Spatial patterns and climatic drivers of phylogenetic structure of regional liverwort assemblages in China.

BACKGROUND AND AIMS: Latitudinal diversity gradients have been intimately linked to the tropical niche conservatism hypothesis, which posits that there has been a strong filter due to the challenges faced by ancestral tropical lineages to adapt to low temperatures and colonize extra-tropical regions. In liverworts, species richness is higher towards the tropics, but the centers of diversity of the basal lineages are distributed across extra-tropical regions, pointing to the colonization of tropical regions by phylogenetically clustered assemblages of species of temperate origin. Here, we test this hypothesis through analyses on the relationship between macroclimatic variation and phylogenetic diversity in Chinese liverworts. METHODS: Phylogenetic diversity metrics and their standardized effect sizes for liverworts in each of the 28 regional floras at the province level in China were related to latitude and six climate variables using regression analysis. We conducted variation partitioning analyses to determine the relative importance of each group of climatic variables. KEY RESULTS: We find that the number of species decreases with latitude, whereas phylogenetic diversity shows the reverse pattern, and that phylogenetic diversity is more strongly correlated with temperature-related variables, compared with precipitation-related variables. CONCLUSIONS: We interpret the opposite patterns observed in phylogenetic diversity and species richness in terms of a more recent origin of tropical diversity coupled with higher extinctions in temperate regions.

Impact of in vitro phytohormone treatments on the metabolome of the leafy liverwort Radula complanata (L.) Dumort.

INTRODUCTION: Liverworts are a group of non-vascular plants that possess unique metabolism not found in other plants. Many liverwort metabolites have interesting structural and biochemical characteristics, however the fluctuations of these metabolites in response to stressors is largely unknown. OBJECTIVES: To investigate the metabolic stress-response of the leafy liverwort Radula complanata. METHODS: Five phytohormones were applied exogenously to in vitro cultured R. complanata and an untargeted metabolomic analysis was conducted. Compound classification and identification was performed with CANOPUS and SIRIUS while statistical analyses including PCA, ANOVA, and variable selection using BORUTA were conducted to identify metabolic shifts. RESULTS: It was found that R. complanata was predominantly composed of carboxylic acids and derivatives, followed by benzene and substituted derivatives, fatty acyls, organooxygen compounds, prenol lipids, and flavonoids. The PCA revealed that samples grouped based on the type of hormone applied, and the variable selection using BORUTA (Random Forest) revealed 71 identified and/or classified features that fluctuated with phytohormone application. The stress-response treatments largely reduced the production of the selected primary metabolites while the growth treatments resulted in increased production of these compounds. 4-(3-Methyl-2-butenyl)-5-phenethylbenzene-1,3-diol was identified as a biomarker for the growth treatments while GDP-hexose was identified as a biomarker for the stress-response treatments. CONCLUSION: Exogenous phytohormone application caused clear metabolic shifts in Radula complanata that deviate from the responses of vascular plants. Further identification of the selected metabolite features can reveal metabolic biomarkers unique to liverworts and provide more insight into liverwort stress responses.

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.

Seasonal Variability of Volatile Components in Calypogeia integristipula.

Liverworts contain a large number of biologically active compounds that are synthesised and stored in their oil bodies. However, knowledge about the chemical composition of individual species is still incomplete. The subject of the study was Calypogeia integristipula, a species representing leafy liverworts. Plant material for chemotaxonomic studies was collected from various locations in Poland. The chemical composition was determined in 74 samples collected from the natural environment in 2021 and 2022 in three growing seasons: spring, summer and autumn, and for comparison with samples originating from in vitro culture. The plants were classified as Calypogeia integristipula on the basis of morphological characteristics, oil bodies, and DNA markers. The volatile organic compounds (VOCs) from the biological material were extracted by headspace solid phase microextraction (HS-SPME). The samples were then analysed by gas chromatography-mass spectrometry (GC-MS). A total of 79 compounds were detected, of which 44 compounds were identified. The remaining compounds were described using the MS fragmentation spectrum. Cyclical changes in the composition of compounds associated with the growing season of Calypogeia integristipula were observed. Moreover, samples from in vitro culture and samples taken from the natural environment were shown to differ in the composition of chemical compounds. In terms of quantity, among the volatile compounds, compounds belonging to the sesquiterpene group (46.54-71.19%) and sesqiuterpenoid (8.12-22.11%) dominate. A smaller number of compounds belong to aromatic compounds (2.30-10.96%), monoterpenes (0.01-0.07%), monoterpenoids (0.02-0.33%), and aliphatic hydrocarbons (1.11-6.12%). The dominant compounds in the analysed liverworts were: anastreptene (15.27-31.14%); bicyclogermacrene (6.99-18.09%), 4,5,9,10-dehydro-isolongifolene (2.00-8.72%), palustrol (4.95-9.94%), spathulenol (0.44-5.11%).

Centromere organization and UU/V sex chromosome behavior in a liverwort.

In 1917, sex chromosomes in plants were discovered in a liverwort with hetermorphic U and V chromosomes. Such heteromorphy is unexpected because, unlike the XY chromosomes in diploid-dominant plants, in haploid-dominant plants the female U and the male V chromosomes experience largely symmetrical potential recombination environments. Here we use molecular cytogenetics and super-resolution microscopy to study Frullania dilatata, a liverwort with one male and two female sex chromosomes. We applied a pipeline to Illumina sequences to detect abundant types of repetitive DNA and developed FISH probes to microscopically distinguish the sex chromosomes. We also determined the phenotypic population sex ratio because biased ratios have been reported from other liverworts with heteromorphic sex chromosomes. Populations had male-biased sex ratios. The sex chromosomes are monocentric, and of 14 probes studied (eight satellites, five transposable elements and one plastid region), four resulted in unique signals that differentiated the sex chromosomes from the autosomes and from each other. One FISH probe selectively marked the centromeres of both U chromosomes, so we could prove that during meiosis each U chromosome associates with one of the opposite telomeres of the V chromosome, resulting in a head-to-head trivalent. The similarity of the two U chromosomes to each other in size and in their centromere FISH signal positions points to their origin via a non-disjunction event (aneuploidy), which would fit with the general picture of sex chromosomes rarely crossing-over and being prone to suffer from non-disjunction.

Diversity, phylogeny, and adaptation of bryophytes: insights from genomic and transcriptomic data.

Bryophytes including mosses, liverworts, and hornworts are among the earliest land plants, and occupy a crucial phylogenetic position to aid in the understanding of plant terrestrialization. Despite their small size and simple structure, bryophytes are the second largest group of extant land plants. They live ubiquitously in various habitats and are highly diversified, with adaptive strategies to modern ecosystems on Earth. More and more genomes and transcriptomes have been assembled to address fundamental questions in plant biology. Here, we review recent advances in bryophytes associated with diversity, phylogeny, and ecological adaptation. Phylogenomic studies have provided increasing supports for the monophyly of bryophytes, with hornworts sister to the Setaphyta clade including liverworts and mosses. Further comparative genomic analyses revealed that multiple whole-genome duplications might have contributed to the species richness and morphological diversity in mosses. We highlight that the biological changes through gene gain or neofunctionalization that primarily evolved in bryophytes have facilitated the adaptation to early land environments; among the strategies to adapt to modern ecosystems in bryophytes, desiccation tolerance is the most remarkable. More genomic information for bryophytes would shed light on key mechanisms for the ecological success of these 'dwarfs' in the plant kingdom.

Desiccation-rehydration measurements in bryophytes: current status and future insights.

Desiccation-rehydration experiments have been employed over the years to evaluate desiccation tolerance of bryophytes (Bryophyta, Marchantiophyta, and Anthocerotophyta). Researchers have applied a spectrum of protocols to induce desiccation and subsequent rehydration, and a wide variety of techniques have been used to study desiccation-dependent changes in bryophyte molecular, cellular, physiological, and structural traits, resulting in a multifaceted assortment of information that is challenging to synthesize. We analysed 337 desiccation-rehydration studies, providing information for 351 species, to identify the most frequent methods used, analyse the advances in desiccation studies over the years, and characterize the taxonomic representation of the species assessed. We observed certain similarities across methodologies, but the degree of convergence among the experimental protocols was surprisingly low. Out of 52 bryophyte orders, 40% have not been studied, and data are lacking for multiple remote or difficult to access locations. We conclude that for quantitative interspecific comparisons of desiccation tolerance, rigorous standardization of experimental protocols and measurement techniques, and simultaneous use of an array of experimental techniques are required for a mechanistic insight into the different traits modified in response to desiccation. New studies should also aim to fill gaps in taxonomic, ecological, and spatial coverage of bryophytes.

An overview of bioinformatics, genomics, and transcriptomics resources for bryophytes.

Bryophytes are useful models for the study of plant evolution, development, plant-fungal symbiosis, stress responses, and gametogenesis. Additionally, their dominant haploid gametophytic phase makes them great models for functional genomics research, allowing straightforward genome editing and gene knockout via CRISPR or homologous recombination. Until 2016, however, the only bryophyte genome sequence published was that of Physcomitrium patens. Throughout recent years, several other bryophyte genomes and transcriptome datasets became available, enabling better comparative genomics in evolutionary studies. The increase in the number of bryophyte genome and transcriptome resources available has yielded a plethora of annotations, databases, and bioinformatics tools to access the new data, which covers the large diversity of this clade and whose biology comprises features such as association with arbuscular mycorrhiza fungi, sex chromosomes, low gene redundancy, or loss of RNA editing genes for organellar transcripts. Here we provide a guide to resources available for bryophytes with regards to genome and transcriptome databases and bioinformatics tools.

Bryophyte ultraviolet-omics: from genes to the environment.

Ultraviolet (UV) radiation has contributed to the evolution of organisms since the origins of life. Bryophytes also have evolutionary importance as the first clearly identified lineage of land plants (embryophytes) colonizing the terrestrial environment, thus facing high UV and water scarcity, among other new challenges. Here we review bryophyte UV-omics, the discipline relating bryophytes and UV, with an integrative perspective from genes to the environment. We consider species and habitats investigated, methodology, response variables, protection mechanisms, environmental interactions, UV biomonitoring, molecular and evolutionary aspects, and applications. Bryophyte UV-omics shows convergences and divergences with the UV-omics of other photosynthetic organisms, from algae to tracheophytes. All these organisms converge in that UV damage may be limited under realistic UV levels, due to structural protection and/or physiological acclimation capacity. Nevertheless, bryophytes diverge because they have a unique combination of vegetative and reproductive characteristics to cope with high UV and other concomitant adverse processes, such as desiccation. This interaction has both evolutionary and ecological implications. In addition, UV effects on bryophytes depend on the species and the evolutionary lineage considered, with mosses more UV-tolerant than liverworts. Thus, bryophytes do not constitute a homogeneous functional type with respect to their UV tolerance.

Liverwort oil bodies: diversity, biochemistry, and molecular cell biology of the earliest secretory structure of land plants.

Liverworts are known for their large chemical diversity. Much of this diversity is synthesized and enclosed within oil bodies (OBs), a synapomorphy of the lineage. OBs contain the enzymes to biosynthesize and store large quantities of sesquiterpenoids and other compounds while limiting their cytotoxicity. Recent important biochemical and molecular discoveries related to OB formation, diversity, and biochemistry allow comparison with other secretory structures of land plants from an evo-devo perspective. This review addresses and discusses the most recent advances in OB origin, development, and function towards understanding the importance of these organelles in liverwort physiology and adaptation to changing environments. Our mapping of OB types and chemical compounds to the current liverwort phylogeny suggests that OBs were present in the most recent common ancestor of liverworts, supporting that OBs evolved as the first secretory structures in land plants. Yet, we require better sampling to define the macroevolutionary pattern governing the ancestral type of OB. We conclude that current efforts to find molecular mechanisms responsible for the morphological and chemical diversity of secretory structures will help understand the evolution of each major group of land plants, and open new avenues in biochemical research on bioactive compounds in bryophytes and vascular plants.

Biogenesis, conservation, and function of miRNA in liverworts.

MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules, 18-24 nucleotides long, that control multiple gene regulatory pathways via post-transcriptional gene silencing in eukaryotes. To develop a comprehensive picture of the evolutionary history of miRNA biogenesis and action in land plants, studies on bryophyte representatives are needed. Here, we review current understanding of liverwort MIR gene structure, miRNA biogenesis, and function, focusing on the simple thalloid Pellia endiviifolia and the complex thalloid Marchantia polymorpha. We review what is known about conserved and non-conserved miRNAs, their targets, and the functional implications of miRNA action in M. polymorpha and P. endiviifolia. We note that most M. polymorpha miRNAs are encoded within protein-coding genes and provide data for 23 MIR gene structures recognized as independent transcriptional units. We identify M. polymorpha genes involved in miRNA biogenesis that are homologous to those identified in higher plants, including those encoding core microprocessor components and other auxiliary and regulatory proteins that influence the stability, folding, and processing of pri-miRNAs. We analyzed miRNA biogenesis proteins and found similar domain architecture in most cases. Our data support the hypothesis that almost all miRNA biogenesis factors in higher plants are also present in liverworts, suggesting that they emerged early during land plant evolution.

Phylotranscriptomics of liverworts: revisiting the backbone phylogeny and ancestral gene duplications.

BACKGROUND AND AIMS: With some 7300 extant species, liverworts (Marchantiophyta) represent one of the major land plant lineages. The backbone relationships, such as the phylogenetic position of Ptilidiales, and the occurrence and timing of whole-genome duplications, are still contentious. METHODS: Based on analyses of the newly generated transcriptome data for 38 liverworts and complemented with those publicly available, we reconstructed the evolutionary history of liverworts and inferred gene duplication events along the 55 taxon liverwort species tree. KEY RESULTS: Our phylogenomic study provided an ordinal-level liverwort nuclear phylogeny and identified extensive gene tree conflicts and cyto-nuclear incongruences. Gene duplication analyses based on integrated phylogenomics and Ks distributions indicated no evidence of whole-genome duplication events along the backbone phylogeny of liverworts. CONCLUSIONS: With a broadened sampling of liverwort transcriptomes, we re-evaluated the backbone phylogeny of liverworts, and provided evidence for ancient hybridizations followed by incomplete lineage sorting that shaped the deep evolutionary history of liverworts. The lack of whole-genome duplication during the deep evolution of liverworts indicates that liverworts might represent one of the few major embryophyte lineages whose evolution was not driven by whole-genome duplications.

Unprecedented 4,9-Seco-Oplopanane and Seven Drimane Sesquiterpenoids from the Chinese Liverwort Lejeunea flava (Sw.) Nees.

An unprecedented 4,9-seco-oplopanane (1), two undescribed drimane epimers (2 and 3), and five known drimane sesquiterpenoids (4-8) were isolated from the Chinese liverwort Lejeunea flava (Sw.) Nees. The structures of the new sesquiterpenoids were determined using nuclear magnetic resonance spectroscopy, electronic circular dichroism calculations, and single-crystal X-ray diffraction measurements. The inhibitory capacity of the new compounds against nitric oxide production in lipopolysaccharide-induced RAW 264.7 murine macrophages, along with the cytotoxicity of the new compounds against A549 and HepG-2 human cancer cell lines, were discussed.

Super-Mitobarcoding in Plant Species Identification? It Can Work! The Case of Leafy Liverworts Belonging to the Genus Calypogeia.

Molecular identification of species is especially important where traditional taxonomic methods fail. The genus Calypogeia belongs to one of the tricky taxons. The simple morphology of these species and a tendency towards environmental plasticity make them complicated in identification. The finding of the universal single-locus DNA barcode in plants seems to be 'the Holy Grail'; therefore, researchers are increasingly looking for multiloci DNA barcodes or super-barcoding. Since the mitochondrial genome has low sequence variation in plants, species delimitation is usually based on the chloroplast genome. Unexpectedly, our research shows that super-mitobarcoding can also work! However, our outcomes showed that a single method of molecular species delimitation should be avoided. Moreover, it is recommended to interpret the results of molecular species delimitation alongside other types of evidence, such as ecology, population genetics or comparative morphology. Here, we also presented genetic data supporting the view that C. suecica is not a homogeneous species.

Seasonal Variability and Effect of Sample Storage on Volatile Components in Calypogeia azurea.

A change in the composition of specialized metabolites is often observed in stressed plants. Phytochemicals play an important role in adapting plants to the environment, particularly overcoming stress conditions such as temperature, humidity, and light intensity. In this study, seasonal variations in the concentrations of volatile organic compounds (VOCs) were analysed in species of Calypogeia azurea. The article presents the effect of sample storage on volatile organic compounds present in Calypogeia liverwort cells and whether the collection habitats of the sample affect the content of phytochemicals. The VOCs of the species within the liverwort Calypogeia azurea were analysed by GC-MS. Compounds were isolated from plant material using the HS-SPME technique. The samples were collected over several years (2019-2021). Of the several dozen samples collected, 79 compounds were isolated, of which 47 were identified.

Comparison of Extraction Techniques for the Determination of Volatile Organic Compounds in Liverwort Samples.

This article focuses on the comparison of four popular techniques for the extraction of volatile organic compounds (VOCs) from liverworts of the Calypogeia azurea species. Since extraction is the most important step in the sample analysis of ingredients present in botanical preparations, their strengths, and weaknesses are discussed. In order to determine the VOCs present in plants, selecting the appropriate one is a key step of the extraction technique. Extraction should ensure the isolation of all components present in the oily bodies of Calypogeia azurea without the formation of any artifacts during treatment. The best extraction method should yield the determined compounds in detectable amounts. Hydrodistillation (HD), applying Deryng apparatus and solid-liquid extraction (SLE), microwave-assisted extraction (MAE), and headspace solid-phase microextraction (HS-SPME) were used for volatile extraction. The extracts obtained were analysed by gas chromatography coupled to mass spectrometry (GC-MS) to determine the compounds.

Bryophyte C:N:P stoichiometry, biogeochemical niches and elementome plasticity driven by environment and coexistence.

Ecological stoichiometry and studies of biogeochemical niches have mainly focused on plankton and vascular plants, but the phenotypically closest modern relatives of early plants, bryophytes, have been largely neglected. We analysed C:N:P stoichiometries and elemental compositions (K, Na, Mg, Ca, S, Fe) of 35 widely distributed bryophyte species inhabiting springs. We estimated bryophyte C:N:P ratios and their biogeochemical niches, investigated how elementomes respond to the environment and determined whether they tend to diverge more for coexisting than non-coexisting individuals and species. The median C:N:P was 145:8:1, intermediate between Redfield's ratio for marine plankton and those for vascular plants. Biogeochemical niches were differentiated amongst species and were phylogenetically conserved. Differences in individual and species-specific elementomes increased with coexistence between species. Our results provide an evolutionary bridge between the ecological stoichiometries of algae and vascular plants and suggest that differences in elementomes could be used to understand community assemblages and functional diversity.

Plastid genomes and phylogenomics of liverworts (Marchantiophyta): Conserved genome structure but highest relative plastid substitution rate in land plants.

With some 7300 species of small nonvascular spore-producing plants, liverworts represent one of the major lineages of land plants. Although multi-locus molecular phylogenetic studies have elucidated relationships of liverworts at different taxonomic categories, the backbone phylogeny of liverworts is still to be fully resolved, especially for the placement of Ptilidiales and the relationships within Jungermanniales and Marchantiales. Here, we provided phylogenomic inferences of liverworts based on 42 newly sequenced and 24 published liverwort plastid genomes representing all but two orders of liverworts, and characterized the evolution of the plastome in liverworts. The structure of the plastid genome is overall conserved across the phylogeny of liverworts, with only two structural variants detected from simple thalloids, besides 18 out of 43 liverwort genera showing intron variations in their plastomes. Complex thalloid liverworts maintain the most plastid genes, and seem to undergo fewer gene deletions and pseudogenization events than other liverworts. Plastid phylogenetic inferences yielded mostly robustly supported relationships, and consistently resolved Ptilidiales as the sister to Porellales. The relative ratio of silent substitutions across the three genetic compartments (i.e., 1:15:10, for mitochondrial:plastid:nuclear) suggests that liverwort plastid genes have the potential to evolve faster than their nuclear counterparts, unlike in any other major land plant lineages where the mutation rate of nuclear genes overwhelm those of their plastid and mitochondrial counterparts.

Bryophyte and lichen biomass and nitrogen fixation in a high elevation cloud forest in Cerro de La Muerte, Costa Rica.

Cloud forests have been found to lose more nitrogen in stream discharge than they gain from atmospheric deposition. They also support a large diversity and biomass of tree epiphytes, predominately composed of cryptogams. Since cryptogam epiphytes harbor nitrogen fixing cyanobacteria, they may help make up for the nitrogen loss from ecosystems. We assessed cryptogam biomass on the ground, boles and branches in Quercus costaricensis dominated stands near the tree line in the Cordillera de Talamanca, Costa Rica. Nitrogen fixation was assayed using 15N2 uptake. Total cryptogam biomass was 2 977 kg ha-1, with 67% being found on the lower branches. Bryophytes and chlorolichens made up 53% and 44%, respectively, of the biomass. Half of the bryophyte mass was composed of the liverwort Plagiochila heterophylla, and 66% of the chlorolichen of Lobariella pallida. There were no significant differences in nitrogen fixation rates between the cryptogam species, with a mean rate of 5.04 µg N g-1 day-1 during the predominantly wet condition in the forest. The overall nitrogen input from fixation was 6.1 kg N ha-1 year-1, of which 78% came from bryophytes, 18% from chlorolichens, and 4% from cyanolichens. Only 2.0% of the fixation occurred in cryptogams on the ground, whereas 67%, 24%, and 7% occurred on the lower branches, boles, and upper branches, respectively. These results show that tree epiphytes constitute a significant source of nitrogen for these forests, due to the trees' large surface area, and can make up for the nitrogen lost from these ecosystems.

Carotenoid Biosynthesis in Liverworts.

In higher plants, there are many studies on carotenoid biosynthetic pathways and their relevant genes. On the other hand, few researches exist on carotenoid biosynthesis in early-land plants containing liverworts, mosses, and ferns. Thus, the evolutionary history of carotenoid biosynthesis genes in land plants has remained unclear. A liverwort Marchantia polymorpha is thought to be one of the first land plants, since this plant remains a primitive figure. Moreover, this liverwort is regarded as the model plant of bryophytes due to several reasons. In this chapter, we review carotenoid biosynthesis in liverworts and discuss the functional evolution and evolutionary history of carotenogenic genes in land plants.