0.05) analyses, we found that species compositions across locations were distinct (stress score = 0.15; p = 1e -04, R = 04025). Next we compared the vegetation distribution data to elemental concentrations of dissolved nutrients (Li, Mg, Al, P, Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Si) from adjacent streams using a Mantel test (method = Spearman, permutation = 9999). The variation in vegetation is correlated with the stream elemental composition data (p = 0.0043). The correlation between the two matrices suggests that either plant community composition drives variation in nutrient concentrations, or plant community and nutrient fluxes may both respond to another environmental variable. To distinguish between these alternatives, we are currently conducting mesocosm weathering experiments to test whether tundra plants vary in their ability to promote biogenic weathering, and whether biogenic weathering is sufficiently enhanced to explain variations in nutrient elements dissolved in streams and vegetation in southwestern Greenland.

Currently, a wide range of genomic techniques is available at a relatively affordable price. However, not all of them have been equally explored in bryophyte systematics. In the present study, we apply next-generation sequencing to identify samples that cannot be assigned to a taxon by morphological analysis or by Sanger sequencing methods. These samples correspond to a moss with an enigmatic morphology that has been found throughout Western Europe over the last two decades. They exhibit several anomalies in the gametophyte and, on the rare occasions that they appear, also in the sporophyte. The most significant alterations are related to the shape of the leaves. Morphologically, all specimens correspond to mosses of the genus Lewinskya, and the least modified samples are potentially attributable to the Lewinskya affinis complex. Specimen identifications were first attempted using up to seven molecular markers with no satisfactory results. Thus, we employed data generated from targeted enrichment using the GoFlag 408 flagellate land plant probe set to elucidate their identity. Our results demonstrate that all the enigmatic samples correspond to a single species, L. affinis s.str. This approach provided the necessary resolution to confidently identify these challenging samples and may be a powerful tool for similar cases, especially in bryophytes.

Polyploidy is widely recognized as an important speciation mechanism because it isolates tetraploids from their diploid progenitors. Polyploidy also provides new genetic material that may facilitate adaptive evolution. However, new mutations are more likely to arise after a neopolyploid has already successfully invaded a population. Thus, the role of adaptive forces in establishing a polyploid remains unclear. One solution to this apparent paradox may lie in the capacity of polyploids to suppress recombination among preexisting locally adapted alleles. The local adaptation mechanism requires that spatially heterogeneous selection acts on multiple loci and that gene flow introduces maladapted alleles to the population where the polyploid forms. The mechanism requires neither strong genetic drift nor any intrinsic benefit of genome doubling and can accommodate any mode of gene action. A unique prediction of the mechanism is that adaptive alleles should predate polyploidization, a pattern consistent with observations from a few well-studied polyploids. The mechanism is also consistent with the coexistence of both diploid and tetraploid cytotypes, fitness heterogeneity among independently derived polyploids, and the prevalence of outcrossing among older polyploids. The local adaptation mechanism also makes novel predictions about circumstances favoring polyploid invasions that can be tested using molecular genetic or comparative approaches.

In species with separate sexes, the genome must produce two distinct developmental programs. Sexually dimorphic development may be controlled by either sex-limited loci or biased expression of loci transmitted through both sexes. Variation in the gene content of sex-limited chromosomes demonstrates that eukaryotic species differ markedly in the roles of these two mechanisms in governing sexual dimorphism. The bryophyte model systems Marchantia polymorpha and Ceratodon purpureus provide a particularly striking contrast. Although both species possess a haploid UV sex chromosome system, in which females carry a U chromosome and males carry a V, M. polymorpha relies on biased autosomal expression, while in C. purpureus, sex-linked genes drive dimorphism. Framing these genetic architectures as divergent outcomes of genetic conflict highlights comparative genomic analyses to better understand the evolution of sexual dimorphism.

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.

Introduction Half a century since the creation of the International Association of Bryologists, we carried out a review to identify outstanding challenges and future perspectives in bryology. Specifically, we have identified 50 fundamental questions that are critical in advancing the discipline. Methods We have adapted a deep-rooted methodology of horizon scanning to identify key research foci. An initial pool of 258 questions was prepared by a multidisciplinary and international working group of 32 bryologists. A series of online surveys completed by a broader community of researchers in bryology, followed by quality-control steps implemented by the working group, were used to create a list of top-priority questions. This final list was restricted to 50 questions with a broad conceptual scope and answerable through realistic research approaches. Key results The top list of 50 fundamental questions was organised into four general topics: Bryophyte Biodiversity and Biogeography; Bryophyte Ecology, Physiology and Reproductive Biology; Bryophyte Conservation and Management; and Bryophyte Evolution and Systematics. These topics included 9, 19, 14 and 8 questions, respectively. Conclusions Although many of the research challenges identified are not newly conceived, our horizon-scanning exercise has established a significant foundation for future bryological research. We suggest analytical and conceptual strategies and novel developments for potential use in advancing the research agenda for bryology.

Phylogenomic data increase the possibilities of resolving the evolutionary and systematic relationships among taxa. This is especially valuable in groups with few and homoplasious morphological characters, in which systematic and taxonomical delimitations have been traditionally difficult. Such is the case of several lineages within Bryophyta, like Orthotrichaceae, the second most diverse family of mosses. Members of tribe Orthotricheae are common in temperate and cold regions, as well as in high tropical mountains. In extratropical areas, they represent one of the main components of epiphytic communities, both in dry and oceanic or hyperoceanic conditions. The epiphytic environment is considered a hostile one for plant development, mainly due to its low capacity of moisture retention. Thus, the diversification of the Orthotrichaceae in this environment could be seen as striking. Over the last two decades, great taxonomic and systematic progresses have led to a rearrangement at the generic level in this tribe, providing a new framework to link environment to patterns of diversification. Here, we use nuclear loci targeted with the GoFlag 408 enrichment probe set to generate a well-sampled phylogeny with well-supported suprageneric taxa and increasing the phylogenetic resolution within the two recognized subtribes. Specifically, we show that several genera with Ulota-like morphology jointly constitute an independent lineage. Within this lineage, the recently described Atlantichella from Macaronesia and Western Europe appears as the sister group of Ulota bellii from Zealandia. This latter species is here segregated in the new genus Rehubryum. Assessment of the ecological and biogeographical affinities of the species within the phylogenetic framework suggests that niche adaptation (including climate and substrate) may be a key evolutionary driver that shaped the high diversification of Orthotricheae.

Studying how different plant groups deal with heavy metal exposure is crucial to improve our understanding of the diversity of molecular mechanisms involved in plant stress response. Here, we used RNA sequencing (RNA-seq) and epigenotyping by sequencing (epiGBS) to assess gene expression and DNA methylation changes respectively in plants from four populations of the metallophyte moss Scopelophila cataractae treated with Cd or Cu in the laboratory. We built RNA-seq and epiGBS sequencing libraries from control and treated samples from each population and sequenced them using Illumina HiSeq 3000 (PE-150 bp) and Illumina HiSeq X-Ten System (PE-150 bp) respectively. For the RNA-seq data, we performed a read quality filter, mapped the reads to the de novo transcriptome created with Trinity, and estimated transcript abundance for each sample. For the epiGBS data, we used a custom pipeline (https://doi.org/10.5281/zenodo.7040291) to map the reads to a de novo reference genome and performed strand-specific nucleotide (single nucleotide polymorphisms, SNPs) and methylation (single cytosine methylation polymorphisms, SMPs) variant calling. We filtered out SNPs and SMPs with low coverage within (positions with <10 sequencing reads per sample) and across samples (positions with poor representation on the full set of samples). Finally, we performed pairwise comparisons between control and treated samples from each population and identified differentially expressed genes and differentially methylated cytosines associated to heavy metal exposure. We payed particular attention to the different responses of the more and the less tolerant populations of S. cataractae. These datasets could contribute to future comparative studies of abiotic stress response across plant groups.

Bryophytes’ remarkable capacity to tolerate extreme abiotic conditions allows us to enhance our understanding of the diversity of molecular mechanisms involved in plant stress response. Here, we used next generation sequencing to study DNA methylation and gene expression changes in plants from four populations of the metallophyte moss Scopelophila cataractae experimentally exposed to either Cd or Cu. These populations previously showed differences in tolerance to both metals, so here, we aimed at uncovering the molecular basis of this phenotypic differentiation. We found no evidence of genetic differentiation among the populations studied. The epigenetic data, however, showed limited but significant population-specific changes in DNA methylation in response to both metals. Exposure to acute Cu stress in the laboratory led to the downregulation of genes involved in heavy metal tolerance in both populations regardless of their tolerance level, but this response was quantitatively higher in the most tolerant. We propose that chronic exposure to varying levels of heavy metals in the field led to potentially non-genetically-based intraspecific differentiation for heavy metal tolerance in S. cataractae . The most tolerant plants invested more in constitutive protection and were more efficient in entering a conservative state when faced with acute Cu stress.

Mosses inhabit nearly all terrestrial ecosystems and engage in important interactions with nitrogen-fixing microbes, sperm-dispersing arthropods, and other plants. It is hypothesized that these interactions could be mediated by biogenic volatile organic compounds (BVOCs). Moss BVOCs may play fundamental roles in influencing local ecologies, such as biosphere-atmosphere-hydrosphere communications, physiological and evolutionary dynamics, plant-microbe interactions, and gametophyte stress physiology. Further progress in quantifying the composition, magnitude, and variability of moss BVOC emissions, and their response to environmental drivers and metabolic requirements, is limited by methodological and analytical challenges. We review several sampling techniques with various analytical approaches and describe best practices in generating moss gametophyte BVOC measures. We emphasize the importance of characterizing the composition and magnitude of moss BVOC emissions across a variety of species to better inform and stimulate important cross-disciplinary studies. We conclude by highlighting how current methods could be employed, as well as best practices for choosing methodologies.

In neutral models of quantitative trait evolution, both genetic and phenotypic divergence scale as random walks, producing a correlation between the two measures. However, complexity in the genotype-phenotype map may alter the correlation between genotypic and phenotypic divergence, even when both are evolving neutrally or nearly so. Understanding this correlation between phenotypic and genetic variation is critical for accurately interpreting the fossil record. This study compares the geographic structure and scaling of morphological variation of the shape of the first lower molar of 77 individuals of the northern grasshopper mouse Onychomys leucogaster to genome-wide SNP variation in the same sample. We found strong genetic structure but weak or absent morphological structure indicating that the scaling of each type of variation is decoupled from one another. Low PST values relative to FST values are consistent with a lack of morphological divergence in contrast to genetic divergence between groups. This lack of phenotypic structure and the presence of notable within-sample phenotypic variance are consistent with uniform selection or constraints on molar shape across a wide geographic and environmental range. Over time, this kind of decoupling may result in patterns of phenotypic stasis masking underlying genetic patterns.