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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.

Key message Cinnamic acid 4-hydroxylase from the hornwort Anthoceros agrestis (AaC4H) was functionally expressed in the moss Physcomitrella patens and characterized at biochemical and molecular levels. Abstract Cinnamic acid 4-hydroxylase (C4H), a cytochrome P450-dependent hydroxylase, catalyzes the formation of 4-coumaric acid (=4-hydroxycinnamic acid) from trans-cinnamic acid. In the hornwort Anthoceros agrestis (Aa), this enzyme is supposed to be involved in the biosynthesis of rosmarinic acid (a caffeic acid ester of 3-(3,4-dihydroxyphenyl)lactic acid) and other related compounds. The coding sequence of AaC4H (CYP73A260) was expressed in the moss Physcomitrella patens (Pp_AaC4H). Protein extracts from the transformed moss showed considerably increased C4H activity driven by NADPH:cytochrome P450 reductase of the moss. Since Physcomitrella has own putative cinnamic acid 4-hydroxylases, enzyme characterization was carried out in parallel with the untransformed Physcomitrella wild type (Pp_WT). Apparent Km-values for cinnamic acid and NADPH were determined to be at 17.3 µM and 88.0 µM for Pp_AaC4H and 25.1 µM and 92.3 µM for Pp_WT, respectively. Expression levels of AaC4H as well as two Physcomitrella patens C4H isoforms were analyzed by quantitative real-time PCR. While PpC4H_1 displayed constantly low levels of expression during the whole 21-day culture period, AaC4H and PpC4H_2 increased their expression during the first 6–8 days of the culture period and then decreased again. This work describes the biochemical in vitro characterization of a cytochrome P450-dependent enzyme, namely C4H, heterologously expressed in the haploid model plant Physcomitrella patens.

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. Abstract 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.

The presented checklist and red list include 231 liverwort (Marchantiophyta) and two hornwort species (Anthocerotophyta). Overall, 22.9% of liverworts are evaluated as threatened (CR – 4.3%, EN – 7.8%, VU – 10.8%), while 17 of species (7.4%) are categorized as NT, 11.6% as DD and 3.0% as RE. A total of 128 species (55.4% of the liverwort flora of Slovakia) were assessed as LC. For the liverwort Lunularia cruciata the criteria were not applicable (NA), since this species is not native to Slovakia. Currently, only two hornworts are known in Slovakia, of which Anthoceros agrestis is assessed as LC and Phaeoceros carolinianus as NT. The main factors of the liverwort and hornwort endangerment and retreat are anthropogenic changes in natural conditions and degradation of the ecosystems caused by agriculture, forestry, aquaculture, climatic changes, industry, and traffic pollution.

We have developed a simple Agrobacterium-mediated method for the stable transformation of the hornwort Anthoceros agrestis, the fifth bryophyte species for which a genetic manipulation technique becomes available. High transformation efficiency was achieved by using thallus tissue grown under low-light conditions. We generated a total of 216 transgenic A. agrestis lines expressing the β-Glucuronidase (GUS), cyan, green, and yellow fluorescent proteins under the 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.

Summary 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 β‐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 hornwort Anthoceros agrestis is emerging as a model system for the study of symbiotic interactions and carbon fixation processes. It is an annual species with a remarkably small and compact genome. Single accessions of the plant have been shown to be related to the cosmopolitan perennial hornwort Anthoceros punctatus. We provide the first detailed insight into the evolutionary history of the two species. Due to the rather conserved nature of organellar loci, we sequenced multiple accessions in the Anthoceros agrestis–A. punctatus complex using three nuclear regions: the ribosomal spacer ITS2, and exon and intron regions from the single-copy coding genes rbcS and phytochrome. We used phylogenetic and dating analyses to uncover the relationships between these two taxa. Our analyses resolve a lineage of genetically near-uniform European A. agrestis accessions and two non-European A. agrestis lineages. In addition, the cosmopolitan species Anthoceros punctatus forms two lineages, one of mostly European accessions, and another from India. All studied European A. agrestis accessions have a single origin, radiated relatively recently (less than 1 million years ago), and are currently strictly associated with agroecosystem habitats.

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. Abstract 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.

Anthoceros agrestis Paton SPECIMEN EXAMINED. — Croatia. Nebojan Village, open ground in a hazelnut plantation, 45.4818°N, 16.1976°E, 190 m a.s.l., 20.IV.2013, leg. Rimac A., det. Rimac A. & Šegota V., ZA[ZA49732]; Ibidem, open ground on the lawn in a yard, 45.4959°N, 16.2038°E, 176 m a.s.l., 7.VIII.2018, leg. Rimac A., det. Rimac A. & Šegota V., ZA[ZA49733], Published as part of Rimac, Anja, Šegota, Vedran, Alegro, Antun, Koletić, Nikola & Vuković, Nina, 2019, Novelties in the hornwort flora of Croatia and Southeast Europe, pp. 289-295 in Cryptogamie, Bryologie 20 (22) on page 290, DOI: 10.5252/cryptogamie-bryologie2019v40a22, http://zenodo.org/record/7822134

Anthoceros agrestis (hornwort) transformation pre-print: https://biorxiv.org/cgi/content/short/2021.01.07.425778v1 This protocol works better for the Oxford strain

Hornworts are crucial to understand the phylogeny of early land plants. The emergence of ‘reverse’ U‐to‐C RNA editing accompanying the widespread C‐to‐U RNA editing in plant chloroplasts and mitochondria may be a molecular synapomorphy of a hornwort–tracheophyte clade. C‐to‐U RNA editing is well understood after identification of many editing factors in models like Arabidopsis thaliana and Physcomitrella patens, but there is no plant model yet to investigate U‐to‐C RNA editing. The hornwort Anthoceros agrestis is now emerging as such a model system. We report on the assembly and analyses of the A. agrestis chloroplast and mitochondrial genomes, their transcriptomes and editomes, and a large nuclear gene family encoding pentatricopeptide repeat (PPR) proteins likely acting as RNA editing factors. Both organelles in A. agrestis feature high amounts of RNA editing, with altogether > 1100 sites of C‐to‐U and 1300 sites of U‐to‐C editing. The nuclear genome reveals > 1400 genes for PPR proteins with variable carboxyterminal DYW domains. We observe significant variants of the ‘classic’ DYW domain, in the meantime confirmed as the cytidine deaminase for C‐to‐U editing, and discuss the first attractive candidates for reverse editing factors given their excellent matches to U‐to‐C editing targets according to the PPR‐RNA binding code.

Agricultural intensification represents one of the major drivers for the dramatic loss of biodiversity worldwide. To halt the decline of farmland biodiversity, Switzerland adopted agri-environment schemes (AES) in 1998. Here, we monitored the occurrence, abundance and habitats of two species of arable bryophyte specialists, the Field hornwort (Anthoceros agrestis) and the Carolina hornwort (Phaeoceros carolinianus), in 28 crop fields in the intensively cultivated Swiss Plateau from 1991–2018, to investigate the effects of arable management, AES directives, and weather conditions on their performance. The target species are characteristic of the specialized short-lived arable bryophyte flora of Central Europe that depend on bare substrate in low-intensively cultivated and regularly ploughed fields. Trends in their occurrence thus reflect in many respects the status of the arable bryophytes in cultivated fields. Hornwort occurrence significantly declined between 1991 and 2018. A strong decrease in stubble fields that remain unmanaged after harvest, the favourite habitat for many arable specialists in the study area, largely accounted for the decline. Stubble fields nearly disappeared in the study area because of a gradual reduction in the cultivation area of cereals and the increasing practice of immediate post-harvest tillage. The latter is common in intensive arable farming and was accentuated by AES directives amended in 2005. Hornwort occurrences were positively affected by high air humidity during summer, but weather effects were subordinate to management effects. We propose tailored amendments of AES regulations, that are implemented at selected sites, to maintain the characteristic arable specialist bryophytes in the Swiss Plateau: crop rotation with adequate proportions of cereals that are regularly ploughed but not before the end of October, no post-harvest processing of stubble fields, and optimization of the existing instrument ‘Biodiversity Promoting Areas’, e.g., short-term fallows in crop fields. Late-autumn or overwintering stubbles and short-term fallows will benefit many other organisms that depend on extensively managed open habitats, for example arable wildflowers, farmland breeding birds and specialized arthropods.

Hornworts are globally the least diverse bryophyte group with respect to species number, with only eight species known from Europe. As for Croatia, only two species are listed in the available literature, with most recent record being more than 85 years old. Our study carried out in Central Croatia identified three new species for the hornwort flora, including two new genera – Notothylas Sull. ex. A. Gray and Anthoceros L. Our finding of very rare Natura 2000 species Notothylas orbicularis (Schwein.) A. Gray, reported hitherto only from four Central European countries, is the first in Southeast Europe and the southernmost in Europe. Furthermore, we recorded Anthoceros agrestis Paton and Phaeoceros carolinianus (Michx.) Prosk., which are either very rare or, more likely, overlooked and understudied in Southeast Europe.

The gametophytic tissue of hornworts is rich in polysaccharides (Renzaglia et al., 2009) and it also seems to be rich in polyphenolics. Both compounds pose a problem for DNA. A modified CTAB protocol was found to be optimal for hornwort genomic DNA extraction (adapted from Porebski et al. 1997). This protocol uses polyvinyl pyrrolidone to remove polyphenolics and contains an extra ethanol precipitation step with a relatively high NaCl concentration compared standard DNA extraction protocols. At NaCl concentrations higher than 0.5 M,polysaccharides remain in solution and they do not co-precipitate with DNA.

� Background and Aims During evolution, plants have acquired and/or lost diverse sugar residues as cell-wall constituents. Of particular interest are primordial cell-wall features that existed, and in some cases abruptly changed, during the momentous step whereby land-plants arose from charophytic algal ancestors. � Methods Polysaccharides were extracted from four charophyte orders [Chlorokybales (Chlorokybus atmophyticus), Klebsormidiales (Klebsormidium fluitans, K. subtile), Charales (Chara vulgaris, Nitella flexilis), Coleochaetales (Coleochaete scutata)] and an early-diverging land-plant (Anthoceros agrestis). ‘Pectins’ and ‘hemicelluloses’, operationally defined as extractable in oxalate (100 � C) and 6 M NaOH (37 � C), respectively, were acid- or Driselase-hydrolysed, and the monosaccharides analysed chromatographically. One unusual monosaccharide, ‘U’, was characterized by 1 H/ 13 C-nuclear magnetic resonance spectroscopy and also enzymically. � Key Results ‘U’ was identified as 3-O-methyl-D-galactose (3-MeGal). All pectins, except in Klebsormidium ,c ontained acid- and Driselase-releasable galacturonate, suggesting homogalacturonan. All pectins, without exception, released rhamnose and galactose on acid hydrolysis; however, only in ‘higher’ charophytes (Charales, Coleochaetales) and Anthoceros were these sugars also efficiently released by Driselase, suggesting rhamnogalacturonan-I. Pectins of ‘higher’ charophytes, especially Chara, contained little arabinose, instead possessing 3-MeGal. Anthoceros hemicelluloses were rich in glucose, xylose, galactose and arabinose (suggesting xyloglucan and arabinoxylan), none of which was consistently present in charophyte hemicelluloses. � Conclusions Homogalacturonan is an ancient streptophyte feature, albeit secondarily lost in Klebsormidium. When conquering the land, the first embryophytes already possessed rhamnogalacturonan-I. In contrast, charophyte and land-plant hemicelluloses differ substantially, indicating major changes during terrestrialization. The presence of 3-MeGal in charophytes and lycophytes but not in the ‘intervening’ bryophytes confirms that cell-wall chemistry changed drastically between major phylogenetic grades.

A study of the ITS1 nucleotide sequences of 1000 moss species of 62 families, 11 liverwort species from five orders, and one hornwort Anthoceros agrestis identified five highly conserved motifs (CM1-CM5), which are presumably involved in pre-rRNA processing. Although the ITS1 sequences substantially differ in length and the extent of divergence, the conserved motifs are found in all of them. ITS1 secondary structures were constructed for 76 mosses, and main regularities at conserved motif positioning were observed. The positions of processing sites in the ITS1 secondary structure of the yeast Saccharomyces cerevisiae were found to be similar to the positions of the conserved motifs in the ITS1 secondary structures of mosses and liverworts. In addition, a potential hairpin formation in the putative secondary structure of a pre-rRNA fragment was considered for the region between ITS1 CM4-CM5 and a highly conserved region between hairpins 49 and 50 (H49 and H50) of the 18S rRNA.

Hornworts are the least species-rich bryophyte lineage, but represent a key group to understand the evolution of plants because, together with the remaining bryophyte lineages (mosses and liverworts), they constitute the earliest diverging land plants. The responses of hornworts to ultraviolet (UV) radiation are unknown, but they may be important to infer how primitive hornworts (and bryophytes in general) coped with UV upon land colonization. In this context, our aim was to show the first data on the effects of UV radiation on the accumulation patterns of phenolic UV-absorbing compounds (UVACs) in the emerging model hornwort Anthoceros agrestis. Thalli of 52 days age were exposed to photosynthetically active radiation (PAR) alone (P regime) and to a combination of PAR + UV-A + UV-B radiation (PAB regime) for 21 days, using realistic UV doses (equivalent to the natural ambient doses received in summer at mid-latitudes). At the end of the culture period, we measured the bulk levels and individual contents of phenolic UV-absorbing compounds (UVACs), differentiating in both cases the UVACs located in the methanol-soluble (mainly vacuolar) and -insoluble (cell wall-bound) fractions (SUVACs and IUVACs, respectively). Three soluble and one insoluble compounds were identified, among which the soluble rosmarinic and anthocerotonic acids are not present in any other bryophyte lineage. The bulk levels of SUVACs were higher than those of IUVACs, a physiological trait more typical of liverworts than of mosses. None of the variables measured responded significantly to UV exposure, but all of them showed an increasing trend under the PAB regime. Given that UV responsiveness of phenolic compounds depends on the UV levels used and the thallus age (with decreasing responsiveness as age increases), further research using higher UV levels and younger thalli should be conducted to more reliably establish the UV reactiveness of Anthoceros agrestis. © 2018 Adac. Tous droits réservés.

Microbial terpene synthase-like (MTPSL) genes are a type of terpene synthase genes only recently identified in plants. In contrast to typical plant terpene synthase genes, which are ubiquitous in land plants, MTPSL genes appear to occur only in nonseed plants. Our knowledge of catalytic functions of MTPSLs is very limited. Here we report biochemical characterization of the enzymes encoded by MTPSL genes from two closely related species of hornworts, Anthoceros punctatus and Anthoceros agrestis. Seven full-length MTPSL genes were identified in A. punctatus (ApMTPSL1-7) based on the analysis of its genome sequence. Using homology-based cloning, the apparent orthologs for six of the ApMTPSL genes, except ApMTPSL2, were cloned from A. agrestis. They were designated AaMTPSL1, 3–7. The coding sequences for each of the 13 Anthoceros MTPSL genes were cloned into a protein expression vector. Escherichia coli-expressed recombinant MTPSLs from hornworts were assayed for terpene synthase activities. Six ApMTPSLs and five AaMTPSLs, except for ApMTPSL5 and AaMTPSL5, showed catalytic activities with one or more isoprenyl diphosphate substrates. All functional MTPSLs exhibited sesquiterpene synthase activities. In contrast, only ApMTPSL7 and AaMTPSL7 showed monoterpene synthase activity and only ApMTPSL2, ApMTPSL6 and AaMTPSL6 showed diterpene synthase activity. Most MTPSLs from Anthoceros contain uncanonical aspartate-rich motif in the form of either ‘DDxxxD’ or ‘DDxxx’. Homology-based structural modeling analysis of ApMTPSL1 and ApMTPSL7, which contain ‘DDxxxD’ and ‘DDxxx’ motif, respectively, showed that ‘DDxxxD’ and ‘DDxxx’ motifs are localized in the similar positions as the canonical ‘DDxxD’ motif in known terpene synthases. To further understand the role of individual aspartate residues in the motifs, ApMTPSL1 and ApMTPSL7 were selected as two representatives for site-directed mutagenesis studies. No activities were detected when any of the conserved aspartic acid was mutated into alanine. This study provides new information about the catalytic functions of MTPSLs and the functionality of their uncanonical aspartate-rich motifs, and builds a knowledge base for studying the biological importance of MTPSL genes and their terpene products in nonseed plants.

Anthoceros punctatus L. and Jungermannia borealis Damsh. et Vaňa have been erroneously reported from Romania and are hereby rejected from the bryophyte checklist of Romania, until new reports. The presence in Romania of Anthoceros agrestis Paton, Cephaloziella massalongi (Spruce) Mull. Frib., Cephaloziella spinigera (Lindb.) Warnst., Marsupella apiculata Schiffn., Scapania cuspiduligera (Nees) Mull. Frib and Scapania praetervisa Meyl. is confirmed, and is commented for Nardia breidleri (Limpr.) Lindb.

Traditionally managed arable fields host a specialised flora adapted to regular disturbance through tillage. Agricultural intensification during the 20th century resulted in a pronounced biodiversity decline in European agroecosystems. Anthoceros agrestis and Phaeoceros carolinianus, both largely confined to cultivated land in Central Europe and the only representatives of hornworts in northern Switzerland, are examples of species that decreased in Central Europe during the last century. A repeated survey of 28 arable fields in the Swiss Plateau from 1989 to 1995 demonstrated that crop type and associated farming routines were critical in determining hornwort occurrences. During the 10 years following the completion of this survey, agri-environment schemes were introduced aiming at a more environmental-friendly agricultural production. We re-investigated the selected sites in 2005–2007 to examine whether these programmes favoured hornworts. We found a significant decrease of untilled autumn stubble-fields, which accounted for a decline of hornworts. High relative summer air humidity positively affected hornwort occurrence. Gametophytic populations of both taxa regenerated from the persistent diaspore bank after years of unfavourable conditions. To ensure the long-term persistence of hornworts in the Swiss Plateau, we recommend three alternative modifications of current Swiss agri-environment schemes to be implemented in cereal fields with known hornwort occurrences: (1) Tailored conservation headlands, (2) Autumn stubble-fields and (3) Field margin strips sown with arable flora but without crop plants. The proposed practices will promote other typically ephemeral arable bryophytes and seed plants and are likely to be advantageous also for other organisms, such as farmland birds.

The monophyletic group of hornworts is believed to represent the immediate sister group of all vascular land plants. However, this traditional view is still debated and cannot be satisfactorily resolved owing to the lack of detailed knowledge on the general biology and genomic features of hornworts. Until now, advancement in this field was primarily hindered by the lack of genomic resources for a hornwort model species. Here we provide the first insight into the major features of the draft genome sequence of the model hornwort, Anthoceros agrestis. We show that A. agrestis has a remarkably small genome, with few recent paralogues, which makes it appropriate for genetic analysis. Finally, we also report on the genomic features of the chloroplast, mitochondrion and nuclear genomes and compare those with algal and vascular land plant genomes. At the very end of the chapter we summarize our achievements and provide a list of issues that need to be resolved in the future.

Background Plants colonized terrestrial environments approximately 480 million years ago and have contributed significantly to the diversification of life on Earth. Phylogenetic analyses position a subset of charophyte algae as the sister group to land plants, and distinguish two land plant groups that diverged around 450 million years ago – the bryophytes and the vascular plants. Relationships between liverworts, mosses hornworts and vascular plants have proven difficult to resolve, and as such it is not clear which bryophyte lineage is the sister group to all other land plants and which is the sister to vascular plants. The lack of comparative molecular studies in representatives of all three lineages exacerbates this uncertainty. Such comparisons can be made between mosses and liverworts because representative model organisms are well established in these two bryophyte lineages. To date, however, a model hornwort species has not been available. Results Here we report the establishment of Anthoceros agrestis as a model hornwort species for laboratory experiments. Axenic culture conditions for maintenance and vegetative propagation have been determined, and treatments for the induction of sexual reproduction and sporophyte development have been established. In addition, protocols have been developed for the extraction of DNA and RNA that is of a quality suitable for molecular analyses. Analysis of haploid-derived genome sequence data of two A. agrestis isolates revealed single nucleotide polymorphisms at multiple loci, and thus these two strains are suitable starting material for classical genetic and mapping experiments. Conclusions Methods and resources have been developed to enable A. agrestis to be used as a model species for developmental, molecular, genomic, and genetic studies. This advance provides an unprecedented opportunity to investigate the biology of hornworts.

A new alkaloid, anthocerodiazonin, was isolated from in vitro cultures of the hornwort, Anthoceros agrestis . Furthermore, six glutamic acid amides, N -(4-hydroxybenzoyl)-glutamic acid, N -(3,4-dihydroxybenzoyl)-glutamic acid, N -(4-hydroxy-3-methoxybenzoyl)-glutamic acid, ( E )- N -( iso feruloyl)-glutamic acid, ( Z )- N -(isoferuloyl)-glutamic acid and ( Z )- N -( p -coumaroyl)-glutamic acid were obtained as natural products. Structures were elucidated from spectroscopic data.

Cell suspension cultures of the hornwort Anthoceros agrestis Paton (Anthocerotaceae) were cultivated and characterized in CB-media containing 2 and 4% sucrose. The suspension cells accumulated rosmarinic acid up to 5.1% of the cell dry weight as well as caffeoyl-4'-hydroxyphenyllactate. Moreover, a more hydrophilic compound was detected which was isolated and identified as rosmarinic acid 3'-O-beta-D-glucoside, a new rosmarinic acid derivative. This new rosmarinic acid derivative was found up to 1.0% of the cell dry weight in suspension cells of A. agrestis.

In phylogenetic trees generated from partial trnL(UAA) intron sequences, the hornworts (represented by nine species from the genera Anthoceros, Dendroceros, Megaceros, Notothylas and Phaeoceros) are resolved as a monophyletic group and are separated from the clades of mosses, liverworts and tracheophytes. A secondary structure of the trnL(UAA) intron of Anthoceros agrestis is presented, displaying the arrangement of the stem-loop regions P1-P9. Compensatory base-pair changes (coevolutionary sites) are detected in regions P4/5 and P9 within the hornwort sequences. The original homology of the most variable region, P8, cannot be detected anymore due to the extremely fast divergent evolution of this segment in the major land plant groups. Similarly, a high sequence divergence occurs in the trnL-trnF intergenic spacer. Apart from synapomorphic substitutions in the trnL(UAA) intron, the hornworts are characterised by a large P6 region consisting of many repetitive elements. The molecular data therefore support the hornworts as representing an independent land plant lineage (Anthocerotophyta). Although relationships between hornworts and the other land plant groups remain unresolved in the trnL(UAA) intron trees, it is rather unlikely that bryophytes are monophyletic in their traditional circumscription, i.e. comprising hornworts, mosses and liverworts.

Cinnamic acid 4-hydroxylase (EC 1.14.13.11), a cytochrome P450-dependent hydroxylase was for the first time characterized from a hornwort, Anthoceros agrestis Paton (Anthocerotaceae). In suspension cultures of A. agrestis up to 5% of the dry weight was accumulated as rosmarinic acid, a natural product commonly known from higher plants (e.g. species of the Lamiaceae and Boraginaceae). Cinnamic acid 4-hydroxylase is involved in the biosynthesis of rosmarinic acid. The participation of cytochrome P450 was demonstrated by the inhibition of hydroxylase activity by cytochrome c and the inhibition of cinnamic acid hydroxylation in a CO-containing atmosphere, which is partially released by illumination with blue light. The apparent K m values were determined to be at 60 μM and 5 μM for NADPH and cinnamic acid, respectively. A comparatively high hydroxylation activity was seen with NADH as electron donor. While the hydroxylase activity with NADPH was strongly inhibited by the competitive electron acceptor cytochrome c, the activity with NADH was less susceptible, indicating the possibility of different electron-transfer pathways.

When Linnaeus (Sp. PI.: 1139. 1753) named the genus Anthoceros he included three species, namely, A. punctatus, A. laevis, and A. multifidus. Anthoceros multifidus was later removed from the genus and transferred to Riccardia by Gray (Nat. Arr. Brit. PI.: 684. 1821 'Riccardius') leaving two of the original species in the genus Anthoceros. Anthoceros punctatus was selected as the generitype by Proskauer (Ann. Bot., n.s. 12: 259. 1948). His selection remained uncontested until Schuster (J. Hattori Bot. Lab. 26: 299. 1963) suggested that Proskauer should have selected A. laevis as the type rather than A. punctatus. That is because Proskauer later decided (Bull. Torrey Bot. Club 78: 346, 1951) that these two original species of Anthoceros actually represented two different genera. Since he had selected A. punctatus as the type, it had to remain with Anthoceros, so he transferred A. laevis to his new genus Phaeoceros (Proskauer I.c., 1951). Anthoceros punctatus had long been considered to be conspecific with Aspiromitus husnotii Steph., the generitype of Aspiromitus Steph. (Proskauer, I.c., 1948: 262), and Schuster (I.c., 1963) stated that "Aspiromitus, typified by A. husnotii (= A. punctatus) must be used for one of the two elements originally included in Anthoceros by Linnaeus, and Anthoceros must be retained for the other ('law of residues')". Following his logic, the two species of Linnaeus would then be Anthoceros laevis and Aspiromitus punctatus; he did not, however, make any new combinations in 1963. Schljakov (Novosti Sist. Niz. Rast. 13: 225. 1976) agreed with Schuster (I.c., 1963) and implicitly lectotypified Anthoceros with Anthoceros laevis, making the new combination Aspiromitus punctatus (L.) Schljak. Grolle (Acta Bot. Fenn. 121: 5. 1983) pointed out that such a later lectotypification was contrary to the provisions of the ICBN, particularly with the removal of any appeal to the "law of residues" in the edition adopted at the Sydney Congress of 1981 (Voss & al., Regnum Veg. 111. 1983) and had to be rejected. That would have been the case except for the provision of the ICBN, first adopted at the Seattle Congress in 1969 (cf. Art. 8 (last clause) in Stafleu & al., Regnum Veg. 82. 1972), and now expressed as Article 10.5(b) of the ICBN (Greuter & al., Regnum Veg. 138. 2000) that a choice of lectotype for the name of a genus or subdivision of a genus may be superseded if ". . . it was based on a largely mechanical method of selection". The selection by Proskauer (l.c., 1948: 262) was obviously mechanical since he wrote: "Anthoceros punctatus was described by Linne (1753) as the first species of Anthoceros. It is here, therefore, considered as the type of the genus". Although the actions of Schuster (l.c., 1963) and Schljakov (l.c.) do not represent formal designation of a type as required by Art. 7.11 of the ICBN, more recently Schuster (Hepaticae and Anthocerotae of North America 6: 745. 1992), following his description of Anthoceros, wrote: "Type. Anthoceros laevis L. The reasons for the present type designation are discussed at length, below". Although this later designation was based solely upon the "law of residues" with no mention whatsoever of the mechanical choice by Proskauer (l.c. 1948: 262), Art. 10 Ex. 7, a "voted example", makes clear that any later type designation that meets the requirements of Art. 7.11 supersedes a choice "based on a largely mechanical method of selections". Therefore, unless the Proskauer designation of A. punctatus is conserved as the type of the name Anthoceros, it must be considered to have been superseded by Schuster's (I.c. 1992: 745) choice of A. laevis. With A. laevis as the generitype, the species of Phaeoceros must either be returned to or transferred to Anthoceros and all species now considered to be members of Anthoceros must be transferred to Aspiromitus. Schuster (l.c., 1992) and Schljakov (l.c.) have already begun this lengthy task to a very limited extent. Not only will the number of new combinations be high, including well over 100 of the 293 binomials listed by Bonner (in Index Hepat. 2. 1962) under Anthoceros, the confusion this will cause will be unprecedented. This will likewise result in changing the generic name for 131 sequences

Anthoceros punctatus var. cavernosus Prosk. homon. illeg. is renamed A. agrestis Paton, redescribed, illustrated and compared with A. punctatus L. (A. husnotii Steph.). The thalli and sporophytes are usually smaller in A. agrestis than in A. punctatus and the antheridial bodies are significantly smaller. World distributions and brief descriptions of British habitats are included for both species showing that A. agrestis is more restricted than A. punctatus in its distribution and ecology.

While angiosperm clocks can be described as an intricate network of interlocked transcriptional feedback loops, clocks of green algae have been modelled as a loop of only two genes. To investigate the transition from a simple clock in algae to a complex one in angiosperms, we performed an inventory of circadian clock genes in bryophytes and charophytes. Additionally, we performed functional characterization of putative core clock genes in the liverwort Marchantia polymorpha and the hornwort Anthoceros agrestis. Phylogenetic construction was combined with studies of spatiotemporal expression patterns and analysis of M. polymorpha clock gene mutants. Homologues to core clock genes identified in Arabidopsis were found not only in bryophytes but also in charophytes, albeit in fewer copies. Circadian rhythms were detected for most identified genes in M. polymorpha and A. agrestis, and mutant analysis supports a role for putative clock genes in M. polymorpha. Our data are in line with a recent hypothesis that adaptation to terrestrial life occurred earlier than previously expected in the evolutionary history of charophyte algae. Both gene duplication and acquisition of new genes was important in the evolution of the plant circadian clock, but gene loss has also contributed to shaping the clock of bryophytes.