Dioecious Liverwort Research Articles

Telomere Length Variation in Model Bryophytes.

The ends of linear chromosomes of most eukaryotes consist of protein-bound DNA arrays called telomeres, which play essential roles in protecting genome integrity. Despite general evolutionary conservation in function, telomeric DNA is known to drastically vary in length and sequence between different eukaryotic lineages. Bryophytes are a group of early diverging land plants that include mosses, liverworts, and hornworts. This group of ancient land plants recently emerged as a new model for important discoveries in genomics and evolutionary biology, as well as for understanding plant adaptations to a terrestrial lifestyle. We measured telomere length in different ecotypes of model bryophyte species, including Physcomitrium patens, Marchantia polymorpha, Ceratodon purpureus, and in Sphagnum isolates. Our data indicate that all analyzed moss and liverwort genotypes have relatively short telomeres. Furthermore, all analyzed ecotypes and isolates of model mosses and liverworts display evidence of substantial natural variation in telomere length. Interestingly, telomere length also differs between male and female strains of the dioecious liverwort M. polymorpha and dioecious moss C. purpureus. Given that bryophytes are extraordinarily well adapted to different ecological niches from polar to tropical environments, our data will contribute to understanding the impact of natural telomere length variation on evolutionary adaptations in this ancient land plant lineage.

HAG1 and SWI3A/B control of male germ line development in P. patens suggests conservation of epigenetic reproductive control across land plants

Key messageBryophytes as models to study the male germ line: loss-of-function mutants of epigenetic regulators HAG1 and SWI3a/b demonstrate conserved function in sexual reproduction.With the water-to-land transition, land plants evolved a peculiar haplodiplontic life cycle in which both the haploid gametophyte and the diploid sporophyte are multicellular. The switch between these phases was coined alternation of generations. Several key regulators that control the bauplan of either generation are already known. Analyses of such regulators in flowering plants are difficult due to the highly reduced gametophytic generation, and the fact that loss of function of such genes often is embryo lethal in homozygous plants. Here we set out to determine gene function and conservation via studies in bryophytes. Bryophytes are sister to vascular plants and hence allow evolutionary inferences. Moreover, embryo lethal mutants can be grown and vegetatively propagated due to the dominance of the bryophyte gametophytic generation. We determined candidates by selecting single copy orthologs that are involved in transcriptional control, and of which flowering plant mutants show defects during sexual reproduction, with a focus on the under-studied male germ line. We selected two orthologs, SWI3a/b and HAG1, and analyzed loss-of-function mutants in the moss P. patens. In both mutants, due to lack of fertile spermatozoids, fertilization and hence the switch to the diploid generation do not occur. Pphag1 additionally shows arrested male and impaired female gametangia development. We analyzed HAG1 in the dioecious liverwort M. polymorpha and found that in Mphag1 the development of gametangiophores is impaired. Taken together, we find that involvement of both regulators in sexual reproduction is conserved since the earliest divergence of land plants.

The impact of asexual and sexual reproduction in spatial genetic structure within and between populations of the dioecious plant Marchantia inflexa (Marchantiaceae).

In dioecious plants, sexual reproduction requires close proximity to potential mates, but clonal growth can increase this distance and, therefore, reduce the probability of mating. Reduction in sexual propagules can lead to decreased dispersal and gene flow between populations. Gene flow and clonal growth may be further influenced by the size of the habitat patch. The effects of habitat size and reproductive mode (sexual or asexual reproduction) on spatial genetic structure and segregation of the sexes were tested by quantifying the distributions of genotypes and the sexes using the dioecious liverwort Marchantia inflexa. Plants were sampled from five pairs of small-large habitat patches to identify within- and among-population spatial genetic structure using 12 microsatellite markers. Spatial distributions were calculated as the likelihood that pairs of individuals were the same sex or genotype, and it was determined how that likelihood was affected by habitat patch size (small/large). Asexual reproduction dominates within populations, and asexual dispersal also occurred across populations. Spatial segregation of the sexes was observed within populations; males were more likely to be near individuals of the same sex than were females. Although the likelihood of both sexes being near members of the same sex was similarly greater on small habitat patches, on large habitat patches male genotypes were almost 15 % more likely to be near clonemates than were female genotypes. The results show a sex difference in clonal clumping that was dependent upon habitat size, suggesting differential colonization and/or survival between males and females. The sexes and genotypes being structured differently within and among populations have implications for the persistence of populations and the interactions between them. This study demonstrates that studying only the sexes and not their genotypes (or vice versa) can limit our understanding of the extent to which reproductive modes (sexual or asexual) influence genetic structure both within and between populations.

Molecular Genetic Tools and Techniques for Marchantia polymorpha Research.

Liverworts occupy a basal position in the evolution of land plants, and are a key group to address a wide variety of questions in plant biology. Marchantia polymorpha is a common, easily cultivated, dioecious liverwort species, and is emerging as an experimental model organism. The haploid gametophytic generation dominates the diploid sporophytic generation in its life cycle. Genetically homogeneous lines in the gametophyte generation can be established easily and propagated through asexual reproduction, which aids genetic and biochemical experiments. Owing to its dioecy, male and female sexual organs are formed in separate individuals, which enables crossing in a fully controlled manner. Reproductive growth can be induced at the desired times under laboratory conditions, which helps genetic analysis. The developmental process from a single-celled spore to a multicellular body can be observed directly in detail. As a model organism, molecular techniques for M. polymorpha are well developed; for example, simple and efficient protocols of Agrobacterium-mediated transformation have been established. Based on them, various strategies for molecular genetics, such as introduction of reporter constructs, overexpression, gene silencing and targeted gene modification, are available. Herein, we describe the technologies and resources for reverse and forward genetics in M. polymorpha, which offer an excellent experimental platform to study the evolution and diversity of regulatory systems in land plants.

AgarTrap-mediated genetic transformation using intact gemmae/gemmalings of the liverwort Marchantia polymorpha L.

The dioecious liverwort, Marchantia polymorpha L., is an emerging model plant. Various molecular biological techniques have been optimized for M. polymorpha for the past several years, and recently we reported a simplified Agrobacterium-mediated transformation method using sporelings (immature thalli from spores) of M. polymorpha. This method, termed AgarTrap (Agar-utilized Transformation with Pouring Solutions), completed by exchanging appropriate solutions on a single Petri dish to produce a sufficient number of independent transgenic sporelings. However, because spores are produced by crosses between males and females, the genetic backgrounds of resulting transgenic sporelings are not uniform. To easily produce transgenic liverworts with a uniform genetic background using AgarTrap, we developed an AgarTrap-mediated transformation method using intact gemmae/gemmalings produced by asexual reproduction. Using AgarTrap with male and female gemmae/gemmalings produced a sufficient number of independent transgenic gemmalings with uniform genetic backgrounds. The optimized transformation efficiencies were approximately 30 and 50 % in males and females, respectively. As with AgarTrap using sporelings, AgarTrap using intact gemmae/gemmalings will be useful in promoting studies of the molecular biology of M. polymorpha.

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

BackgroundIn 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.ResultsThe 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.ConclusionsWe 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.

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.

Gene content, organization and molecular evolution of plant organellar genomes and sex chromosomes - Insights from the case of the liverwort Marchantia polymorpha

The complete nucleotide sequence of chloroplast DNA (121,025 base pairs, bp) from a liverwort, Marchantia polymorpha, has made clear the entire gene organization of the chloroplast genome. Quite a few genes encoding components of photosynthesis and protein synthesis machinery have been identified by comparative computer analysis. We also determined the complete nucleotide sequence of the liverwort mitochondrial DNA and deduced 96 possible genes in the sequence of 186,608 bp. The complete chloroplast genome encodes twenty introns (19 group II and 1 group I) in 18 different genes. One of the chloroplast group II introns separates a ribosomal protein gene in a trans-position. The mitochondrial genome contains thirty-two introns (25 group II and 7 group I) in the coding regions of 17 genes. From the evolutionary point of view, we describe the origin of organellar introns and give evidence for vertical and horizontal intron transfers and their intragenomic propagation. Furthermore, we describe the gene organization of the Y chromosome in the dioecious liverwort M. polymorpha, the first detailed view of a Y chromosome in a haploid organism. On the 10 megabase (Mb) Y chromosome, 64 genes are identified, 14 of which are detected only in the male genome. These 14 genes are expressed in reproductive organs but not in vegetative thalli, suggesting their participation in male reproductive functions. These findings indicate that the Y and X chromosomes share the same ancestral autosome and support the prediction that in a haploid organism essential genes on sex chromosomes are more likely to persist than in a diploid organism.

Overgrowth competition, fragmentation and sex-ratio dynamics: a spatially explicit, sub-individual-based model

Sessile organisms that compete for access to resources by overgrowing each other may risk the local elimination of one sex or the other, as frequently happens within clumps of the dioecious liverwort Marchantia inflexa. A multi-stage, spatially implicit differential-equation model of M. inflexa growing in an isolated patch, analysed in a previous study, indicated that long-term coexistence of the sexes within such patches may be only temporary. Here we derive a spatially explicit, sub-individual-based model to reconsider this interpretation when much more ecological realism is taken into account, including the process of fragmentation. The model tracks temporally discrete growth increments in continuous space, representing growth architecture and the overgrowth process in significant geometric detail. Results remain generally consistent with the absence of long-term coexistence of the sexes in individual patches of Marchantia. Dynamics of sex-specific growth qualitatively resemble those generated by differential-equation models, suggesting that this much simpler framework may be adequate for multi-patch metapopulation models. Direct competition between fragmenting and non-fragmenting clones demonstrates the importance of fragmentation in overgrowth competition. The results emphasize the need for empirical work on mechanisms of overgrowth and for modeling and empirical studies of life history tradeoffs and sex-ratio dynamics in multi-patch systems.

A mutant with constitutive sexual organ development in Marchantia polymorpha L.

In lower land plants, genes controlling the transition from vegetative growth to sexual reproduction have not yet been identified. In the dioecious liverwort Marchantia polymorpha, the transition to sexual reproduction accompanied by the formation of sexual organs on the gametophytic thallus is initiated under long-day conditions. By particle bombardment-mediated mutagenesis, we generated a mutant of M. polymorpha that constitutively forms sexual organs. This mutant is fully fertile, showing that the mutation does not affect formation of male or female sexual organs per se. Genetic analysis reveals that this phenotype is caused by mutation of a single autosomal locus, suggesting that this mutation defines or controls a gene regulating the transition to sexual reproduction in M. polymorpha.

Evolution of ribosomal DNA unit on the X chromosome independent of autosomal units in the liverwort Marchantia polymorpha.

In the haploid dioecious liverwort, Marchantia polymorpha, the X chromosome, but not the Y, carries a cluster of ribosomal RNA genes (rDNAs). Here we show that sequences of 5S, 17S, 5.8S and 26S rDNAs are highly conserved (>99% identity) between the X chromosomal and autosomal rDNA repeat units, but the intergenic spacer sequences differ considerably. The most prominent difference is the presence of a 615-bp DNA fragment in the intergenic spacer, X615, which has accumulated predominantly in the rDNA cluster of the X chromosome. These observations suggest that the rDNA repeat unit on the X chromosome evolved independently of that on autosomes, incorporating sex chromosome-specific sequences.

Local sex-ratio dynamics: a model for the dioecious liverwort Marchantia inflexa

Local sex-ratio dynamics: a model for the dioecious liverwort Marchantia inflexa

Construction of male and female PAC genomic libraries suitable for identification of Y-chromosome-specific clones from the liverwort, Marchantia polymorpha.

Unlike higher plants, the dioecious liverwort, Marchantia polymorpha, has uniquely small sex chromosomes, with X chromosomes present only in female gametophytes and Y chromosomes only in male gametophytes. We have constructed respective genomic libraries for male and female plantlets using a P1-derived artificial chromosome (pCYPAC2). With an average insert size of approximately 90 kb, each PAC library is estimated to cover the entire genome with a probability of more than 99.9%. Male-specific PAC clones were screened for by differential hybridization using male and female genomic DNAs as separate probes. Seventy male-specific PAC clones were identified. The male specificity of one of the clones, pMM4G7, was verified by Southern hybridization and PCR analysis. This clone was indeed located on the Y chromosome as verified by fluorescence in situ hybridization (FISH). This result shows that the Y chromosome contains unique sequences that are not present either on the X chromosome or any of the autosomes. Thus, the respective male and female libraries for M. polymorpha offer an opportunity to identify key genes involved in the process of sex differentiation and this unique system of sex determination.

Population sex ratios, sex‐specific clonal traits and tradeoffs among these traits in the liverwort Marchantia inflexa

Unisexual, female‐ or male‐biased populations are common among some clonal plants. Within and among populations, the relative frequencies of males and females can be influenced by sex‐specific demographic patterns that, in turn, can be a consequence of life‐history characteristics. The objectives of this study were to describe (1) population sex‐ratio patterns among habitat patches along a river, and (2) sex‐specific patterns of and correlations among life‐history clonal traits including: growth rates, number of meristematic tips and asexual reproduction in the dioecious liverwort, Marchantia inflexa. In Trinidad, a section of a stream was surveyed for the occurrence of female and male sex expressing thalli among habitat patches, and habitat characteristics (canopy openness and patch size) were recorded. The numbers of female and male inflorescences were obtained also. Of the 209 patches observed in the field, 83% did not contain sex expressing thalli, 9% contained thalli of both sexes, and the rest contained only female‐ (4%) or male‐expressing (4%) thalli. Sex expression was less common among small patches, and there was a tendency for sex expression to be less likely among patches with the lowest canopy openness. The proportion of male inflorescences among the bisexual patches ranged from 0.22 to 0.80. In a greenhouse, we planted gemmae (asexual propagules) from field‐collected isolates: ten female and eight male. On six occasions, we harvested replicates of each isolate to estimate isolate trait means. Females grew faster, produced more meristematic tips and had lower levels of asexual reproduction than males. Number of meristematic tips was negatively correlated with asexual reproduction and positively correlated with growth rate. These sex‐specific patterns may lead to the high frequency of single sex patches as well as biased adult population sex ratios that are observed in the field.

Sperm limitation and genetic effects on fecundity in the dioecious liverwort Sphaerocarpos texanus

Sperm limitation and genetic effects on fecundity in the dioecious liverwort Sphaerocarpos texanus

Sex Ratios ofRiccia frostiiAust. in Khartoum Province, Sudan

Spore dispersal of the dioecious liverwort, Riccia frostii Aust., in Khartoum Province Sudan, is by the Blue Nile in flood. Although a 1 : 1 sex ratio is to be expected, sample counts have shown that females always outnumber males and the actual ratio varies from site to site. Shading has been shown to suppress the development of male thalli, while a pattern analysis and covariance analysis of an apparently homogeneous site showed that male and female thalli reacted differently to microtopographical/substrate differences on the Nile bank. Extreme age of the population produces a change in sex ratio through the earlier death of male thalli; a quickly drying substrate also produces a differential mortality of male thalli. The effect of the high summer temperatures on the dormant spores which remain in situ on the Nile banks is not known but may produce a change in the sex ratio through the differential mortality of the spores.