An optimized transformation protocol for Anthoceros agrestis and three more hornwort species.

Land plants comprise two large monophyletic lineages, the vascular plants and the bryophytes, which diverged from their most recent common ancestor approximately 480 million years ago. Of the three lineages of bryophytes, only the mosses and the liverworts are systematically investigated, while the hornworts are understudied. Despite their importance for understanding fundamental questions of land plant evolution, they only recently became amenable to experimental investigation, with Anthoceros agrestis being developed as a hornwort model system. Availability of a high-quality genome assembly and a recently developed genetic transformation technique makes A. agrestis an attractive model species for hornworts. Here we describe an updated and optimized transformation protocol for A. agrestis, which can be successfully used to genetically modify one more strain of A. agrestis and three more hornwort species, Anthoceros punctatus, Leiosporoceros dussii, and Phaeoceros carolinianus. The new transformation method is less laborious, faster, and results in the generation of greatly increased numbers of transformants compared with the previous method. We have also developed a new selection marker for transformation. Finally, we report the development of a set of different cellular localization signal peptides for hornworts providing new tools to better understand the hornwort cell biology.

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.

The first step into phenolic metabolism in the hornwort Anthoceros agrestis: molecular and biochemical characterization of two phenylalanine ammonia-lyase isoforms.

MAIN CONCLUSION: Two isoforms of phenylalanine ammonia-lyase (PAL) have been isolated as cDNA sequences from the hornwort Anthoceros agrestis. The encoded enzymes convert L-phenylalanine and to lower extents L-tyrosine and L-histidine. Thus, the functional presence of the general phenylpropanoid pathway in one of the earliest land plant groups is established. The hornwort Anthoceros agrestis has an elaborated phenolic metabolism resulting in phenolic compounds, such as rosmarinic acid or megacerotonic acid. The general phenylpropanoid pathway is involved in the biosynthesis of these compounds. Two phenylalanine ammonia-lyase (PAL) genes, AaPAL1 and AaPAL2, have been identified in Anthoceros agrestis and the protein with an N-terminal 6xHis-tag heterologously synthesized in Escherichia coli for a full biochemical characterization. Both PAL proteins accept L-phenylalanine, L-tyrosine as well as L-histidine as substrates, although the activity is explicitly the highest with L-phenylalanine. Km values as well as catalytic efficiencies were determined for phenylalanine (Km AaPAL1 39 µM, AaPAL2 18 µM) and tyrosine (Km AaPAL1 3.3 mM, AaPAL2 3.5 mM). In suspension cultures of Anthoceros agrestis, PAL genes were transcribed in parallel to rosmarinic acid (RA) accumulation and both showed highest abundance in the early growth phase. In a phylogenetic tree, both AaPAL amino acid sequences grouped within a clade with PAL amino acid sequences of diverse origin ranging from non-vascular to vascular plants, while most PALs from eudicots and monocots were mainly found in two other clades. The similarity of the hornwort PAL amino acid sequences to PAL sequences from vascular plants is more than 80% showing a strong conservation within the land plants. With this characterization of PALs from Anthoceros agrestis together with former investigations concerning cinnamic acid 4-hydroxylase and 4-coumaric acid CoA-ligase, the functional presence of the general phenylpropanoid pathway in this hornwort is proven.

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.

Insight into early diversification of leucine-rich repeat receptor-like kinases provided by the sequenced moss and hornwort genomes.

KEY MESSAGE: Identification of the subfamily X leucine-rich repeat receptor-like kinases in the recently sequenced moss and hornwort genomes points to their diversification into distinct groups during early evolution of land plants. Signal transduction mediated through receptor-ligand interactions plays key roles in controlling developmental and physiological processes of multicellular organisms, and plants employ diverse receptors in signaling. Leucine-rich repeat receptor-like kinases (LRR-RLKs) represent one of the largest receptor classes in plants and are structurally classified into subfamilies. LRR-RLKs of the subfamily X are unique in the variety of their signaling roles; they include receptors for steroid or peptide hormones as well as negative regulators of signaling through binding to other LRR-RLKs, raising a question as to how they diversified. However, our understanding of diversification processes of LRR-RLKs has been hindered by the paucity of genomic data in non-seed plants and limited taxa sampling in previous phylogenetic analyses. Here we analyzed the phylogeny of LRR-RLK X sequences collected from all major land plant lineages and show that this subfamily diversified into six major clades before the divergence between bryophytes and vascular plants. Notably, we have identified homologues of the brassinosteroid receptor, BRASSINOSTEROID INSENSITIVE 1 (BRI1), in the genomes of Sphagnum mosses, hornworts, and ferns, contrary to earlier reports that postulate the origin of BRI1-like LRR-RLKs in the seed plant lineage. The phylogenetic distribution of major clades illustrates that the current receptor repertoire was shaped through lineage-specific gene family expansion and independent gene losses, highlighting dynamic changes in the evolution of LRR-RLKs.

Identification and biochemical characterisation of tyrosine aminotransferase from Anthoceros agrestis unveils the conceivable entry point into rosmarinic acid biosynthesis in hornworts.

MAIN CONCLUSION: Tyrosine aminotransferase (AaTAT) from the hornwort Anthoceros agrestis Paton (Anthocerotaceae) was amplified and expressed in E. coli. The active enzyme is able to accept a wide range of substrates with distinct preference for L-tyrosine, therefore, possibly catalysing the initial step in rosmarinic acid biosynthesis. The presence of rosmarinic acid (RA) in the hornwort A. agrestis is well known, and some attempts have been made to clarify the biosynthesis of this caffeic acid ester in lower plants. Parallel to the biosynthesis in vascular plants, the involvement of tyrosine aminotransferase (EC 2.6.1.5; TAT) as the initial step was assumed. The amplification of a nucleotide sequence putatively encoding AaTAT (Genbank MN922307) and expression in E. coli were successful. The enzyme proved to have a high acceptance of L-tyrosine (Km 0.53 mM) whilst slightly preferring 2-oxoglutarate over phenylpyruvate as co-substrate. Applying L-phenylalanine as a potential amino donor or using oxaloacetate or pyruvate as a replacement for 2-oxoglutarate as amino acceptor resulted in significantly lower catalytic efficiencies in each of these cases. To facilitate further substrate search, two methods were introduced, one using ninhydrin after thin-layer chromatography and the other using derivatisation with o-phthalaldehyde followed by HPLC or LC-MS analysis. Both methods proved to be well applicable and helped to confirm the acceptance of further aromatic and aliphatic amino acids. This work presents the first description of a heterologously expressed TAT from a hornwort (A. agrestis) and describes the possible entry into the biosynthesis of RA and other specialised compounds in a so far neglected representative of terrestrial plants and upcoming new model organism.

An Agrobacterium-mediated stable transformation technique for the hornwort model Anthoceros agrestis.

Despite their key phylogenetic position and their unique biology, hornworts have been widely overlooked. Until recently there was no hornwort model species amenable to systematic experimental investigation. Anthoceros agrestis has been proposed as the model species to study hornwort biology. We have developed an Agrobacterium-mediated method for the stable transformation of A. agrestis, a hornwort model species for which a genetic manipulation technique was not yet available. High transformation efficiency was achieved by using thallus tissue grown under low light conditions. We generated a total of 274 transgenic A. agrestis lines expressing the ß-glucuronidase (GUS), cyan, green, and yellow fluorescent proteins under control of the CaMV 35S promoter and several endogenous promoters. Nuclear and plasma membrane localization with multiple color fluorescent proteins was also confirmed. The transformation technique described here should pave the way for detailed molecular and genetic studies of hornwort biology, providing much needed insight into the molecular mechanisms underlying symbiosis, carbon-concentrating mechanism, RNA editing and land plant evolution in general.

The hornworts: morphology, evolution and development.

Extant land plants consist of two deeply divergent groups, tracheophytes and bryophytes, which shared a common ancestor some 500 million years ago. While information about vascular plants and the two of the three lineages of bryophytes, the mosses and liverworts, is steadily accumulating, the biology of hornworts remains poorly explored. Yet, as the sister group to liverworts and mosses, hornworts are critical in understanding the evolution of key land plant traits. Until recently, there was no hornwort model species amenable to systematic experimental investigation, which hampered detailed insight into the molecular biology and genetics of this unique group of land plants. The emerging hornwort model species, Anthoceros agrestis, is instrumental in our efforts to better understand not only hornwort biology but also fundamental questions of land plant evolution. To this end, here we provide an overview of hornwort biology and current research on the model plant A. agrestis to highlight its potential in answering key questions of land plant biology and evolution.

The diversity and community structure of symbiotic cyanobacteria in hornworts inferred from long-read amplicon sequencing.

PREMISE: Nitrogen-fixing endosymbioses with cyanobacteria have evolved independently in five very different plant lineages. Expanding knowledge of these symbioses promises to improve the understanding of symbiosis evolution and broaden the toolkit for agricultural engineering to reduce artificial fertilizer use. Here we focused on hornworts, a bryophyte lineage in which all members host cyanobacteria, and investigated factors shaping the diversity of their cyanobiont communities. METHODS: We sampled hornworts and adjacent soils in upstate New York throughout the hornwort growing season. We included all three sympatric hornwort species in the area, allowing us to directly compare partner selectivity. To profile cyanobacteria communities, we established a metabarcoding protocol targeting rbcL-X with PacBio long reads. RESULTS: The hornwort cyanobionts detected were phylogenetically diverse, including clades that do not contain other known plant symbionts. We found significant overlap between hornwort cyanobionts and soil cyanobacteria, a pattern not previously reported in other plant-cyanobacteria symbioses. Cyanobiont communities differed between host plants only centimeters apart, but we did not detect an effect of sampling time or host species on the cyanobacterial community structure. CONCLUSIONS: This study expands the phylogenetic diversity of known symbiotic cyanobacteria. Our analyses suggest that hornwort cyanobionts have a tight connection to the soil background, and we found no evidence that time within growing season, host species, or distance at the scale of meters strongly govern cyanobacteria community assembly. This study provides a critical foundation for further study of the ecology, evolution, and interaction dynamics of plant-cyanobacteria symbiosis.

Organellomic data sets confirm a cryptic consensus on (unrooted) land-plant relationships and provide new insights into bryophyte molecular evolution.

PREMISE: Phylogenetic trees of bryophytes provide important evolutionary context for land plants. However, published inferences of overall embryophyte relationships vary considerably. We performed phylogenomic analyses of bryophytes and relatives using both mitochondrial and plastid gene sets, and investigated bryophyte plastome evolution. METHODS: We employed diverse likelihood-based analyses to infer large-scale bryophyte phylogeny for mitochondrial and plastid data sets. We tested for changes in purifying selection in plastid genes of a mycoheterotrophic liverwort (Aneura mirabilis) and a putatively mycoheterotrophic moss (Buxbaumia), and compared 15 bryophyte plastomes for major structural rearrangements. RESULTS: Overall land-plant relationships conflict across analyses, generally weakly. However, an underlying (unrooted) four-taxon tree is consistent across most analyses and published studies. Despite gene coverage patchiness, relationships within mosses, liverworts, and hornworts are largely congruent with previous studies, with plastid results generally better supported. Exclusion of RNA edit sites restores cases of unexpected non-monophyly to monophyly for Takakia and two hornwort genera. Relaxed purifying selection affects multiple plastid genes in mycoheterotrophic Aneura but not Buxbaumia. Plastid genome structure is nearly invariant across bryophytes, but the tufA locus, presumed lost in embryophytes, is unexpectedly retained in several mosses. CONCLUSIONS: A common unrooted tree underlies embryophyte phylogeny, [(liverworts, mosses), (hornworts, vascular plants)]; rooting inconsistency across studies likely reflects substantial distance to algal outgroups. Analyses combining genomic and transcriptomic data may be misled locally for heavily RNA-edited taxa. The Buxbaumia plastome lacks hallmarks of relaxed selection found in mycoheterotrophic Aneura. Autotrophic bryophyte plastomes, including Buxbaumia, hardly vary in overall structure.

Phenolic metabolism in the hornwort Anthoceros agrestis: 4-coumarate CoA ligase and 4-hydroxybenzoate CoA ligase.

KEY MESSAGE: 4-Coumarate coenzyme A ligase and 4-hydroxybenzoate coenzyme A ligase from the hornwort Anthoceros agrestis expressed in E. coli were characterized on biochemical and molecular levels and showed interesting substrate specificities. Acyl-activating enzymes are associated with the biosynthesis or degradation of various metabolic products such as lipids, amino acids, sugars, and natural compounds. In this work, cDNA sequences encoding 4-coumarate coenzyme A ligase (4CL) and 4-hydroxybenzoate coenzyme A ligase (4HBCL) were amplified from the hornwort Anthoceros agrestis. The coding sequences were expressed in E. coli and purified by Ni-chelate chromatography. The CoA ligases exhibited different substrate specificities. 4CL catalyzed the activation of 4-coumaric acid, 3-coumaric acid, 2-coumaric acid, caffeic acid, isoferulic acid, ferulic acid, and cinnamic acid but lacked activities towards sinapic acid and benzoic acids. In contrast, 4HBCL preferred 4-hydroxybenzoic acid and benzoic acid, but also accepted other benzoic acid derivatives except salicylic acid and 3-aminosalicylic acid. Furthermore, 4HBCL also activated isoferulic acid, cinnamic acid, 2-coumaric acid, 3-coumaric acid, 4-coumaric acid and caffeic acid, but lacked affinity for ferulic acid and sinapic acid. These substrate specificities could be related to the phenolic compounds identified in Anthoceros agrestis.

The distribution and evolution of fungal symbioses in ancient lineages of land plants.

An accurate understanding of the diversity and distribution of fungal symbioses in land plants is essential for mycorrhizal research. Here we update the seminal work of Wang and Qiu (Mycorrhiza 16:299-363, 2006) with a long-overdue focus on early-diverging land plant lineages, which were considerably under-represented in their survey, by examining the published literature to compile data on the status of fungal symbioses in liverworts, hornworts and lycophytes. Our survey combines data from 84 publications, including recent, post-2006, reports of Mucoromycotina associations in these lineages, to produce a list of at least 591 species with known fungal symbiosis status, 180 of which were included in Wang and Qiu (Mycorrhiza 16:299-363, 2006). Using this up-to-date compilation, we estimate that fewer than 30% of liverwort species engage in symbiosis with fungi belonging to all three mycorrhizal phyla, Mucoromycota, Basidiomycota and Ascomycota, with the last being the most widespread (17%). Fungal symbioses in hornworts (78%) and lycophytes (up to 100%) appear to be more common but involve only members of the two Mucoromycota subphyla Mucoromycotina and Glomeromycotina, with Glomeromycotina prevailing in both plant groups. Our fungal symbiosis occurrence estimates are considerably more conservative than those published previously, but they too may represent overestimates due to currently unavoidable assumptions.

Life at the boundary: photosynthesis at the soil-fluid interface. A synthesis focusing on mosses.

Mosses are among the earliest branching embryophytes and probably originated not later than the early Ordovician when atmospheric CO2 was higher and O2 was lower than today. The C3 biochemistry and physiology of their photosynthesis suggests, by analogy with tracheophytes, that growth of extant bryophytes in high CO2 approximating Ordovician values would increase the growth rate. This occurs for many mosses, including Physcomitrella patens in suspension culture, although recently published transcriptomic data on this species at high CO2 and present-day CO2 show down-regulation of the transcription of several genes related to photosynthesis. It would be useful if transcriptomic (and proteomic) data comparing growth conditions are linked to measurements of growth and physiology on the same, or parallel, cultures. Mosses (like later-originating embryophytes) have been subject to changes in bulk atmospheric CO2 and O2 throughout their existence, with evidence, albeit limited, for positive selection of moss Rubisco. Extant mosses are subject to a large range of CO2 and O2 concentrations in their immediate environments, especially aquatic mosses, and mosses are particularly influenced by CO2 generated by, and O2 consumed by, soil chemoorganotrophy from organic C produced by tracheophytes (if present) and bryophytes.

Stomatal density and aperture in non-vascular land plants are non-responsive to above-ambient atmospheric CO2 concentrations.

BACKGROUND AND AIMS: Following the consensus view for unitary origin and conserved function of stomata across over 400 million years of land plant evolution, stomatal abundance has been widely used to reconstruct palaeo-atmospheric environments. However, the responsiveness of stomata in mosses and hornworts, the most basal stomate lineages of extant land plants, has received relatively little attention. This study aimed to redress this imbalance and provide the first direct evidence of bryophyte stomatal responsiveness to atmospheric CO2. METHODS: A selection of hornwort (Anthoceros punctatus, Phaeoceros laevis) and moss (Polytrichum juniperinum, Mnium hornum, Funaria hygrometrica) sporophytes with contrasting stomatal morphologies were grown under different atmospheric CO2 concentrations ([CO2]) representing both modern (440 p.p.m. CO2) and ancient (1500 p.p.m. CO2) atmospheres. Upon sporophyte maturation, stomata from each bryophyte species were imaged, measured and quantified. KEY RESULTS: Densities and dimensions were unaffected by changes in [CO2], other than a slight increase in stomatal density in Funaria and abnormalities in Polytrichum stomata under elevated [CO2]. CONCLUSIONS: The changes to stomata in Funaria and Polytrichum are attributed to differential growth of the sporophytes rather than stomata-specific responses. The absence of responses to changes in [CO2] in bryophytes is in line with findings previously reported in other early lineages of vascular plants. These findings strengthen the hypothesis of an incremental acquisition of stomatal regulatory processes through land plant evolution and urge considerable caution in using stomatal densities as proxies for paleo-atmospheric CO2 concentrations.

The capability to synthesize phytochelatins and the presence of constitutive and functional phytochelatin synthases are ancestral (plesiomorphic) characters for basal land plants.

Bryophytes, a paraphyletic group which includes liverworts, mosses, and hornworts, have been stated as land plants that under metal stress (particularly cadmium) do not synthesize metal-binding peptides such as phytochelatins. Moreover, very little information is available to date regarding phytochelatin synthesis in charophytes, postulated to be the direct ancestors of land plants, or in lycophytes, namely very basal tracheophytes. In this study, it was hypothesized that basal land plants and charophytes have the capability to produce phytochelatins and possess constitutive and functional phytochelatin synthases. To verify this hypothesis, twelve bryophyte species (six liverworts, four mosses, and two hornworts), three charophytes, and two lycophyte species were exposed to 0-36 µM cadmium for 72 h, and then assayed for: (i) glutathione and phytochelatin quali-quantitative content by HPLC and mass spectrometry; (ii) the presence of putative phytochelatin synthases by western blotting; and (iii) in vitro activity of phytochelatin synthases. Of all the species tested, ten produced phytochelatins in vivo, while the other seven did not. The presence of a constitutively expressed and functional phytochelatin synthase was demonstrated in all the bryophyte lineages and in the lycophyte Selaginella denticulata, but not in the charophytes. Hence, current knowledge according to phytochelatins have been stated as being absent in bryophytes was therefore confuted by this work. It is argued that the capability to synthesize phytochelatins, as well as the presence of active phytochelatin synthases, are ancestral (plesiomorphic) characters for basal land plants.

Beyond nutmeg, mace, and cloves: Checklist of the liverworts and hornworts of Maluku Islands (Moluccas), Indonesia.

The first ever liverwort and hornwort checklist is provided for the Maluku Islands (Moluccas/Spice Islands) of Indonesia. We report 355 accepted and 16 doubtful species and reject 22 species previously reported for Maluku Islands. The list is based on the specimens housed in the Herbarium Bogoriense (BO) and reports from over 500 literature references, including monographs, regional studies, and molecular investigations. The Maluku Islands are part of the Wallacea Biodiversity Hotspot with many unique species found only in Wallacea. Publications focusing on liverworts and hornworts of Maluku Islands are few and scattered. Considering regionally widespread species that have been recorded elsewhere, we predict that further fieldwork exploring the diversity of habitats coupled with collections unveiled from regional herbaria, a number of new records remain to be reported.