An ancient tropical origin, dispersals via land bridges and Miocene diversification explain the subcosmopolitan disjunctions of the liverwort genus Lejeunea.

Understanding the biogeographical and diversification processes explaining current diversity patterns of subcosmopolitan-distributed groups is challenging. We aimed at disentangling the historical biogeography of the subcosmopolitan liverwort genus Lejeunea with estimation of ancestral areas of origin and testing if sexual system and palaeotemperature variations can be factors of diversification. We assembled a dense taxon sampling for 120 species sampled throughout the geographical distribution of the genus. Lejeunea diverged from its sister group after the Paleocene-Eocene boundary (52.2 Ma, 95% credibility intervals 50.1-54.2 Ma), and the initial diversification of the crown group occurred in the early to middle Eocene (44.5 Ma, 95% credibility intervals 38.5-50.8 Ma). The DEC model indicated that (1) Lejeunea likely originated in an area composed of the Neotropics and the Nearctic, (2) dispersals through terrestrial land bridges in the late Oligocene and Miocene allowed Lejeunea to colonize the Old World, (3) the Boreotropical forest covering the northern regions until the late Eocene did not facilitate Lejeunea dispersals, and (4) a single long-distance dispersal event was inferred between the Neotropics and Africa. Biogeographical and diversification analyses show the Miocene was an important period when Lejeunea diversified globally. We found slight support for higher diversification rates of species with both male and female reproductive organs on the same individual (monoicy), and a moderate positive influence of palaeotemperatures on diversification. Our study shows that an ancient origin associated with a dispersal history facilitated by terrestrial land bridges and not long-distance dispersals are likely to explain the subcosmopolitan distribution of Lejeunea. By enhancing the diversification rates, monoicy likely favoured the colonisations of new areas, especially in the Miocene that was a key epoch shaping the worldwide distribution.

Transformation of Riccia fluitans, an Amphibious Liverwort Dynamically Responding to Environmental Changes.

The colonization of land by streptophyte algae, ancestors of embryophyte plants, was a fundamental event in the history of life on earth. Bryophytes are early diversifying land plants that mark the transition from freshwater to terrestrial ecosystems. The amphibious liverwort Riccia fluitans can thrive in aquatic and terrestrial environments and thus represents an ideal organism to investigate this major transition. Therefore, we aimed to establish a transformation protocol for R. fluitans to make it amenable for genetic analyses. An Agrobacterium transformation procedure using R. fluitans callus tissue allows to generate stably transformed plants within 10 weeks. Furthermore, for comprehensive studies spanning all life stages, we demonstrate that the switch from vegetative to reproductive development can be induced by both flooding and poor nutrient availability. Interestingly, a single R. fluitans plant can consecutively adapt to different growth environments and forms distinctive and reversible features of the thallus, photosynthetically active tissue that is thus functionally similar to leaves of vascular plants. The morphological plasticity affecting vegetative growth, air pore formation, and rhizoid development realized by one genotype in response to two different environments makes R. fluitans ideal to study the adaptive molecular mechanisms enabling the colonialization of land by aquatic plants.

Full sunlight acclimation mechanisms in Riccia discolor thalli: Assessment at morphological, anatomical, and biochemical levels.

Light occupies a central position in regulating development of plants. Either little or excess of light could be harmful for plants. Since bryophytes are shade loving organisms, they must adapt to function in fluctuating light regimes. Therefore, the aim of this study was to investigate acclimatory responses of Riccia discolor thalli grown under full sunlight, and were compared with shade grown thalli (control). Length, width, and fresh mass of thallus were significantly lower (by 27, 41 and 37%, respectively) but endogenous nitric oxide content (by 81%) and nitric oxide synthase like activity (by 58%) were higher in full sunlight grown thalli than shade grown thalli. Number of rhizoids was greater in shade but length and width of rhizoids were higher (by 36 and 25%, respectively) in full sunlight grown thalli. The content of carotenoids was higher (by 34%) in full sunlight grown thalli. In full sunlight grown thalli, chloroplasts exhibited avoidance movement but in shade grown thalli they exhibited accumulation movement. Photosynthetic yields were higher in shade grown thalli. Among energy fluxes, ABS/RC did not vary but DI0/RC was higher (by 12%) in full sunlight grown thalli. Reactive oxygen species and damage were greater in full sunlight grown thalli despite enhanced levels of antioxidants i.e. superoxide dismutase (by 66%) and catalase (by 34%). Overall results suggest that full sunlight acclimation in Riccia discolor thalli occurred at various levels in which endogenous NO plays a positive role.

The identification of differentially expressed genes in male and female gametophytes of simple thalloid liverwort Pellia endiviifolia sp. B using an RNA-seq approach.

MAIN CONCLUSION: This study shows differences in gene expression between male and female gametophytes of the simple thalloid liverwort with a distinction between the vegetative and reproductive phases of growth. Pellia endiviifolia is a simple thalloid liverwort that, together with hornworts and mosses, represents the oldest living land plants. The limited taxon sampling for genomic and functional studies hampers our understanding of processes governing evolution of these plants. RNA sequencing represents an attractive way to elucidate the molecular mechanisms of non-model species development. In the present study, RNA-seq was used to profile the differences in gene expression between P. endiviifolia male and female gametophytes, with a distinction between the vegetative and reproductive phases of growth. By comparison of the gene expression profiles from individuals producing sex organs with the remaining thalli types, we have determined a set of genes whose expression might be important for the development of P. endiviifolia reproductive organs. The selected differentially expressed genes (DEGs) were categorized into five main pathways: metabolism, genetic information processing, environmental information processing, cellular processes, and organismal systems. A comparison of the obtained data with the Marchantia polymorpha transcriptome resulted in the identification of genes exhibiting a similar expression pattern during the reproductive phase of growth between members of the two distinct liverwort classes. The common expression profile of  87 selected genes suggests a common mechanism governing sex organ development in both liverwort species. The obtained RNA-seq results were confirmed by RT-qPCR for the DEGs with the highest differences in expression level. Five Pellia-female-specific and two Pellia-male-specific DEGs showed enriched expression in archegonia and antheridia, respectively. The identified genes are promising candidates for functional studies of their involvement in liverwort sexual reproduction.

The Increase of Simple Sequence Repeats during Diversification of Marchantiidae, An Early Land Plant Lineage, Leads to the First Known Expansion of Inverted Repeats in the Evolutionarily-Stable Structure of Liverwort Plastomes.

The chloroplast genomes of liverworts, an early land plant lineage, exhibit stable structure and gene content, however the known resources are very limited. The newly sequenced plastomes of Conocephalum, Riccia and Sphaerocarpos species revealed an increase of simple sequence repeats during the diversification of complex thalloid liverwort lineage. The presence of long TA motifs forced applying the long-read nanopore sequencing method for proper and dependable plastome assembly, since the length of dinucleotide repeats overcome the length of Illumina short reads. The accumulation of SSRs (simple sequence repeats) enabled the expansion of inverted repeats by the incorporation of rps12 and rps7 genes, which were part of large single copy (LSC) regions in the previously sequenced plastomes. The expansion of inverted repeat (IR) at the genus level is reported for the first time for non-flowering plants. Moreover, comparative analyses with remaining liverwort lineages revealed that the presence of SSR in plastomes is specific for simple thalloid species. Phylogenomic analysis resulted in trees confirming monophyly of Marchantiidae and partially congruent with previous studies, due to dataset-dependent results of Dumortiera-Reboulia relationships. Despite the lower evolutionary rate of Marchantiales plastomes, significant barcoding gap was detected, even for recently divergent holarctic Conocephalum species. The sliding window analyses revealed the presence of 18 optimal (500 bp long) barcodes that enable the molecular identification of all studied species.

Phytofiltration of chosen metals by aquarium liverwort (Monosoleum tenerum).

Aquatic bryophytes are widely used as indicators of water pollution with various substances, including metals. We present a first study concerning the feasibility of Monosoleum tenerum to remove potentially toxic metals Zn, Cu, Ni, Mn and Fe from water. The novel approach to study the bioaccumulation under controlled conditions and in vitro grown aquarium liverworts was applied. The plants were exposed to 1, 10 and 100 ppm multi-metal solution for 7 days and subsequently the metal content was determined in plant material and media to calculate the bioaccumulation factor (BAF). The study revealed various accumulation behavior (metal and dose dependent), which followed the order of Cu > Zn > Mn > Ni > Fe for 1 ppm; Zn > Cu > Mn > Fe > Ni for 10 ppm and Cu > Fe > Zn > Ni > Mn for 100 ppm solution. However, with increasing Cu, Mn and Ni concentration in the solution, BAF decreased. For Zn the highest BAF value (136) was obtained for the 10 ppm solution. Fe bioaccumulation increased with the increasing solution concentration. After 7 week exposure, the results indicated highest accumulation of Cu (3,25 mg) followed by Fe (1,8 mg) in the plant tissue for the 100 ppm solution. Overall, the bryophyte M. tenerum demonstrate high potential for the removal of toxic metals from the multi-metal solution via accumulation in plant tissue. The BAF values were proper to indicate the use of M. tenerum for phytofiltration of waste waters affected by Zn, Cu, Ni, Mn and Fe.

Comparison of pharmacological properties and phytochemical constituents of in vitro propagated and naturally occurring liverwort Lunularia cruciata.

BACKGROUND: Study of phytochemicals and pharmacological properties of bryophytes has been neglected for a long time because of the three main reasons i.e. (i) difficulty in collection in large amount for analysis; (ii) their availablility only in particular season and (iii) their restricted geographic distribution. So, the aim of this work was to propagate Lunularia cruciata under in vitro condition for comparing its pharmacological properties and phytocmecial constituents with naturally grown counterparts. METHOD: Axenic culture of L. cruciata was established by propagating gemmae under in vitro condition. Appropriate culture conditions, media, and the effect of hormones on growth and development were studied. The phytochemical composition was determined by GC-MS analysis and pharmacological activity was evaluated by assessing the antioxidant and anti-diabetic activities. For the antioxidant activity ABTS+ [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)], DPPH• (2,2-diphenyl-1-picrylhydrazyl) and metal chelating assays were done and for evaluation of the in vitro anti-diabetic activity α-glucosidase and α-amylase inhibitory activities were done. RESULT: Growth of L. cruciata was recorded in half strength MS media. Benzylaminopurine (BAP: 2 mg/L) and 1-Naphtheleneacetic acid (NAA: 0.5 mg/L) were the successful hormonal combination. GC-MS analysis revealed the existence of nine key compounds in both in vitro and naturally grown L. cruciata. Result of antioxidant and anti-diabetic activity showed that in vitro grown L. cruciata has a more or less similar antioxidant and anti-diabetic activities as naturally grown ones. This result confirms the possibility of using in vitro grown plants in place of naturally grown plants for research and clinical purposes.

A cis-acting bidirectional transcription switch controls sexual dimorphism in the liverwort.

Plant life cycles alternate between haploid gametophytes and diploid sporophytes. While regulatory factors determining male and female sexual morphologies have been identified for sporophytic reproductive organs, such as stamens and pistils of angiosperms, those regulating sex-specific traits in the haploid gametophytes that produce male and female gametes and hence are central to plant sexual reproduction are poorly understood. Here, we identified a MYB-type transcription factor, MpFGMYB, as a key regulator of female sexual differentiation in the haploid-dominant dioicous liverwort, Marchantia polymorpha MpFGMYB is specifically expressed in females and its loss resulted in female-to-male sex conversion. Strikingly, MpFGMYB expression is suppressed in males by a cis-acting antisense gene SUF at the same locus, and loss-of-function suf mutations resulted in male-to-female sex conversion. Thus, the bidirectional transcription module at the MpFGMYB/SUF locus acts as a toggle between female and male sexual differentiation in M. polymorpha gametophytes. Arabidopsis thaliana MpFGMYB orthologs are known to be expressed in embryo sacs and promote their development. Thus, phylogenetically related MYB transcription factors regulate female gametophyte development across land plants.

Archetypal Roles of an Abscisic Acid Receptor in Drought and Sugar Responses in Liverworts.

Abscisic acid (ABA) controls seed dormancy and stomatal closure through binding to the intracellular receptor Pyrabactin resistance1 (Pyr1)/Pyr1-like/regulatory components of ABA receptors (PYR/PYL/RCAR) in angiosperms. Genes encoding PYR/PYL/RCAR are thought to have arisen in the ancestor of embryophytes, but the roles of the genes in nonvascular plants have not been determined. In the liverwort Marchantia polymorpha, ABA reduces growth and enhances desiccation tolerance through increasing accumulation of intracellular sugars and various transcripts such as those of Late Embryogenesis Abundant (LEA)-like genes. In this study, we analyzed a gene designated MpPYL1, which is closely related to PYR/PYL/RCAR of angiosperms, in transgenic liverworts. Transgenic lines overexpressing MpPYL1-GFP showed ABA-hypersensitive growth with enhanced desiccation tolerance, whereas Mppyl1 generated by CRISPR-Cas9-mediated genome editing showed ABA-insensitive growth with reduced desiccation tolerance. Transcriptome analysis indicated that MpPYL1 is a major regulator of abiotic stress-associated genes, including all 35 ABA-induced LEA-like genes. Furthermore, these transgenic plants showed altered responses to extracellular Suc, suggesting that ABA and PYR/PYL/RCAR function in sugar responses. The results presented here reveal an important role of PYR/PYL/RCAR in the ABA response, which was likely acquired in the common ancestor of land plants. The results also indicate the archetypal role of ABA and its receptor in sugar response and accumulation processes for vegetative desiccation tolerance in bryophytes.

Composición y distribución de hepáticas (Marchantiophyta) en un intervalo altitudinal en la Cordillera Oriental de Colombia / Composition and distribution of liverworts (Marchantiophyta) in a continuous altitudinal range on the Cordillera Oriental of Colombia

Resumen La composición de especies de hepáticas sobre los Andes del Norte varía con la altitud en relación con diferentes factores bióticos (vegetación) y abióticos (temperatura, humedad y luminosidad). Para probar esta afirmación se determinó la diversidad y distribución de las Marchantiophytas en un rango altitudinal entre los 2 400 y 3 400 m sobre la Cordillera Oriental colombiana, estableciendo cómo éstas varían entre altitudes y zonas de vida con respecto a los factores abióticos. Se realizaron muestreos cada 200 m y se registraron datos ambientales durante un año. Se encontraron 162 especies, la composición de hepáticas varió a lo largo del gradiente altitudinal con un gran número de especies únicas en cada altitud. Se encontró la máxima diversidad en los 3 000 m, con una dominancia del hábito folioso y de especies epifitas, mientras que la mayor riqueza de familias y géneros se presentó entre los 3 200 y 3 400 m. Anoplolejeunea conferta presentó la mayor cobertura en la zona así como el mayor valor del índice de valor de importancia (IVI) y distribución altitudinal más amplia, ubicándose desde los 2 400 hasta 3 000 m en cuatro diferentes sustratos. Las hepáticas se distribuyeron altitudinalmente de manera diferencial, con una diversidad beta alta (0.864) debido al recambio altitudinal de especies, con más del 30 % de disimilaría en la composición de especies cada 200 m altitudinales, dependiendo principalmente de cambios en la luminosidad. Consecuentemente, la temperatura, humedad y luminosidad y su relación con la vegetación son determinantes en la diversidad y distribución de las hepáticas en la Cordillera Oriental de los Andes colombianos.

Abstract The species composition of liverworts varies with altitude in the Northern part of the Andes due to the relationship of biotic (vegetation) and abiotic factors (temperature, humidity, and sunlight brightness). In order to test this affirmation we determined the diversity, species composition, abundance, and distribution of species of Marchantiophyta in an altitudinal range from 2 400 to 3 400 m on the Colombian Cordillera Oriental, and established how these vary with altitude and life-zones, regarding abiotic factors. Samples, taken every 200 m in the altitudinal range, and environmental data were registered during a year. We found 162 species, the composition of liverworts varied throughout the altitudinal gradient with a high number of unique species in each altitude. The maximum diversity was found at 3 000 m, along with a dominance of leafy habit and epiphytic species, while the higher richness of families and genera was found between 3 200 and 3 400 m. Anoplolejeunea conferta had the largest value of coverage in the zone, the highest value of importance value index (IVI), and the widest altitudinal distribution, from 2 400 to 3 000 m in four different substrates. The liverworts were distributed differently with altitude, we found a high beta diversity (0.864) due to the replacement of species, with more that 30 % of dissimilarity in species composition every 200 altitudinal meters, mainly depending on sunlight brightness variation. Accordingly, we found that abiotic factors like temperature, humidity, and sunlight brightness and its relationship vegetation are determinant in the diversity and altitudinal distribution of liverworts in the study area. Rev. Biol. Trop. 66(2): 559-570. Epub 2018 June 01.

Boron and the evolutionary development of roots.

Experimental work has shown that Boron (i.e., Boric acid, B) is an essential and multifunctional microelement for vascular plant development. In addition to its other functions, which include xylem development and lignin biosynthesis, we now know that B is involved in phytohormone-signaling and influences the mechanical properties of intercellular pectins. From these data, we conclude that B played an important role during the evolutionary development of lignified tissues, and that it may have been involved in the evolution of vascular plant roots, as hypothesized by D. H. Lewis in 1980. Herein, we review the data pertaining to Lewis' hypothesis, present experimental results on the role of B in root (vs. rhizoid) formation in sunflower vs. a liverwort, and describe the appearance of roots in the fossil record. Open questions are addressed, notably the lack of our knowledge concerning soil microbes and their interactive roles with the micronutrient B during root formation.

Algal ancestor of land plants was preadapted for symbiosis.

Colonization of land by plants was a major transition on Earth, but the developmental and genetic innovations required for this transition remain unknown. Physiological studies and the fossil record strongly suggest that the ability of the first land plants to form symbiotic associations with beneficial fungi was one of these critical innovations. In angiosperms, genes required for the perception and transduction of diffusible fungal signals for root colonization and for nutrient exchange have been characterized. However, the origin of these genes and their potential correlation with land colonization remain elusive. A comprehensive phylogenetic analysis of 259 transcriptomes and 10 green algal and basal land plant genomes, coupled with the characterization of the evolutionary path leading to the appearance of a key regulator, a calcium- and calmodulin-dependent protein kinase, showed that the symbiotic signaling pathway predated the first land plants. In contrast, downstream genes required for root colonization and their specific expression pattern probably appeared subsequent to the colonization of land. We conclude that the most recent common ancestor of extant land plants and green algae was preadapted for symbiotic associations. Subsequent improvement of this precursor stage in early land plants through rounds of gene duplication led to the acquisition of additional pathways and the ability to form a fully functional arbuscular mycorrhizal symbiosis.

Female-specific gene expression in dioecious liverwort Pellia endiviifolia is developmentally regulated and connected to archegonia production.

BACKGROUND: In flowering plants a number of genes have been identified which control the transition from a vegetative to generative phase of life cycle. In bryophytes representing basal lineage of land plants, there is little data regarding the mechanisms that control this transition. Two species from bryophytes - moss Physcomitrella patens and liverwort Marchantia polymorpha are under advanced molecular and genetic research. The goal of our study was to identify genes connected to female gametophyte development and archegonia production in the dioecious liverwort Pellia endiviifolia species B, which is representative of the most basal lineage of the simple thalloid liverworts. RESULTS: The utility of the RDA-cDNA technique allowed us to identify three genes specifically expressed in the female individuals of P.endiviifolia: PenB_CYSP coding for cysteine protease, PenB_MT2 and PenB_MT3 coding for Mysterious Transcripts1 and 2 containing ORFs of 143 and 177 amino acid residues in length, respectively. The exon-intron structure of all three genes has been characterized and pre-mRNA processing was investigated. Interestingly, five mRNA isoforms are produced from the PenB_MT2 gene, which result from alternative splicing within the second and third exon. All observed splicing events take place within the 5'UTR and do not interfere with the coding sequence. All three genes are exclusively expressed in the female individuals, regardless of whether they were cultured in vitro or were collected from a natural habitat. Moreover we observed ten-fold increased transcripts level for all three genes in the archegonial tissue in comparison to the vegetative parts of the same female thalli grown in natural habitat suggesting their connection to archegonia development. CONCLUSIONS: We have identified three genes which are specifically expressed in P.endiviifolia sp B female gametophytes. Moreover, their expression is connected to the female sex-organ differentiation and is developmentally regulated. The contribution of the identified genes may be crucial for successful liverwort sexual reproduction.

The thalloid liverwort Plagiochasma rupestre supports arbuscular mycorrhiza-like symbiosis in vitro.

In the present study, we obtained in vitro dual cultures between the liverwort Plagiochasma rupestre and two arbuscular mycorrhizal (AM) fungi: Glomus intraradices and Glomus clarum. Four agarized culture media were tested for optimal growth of P. rupestre. Also, a description of the symbiotic association is provided. Plagiochasma rupestre gametophytes profusely grew axenically in MM with sucrose, and thalli were successfully subcultured under these growth conditions. Arbuscular mycorrhizal fungal hyphae colonized P. rupestre thalli through rhizoids or by forming appresoria in the ventral thallus cells. Arbuscules, mycelia and structures resembling intrathallic spores or vesicles were developed in the internal parenchymatic cells. The pattern of AM colonization in P. rupestre was very similar to the Paris-type. After 100 days of dual culture, the external mycelia of both AM fungal strains formed thousands of small viable spores, suggesting that P. rupestre in vitro culture could be a valuable tool for studying the biology of both symbiotic partners and conserving AM fungi in in vitro germplasm collections.

Novel genes specifically expressed during the development of the male thalli and antheridia in the dioecious liverwort Pellia endiviifolia.

In bryophytes (lower plants), sex determination is manifested in the gametophyte generation by the production of egg- and sperm-forming gametangia. We identified four genes specifically expressed in the male thalli of dioecious liverwort Pellia endiviifolia species B using RDA-cDNA method. These are: PenB_TUA1 coding for an α-tubulin family protein, PenB_Raba1/11 coding for a Rab family protein, PenB_HMG-box coding for an HMG-box family protein and PenB_MT coding for an unknown transcript that contains an ORF of 295 amino acid residues. The expression of identified genes shows developmental and environmental regulation. PenB_TUA1 and PenB_Raba1/11 are expressed in the male thalli, regardless of whether they develop antheridia. PenB_HMG-box and PenB_MT are exclusively expressed in the male thalli-producing antheridia while growing in the field. Moreover, two genes PenB_TUA1 and PenB_Raba1/11 are encoded only in the male genome of P. endiviifolia sp B. Our studies show for the first time the specific contribution of identified genes in the liverwort male gametophyte development. In higher plants, correct regulation of α-tubulin and Rab family genes activity is essential for tip-focused membrane trafficking and growth of the male gametophyte. Thus these genes are critical to the reproductive success of these plants. Plant HMG-box proteins bind DNA and may affect chromatin structure, promoting the assembly of nucleoprotein complexes that control DNA-dependent processes including transcription. Our results show that genes connected with the gametogenesis processes are evolutionarily conserved from the liverworts - the oldest living land plants, to higher plants.

Influence of soil fumigation by methyl bromide and methyl iodide on rhizosphere and phyllosphere microbial community structure.

Rhizosphere and phyllosphere microbial communities were evaluated on roots and leaves of growth chamber-grown lettuce (Lactuca sativa (L.) cv. Green Forest) plants by culture-dependent and -independent methods after soil fumigation. Denaturing gradient gel electrophoresis (DGGE) with 16S rRNA primers followed by cloning and sequencing was used to identify major rRNA bands from the rhizosphere and phyllosphere. Three weeks after fumigation, there were no differences (P = 0.16) in rhizosphere microbial communities between the fumigated treatments and the control. The same effect was observed during week seven after fumigation (P=0.49). Also, no significant differences (P=0.49) were found in the phyllosphere microbial communities between the fumigated treatments and the control during the growth period of the plant. A majority of the bands in the rhizosphere were related to known bacterial sequences with a 96 to 100 % sequence similarity. Some of the derived sequences were related to Pseudomonas syringae pv. tomato DC300 and Bradyrhizobium japonicum USDA 110. A total of 23 isolates were identified from leaf surface by both culture-dependent and independent methods, and only Photorhabdus luminescens was found on leaf surface using both techniques. All the Biolog isolates from phyllosphere were from the Proteobacteria phylum compared to the culture-independent bands from the leaves that were from different bacterial phyla. Based on our data, methyl bromide (MeBr) and methyl iodide (MeI) did not have any significant negative effects on rhizosphere and phyllosphere microbial communities throughout the growing period of lettuce.

Conservative ecological and evolutionary patterns in liverwort-fungal symbioses.

Liverworts, the most ancient group of land plants, form a range of intimate associations with fungi that may be analogous to the mycorrhizas of vascular plants. Most thalloid liverworts contain arbuscular mycorrhizal glomeromycete fungi similar to most vascular plants. In contrast, a range of leafy liverwort genera and one simple thalloid liverwort family (the Aneuraceae) have switched to basidiomycete fungi. These liverwort switches away from glomeromycete fungi may be expected to parallel switches undergone by vascular plants that target diverse lineages of basidiomycete fungi to form ectomycorrhizas. To test this hypothesis, we used a cultivation-independent approach to examine the basidiomycete fungi associated with liverworts in varied worldwide locations by generating fungal DNA sequence data from over 200 field collections of over 30 species. Here we show that eight leafy liverwort genera predominantly and consistently associate with members of the Sebacina vermifera species complex and that Aneuraceae thalloid liverworts associate nearly exclusively with Tulasnella species. Furthermore, within sites where multiple liverwort species co-occur, they almost never share the same fungi. Our analyses reveal a strikingly conservative ecological and evolutionary pattern of liverwort symbioses with basidiomycete fungi that is unlike that of vascular plant mycorrhizas.

Sebacinales are associates of the leafy liverwort Lophozia excisa in the southern maritime Antarctic.

The leafy liverwort Lophozia excisa, which is colonised by basidiomycete fungi in other biomes and which evidence suggests may be colonised by mycorrhizal fungi in Antarctica, was sampled from Léonie Island in the southern maritime Antarctic (67 degrees 36' S, 68 degrees 21' W). Microscopic examination of plants indicated that fungal hyphae colonised 78% of the rhizoids of the liverwort, apparently by entering the tips of rhizoids prior to growing into their bases, where they formed hyphal coils. Extensive colonisation of stem medullary cells by hyphae was also observed. DNA was extracted from surface-sterilised liverwort tissues and sequenced following nested PCR, using the primer set ITS1F/TW14, followed by a second round of amplification using the ITSSeb3/TW13 primer set. Neighbour-joining analyses showed that the sequences obtained nested in Sebacinales clade B as a 100% supported sister group to Sebacinales sequences from the leafy liverworts Lophozia sudetica, L. incisa and Calypogeia muelleriana sampled from Europe. Direct PCR using the fungal specific primer set ITS1F/ITS4 similarly identified fungi belonging to Sebacinales clade B as the principal colonists of L. excisa tissues. These observations indicate the presence of a second mycothallus in Antarctica and support the previous suggestion that the Sebacinales has a wide geographical distribution.

Does Lunularia cruciata form symbiotic relationships with either Glomus proliferum or G. intraradices?

The present study was undertaken to investigate whether the behaviour in vitro of Lunularia cruciata grown with Glomus intraradices and G. proliferum, on SRV medium with 29.2 mM sucrose satisfies the requirements of Koch' postulates for mutualistic symbiosis. Hyphae emerging from mycothallus were able to grow over a two-compartment Petri dish barrier and capture and translocate phosphorus into the host liverwort. Thus, there were increases in plant dry weight, higher AM fungi spore production, and higher plant total phosphorus content. Moreover, this colonization of L. cruciata reproduces typical symptoms generally associated with mycorrhizae. These results showed that mycothalli of L. cruciata have available functionalities generally associated with mycorrhizal symbiosis in higher plants; however, the energy/photosynthetic carbon requirements to maintain a mutualistic symbiosis may be a limiting factor in vivo. Features here discussed indicate that, at least in tested experimental conditions, the endophytic association of L. cruciata with both G. intraradices and G. proliferum is a parasitic/opportunistic partnership rather than a mutualistic symbiosis.

Desiccation tolerance in red and green gametophytes of Jamesoniella colorata in relation to photoprotection.

This study tests the hypothesis that red-leaved gametophytes of the liverwort Jamesoniella colorata (Lehm.) Schiffn., which are found in relatively dry habitats, are more desiccation tolerant than their green counterparts, which are found in moister environments, through superior photoprotective systems. The potential role of red foliar pigments in relation to water deficits is investigated by measuring cell water-relations, oxidative damage and photosynthetic responses. The presence of red pigments, or other cellular constituents, did not affect cell water-relations during dehydration and thus appear not to be involved in cell osmotic regulation. During drying, both colour morphs showed a similar non-photochemical quenching activity and did not experience significant oxidative damage, as measured by the amounts of ascorbate, malondialdehyde and photosynthetic pigments. However, the levels of oxidative damage increased directly upon rewetting the gametophytes, especially in low light conditions (25 micromol m(-2) s(-1)). The efficiency of photosystem II only recovered partially after severe water deficits in both phenotypes. However, the red gametophytes recovered faster and more completely from mild water deficits than did the greens. Moreover, they experienced significantly less photobleaching after rehydration in low light. It is suggested that red pigments and/or carotenoids in these gametophytes improve desiccation tolerance by alleviating photooxidative damage.