Rates and patterns of molecular evolution in bryophyte genomes, with focus on complex thalloid liverworts, Marchantiopsida.

Plants commonly referred to as "bryophytes" belong to three major lineages of non-vascular plants: the liverworts, the hornworts and the mosses. They are unique among land plants in having a dominant haploid generation and a short-lived diploid sporophytic generation. The dynamics of selection acting on a haploid genome differs from those acting on a diploid genome: new mutations are directly exposed to selection. The general aim of this paper is to investigate the diversification rateof bryophytes - measured as silent site substitution rate representing neutral evolution (mutation rate) and the nonsynonymous to synonymous substitution rate ratio (dN/dS) representing selective evolution - and compare it with earlier studies on vascular plants. Results show that the silent site substitution rate is lower for liverworts as compared to angiosperms, but not as low as for gymnosperms. The selection pressure, measured as dN/dS, isnot remarkably lower for bryophytes as compared to other diploid dominant plants as would be expected by the masking hypothesis, indicating that other factors are more important than ploidy.

Mechanisms behind species-specific water economy responses to water level drawdown in peat mosses.

BACKGROUND AND AIMS: The ecosystem engineers Sphagnum (peat mosses) are responsible for sequestering a large proportion of carbon in northern peatlands. Species may respond differently to hydrological changes, and water level changes may lead to vegetation shifts in peatlands, causing them to revert from sinks to sources of carbon. We aimed to compare species-specific responses to water level drawdown within Sphagnum, and investigate which traits affect water economy in this genus. METHODS: In a mesocosm experiment, we investigated how water level drawdown affected water content (WC) in the photosynthetically active apex of the moss and maximum quantum yield of photosystem II (i.e. Fv/Fm) of 13 Sphagnum species. Structural traits were measured, and eight anatomical traits were quantified from scanning electron microscopy micrographs. KEY RESULTS: Mixed-effects models indicated that at high water level, large leaves were the most influential predictor of high WC, and at low water level WC was higher in species growing drier in the field, with larger hyaline cell pore sizes and total pore areas associated with higher WC. Higher stem and peat bulk density increased WC, while capitulum mass per area and numerical shoot density did not. We observed a clear positive relationship between Fv/Fm and WC in wet-growing species. CONCLUSIONS: While we found that most hummock species had a relatively high water loss resistance, we propose that some species are able to maintain a high WC at drawdown by storing large amounts of water at a high water level. Our result showing that leaf traits are important warrants further research using advanced morphometric methods. As climate change may lead to more frequent droughts and thereby water level drawdowns in peatlands, a mechanistic understanding of species-specific traits and responses is crucial for predicting future changes in these systems.

Early evolution of the land plant circadian clock.

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.

Population structure and genetic diversity in the nannandrous moss Homalothecium lutescens: does the dwarf male system facilitate gene flow?

BACKGROUND: Nannandry is a sexual system where males ("dwarf males") are much smaller than the conspecific females. Dwarf males occur in a wide range of unrelated organisms but the evolutionary advantages of this condition are poorly understood. The dwarf male sexual system results in differences in the mode of dispersal and establishment as well as the life span between males and females. Such differences must have profound effects on the population dynamics and genetic structures. We have studied four populations of the nannandrous moss Homalothecium lutescens in southern Sweden. We genotyped dwarf males and female shoots with the aim of describing the genetic diversity and structure of the populations. RESULTS: Dwarf males were most related to their host shoot, then their colony (within 0.5 m(2)) and then the rest of the population, which suggests restricted spore dispersal. However, a few dwarf males in each population appeared to originate from other colonies and sometimes even other populations. Genetic diversity of dwarf males was generally high but showed no tendency to be consistently higher or lower than female genetic diversity within the four populations. CONCLUSIONS: Although most dwarf males have local origin, sporadic dispersal events occur. The ability of the dwarf males to establish in high numbers in mature colonies facilitates gene flow between populations as well as increases the potential to accumulate genetic diversity within populations.

Selective spore germination on shoots of Homalothecium lutescens, a moss with dwarf males.

Spores from three bryophyte species with dwarf males (Homalothecium lutescens, Homalothecium sericeum and Isothecium alopecuroides) were sown on shoots of H. lutescens in vitro. After 10 months, presence and fertility of dwarf plants were scored. Spores of the more distantly related I. alopecuroides were unable to develop into dwarf plants on H. lutescens. Spores of both H. lutescens and H. sericeum developed into dwarf plants. In fact, dwarf plants of H. sericeum were both more abundant and more often fertile than those of H. lutescens. The ability of H. sericeum spores to develop into dwarf males on shoots of H. lutescens suggests a possible pathway for hybridization between the two species. On the other hand, the inability of I. alopecuroides to develop into dwarf males on shoots of H. lutescens suggests that regulation of spore germination and dwarf male development on host shoots is associated with the degree of relatedness between species.

Interspecific competition affects spore germination and gametophore development of mosses.

Background: Interactions between moss species in their earliest growth stages have received little attention. To what extent interspecific competition or priority effects influence spore germination, protonemal development and gametophore emergence is unknown. We evaluated such effects in pairwise interaction between six common bryophyte species: Atrichum undulatum, Bryum argenteum, Ceratodon purpureus, Funaria hygrometrica, Hypnum cupressiforme, Leptobryum pyriforme. Methods: Interspecific interactions were assessed in vitro. Spores were sterilized and sown on agar plates in three treatments: 1) as single species cultures (controls), 2) as pairwise species cultures inoculated simultaneously, and 3) with a time lag of 20 days between species. Data on time needed for spore germination, germination rate, the time needed for gametophore differentiation, number of gametophores per germinated spore and average diameter of colonies were collected. We also performed spore germination tests in single-species cultures at the start and end of the study, as well as tests for density-dependency at spore germination and gametophore formation. Results: We observed strong pairwise interactive effects when sowing spores of different species simultaneously or with a delay of 20 days. The results indicate that spore germination is often inhibited by interspecific competition. The first species has an advantage as compared to the later colonizing species, i.e., an apparent priority effect. Interspecific interactions were also evident during gametophore development and included both inhibition and facilitation. Conclusion: We found pronounced differences in the relative performance of species in interaction with other species during spore germination and gametophore formation. Allelopathic effects are the most probable explanation for these observations. Our results under sterile lab conditions are likely to reflect processes that occur in the wild, governing biotic filtering and bryophyte community assembly during primary and secondary colonization.

Lipidomes of Icelandic bryophytes and screening of high contents of polyunsaturated fatty acids by using lipidomics approach.

Bryophytes (mosses, liverworts, and hornworts) have interested researchers because of their high chemical diversity and their potential uses in pharmaceutical, food, and cosmetic industries. Specifically, long-chain polyunsaturated fatty acids (l-PUFA) such as arachidonic acid (AA) and eicosapentaenoic acid (EPA) are commonly found in bryophytes, but not in vascular plants. Bryophytes accumulate PUFAs in cold or even freezing temperature to keep the cell fluidity. Iceland has a long history of bryophyte vegetation. These bryophytes are highly adapted to the harsh environment in Iceland and therefore are expected to produce high amounts of PUFAs. However, despite the fact that hundreds of mosses and liverworts have been found in Iceland, their lipid profiles largely remain unknown. In this study, we performed untargeted lipidomics by using UPLC-ESI-QTOF-MS as a rapid screening strategy to examine the lipid compositions of 39 local bryophyte species in Iceland and aimed to find high AA and EPA producers. A total of 280 lipid molecular species from 15 lipid classes were quantified with isotope-labeled internal standards. AA and EPA were abundantly distributed in the phospholipids (mainly PC and PE) and glycerolipids (MGDG and DGDG) in six moss species, namely Racomotrium lanuginosum, R. ericoides, Bryum psedotriquetrium, Plagiomnium ellipticum, Hylocomium splendens, and Rhytidiadelphus triquetrus. Two of the six species (B. psedotriquetrium and H. splendens) also accumulated high concentrations of PUFA-containing-triacylglycerols.

Genome Evolution in Plants: Complex Thalloid Liverworts (Marchantiopsida).

Why do some genomes stay small and simple, while others become huge, and why are some genomes more stable? In contrast to angiosperms and gymnosperms, liverworts are characterized by small genomes with low variation in size and conserved chromosome numbers. We quantified genome evolution among five Marchantiophyta (liverworts), measuring gene characteristics, transposable element (TE) landscape, collinearity, and sex chromosome evolution that might explain the small size and limited variability of liverwort genomes. No genome duplications were identified among examined liverworts and levels of duplicated genes are low. Among the liverwort species, Lunularia cruciata stands out with a genome size almost twice that of the other liverwort species investigated here, and most of this increased size is due to bursts of Ty3/Gypsy retrotransposons. Intrachromosomal rearrangements between examined liverworts are abundant but occur at a slower rate compared with angiosperms. Most genes on L. cruciata scaffolds have their orthologs on homologous Marchantia polymorpha chromosomes, indicating a low degree of rearrangements between chromosomes. Still, translocation of a fragment of the female U chromosome to an autosome was predicted from our data, which might explain the uniquely small U chromosome in L. cruciata. Low levels of gene duplication, TE activity, and chromosomal rearrangements might contribute to the apparent slow rate of morphological evolution in liverworts.

Genetic Diversity and Population Structure in Bryophyte With Facultative Nannandry.

Among plants, gender dimorphism occurs in about 10% of all angiosperms and more than 50% of all moss taxa, with dwarf males (DM) found exclusively in some unisexual mosses. In this study, we explore the role of male dwarfism as a reproductive strategy in the widespread acrocarpous moss Dicranum scoparium, which has facultative male dwarfism, having both dwarf males (DMs) and normal-sized males (NMs). We retrieved 119 SNP markers from transcriptomes which were used to genotype 403 samples from 11 sites at seven localities in southern Sweden. Our aims were to compare the genetic variability and genetic structure of sexually reproducing populations at different geographic levels (cushion, site, and locality) and compare in particular the relative contribution of females, dwarf males and normal-sized males to the observed genetic diversity. The numbers of DMs differed strongly between sites, but when present, they usually outnumbered both females and NMs. Low genetic differentiation was found at locality level. Genetic differentiation was strongest between cushions for females and NMs and within cushions for DMs indicating small scale structuring and sometimes inbreeding. NMs were more clonal than either DMs or females. Genetic diversity was similar between females and DMs, but lower for NMs. Two haplotypes were shared between females and DMs and one haplotype was shared between a DM and a NM. In conclusion, our results show that DMs and NMs play different roles in reproduction, inbreeding may occur at cushion level, but gene flow is high enough to prevent substantial genetic drift.

Evolutionary History of the Marchantia polymorpha Complex.

The potential role of introgression in evolution has gained increased interest in recent years. Although some fascinating examples have been reported, more information is needed to generalize the importance of hybridization and introgression for adaptive divergence. As limited data exist on haploid dominant species, we analyzed genomes of three subspecies of the liverwort Marchantia polymorpha. We used available genomic data for subsp. ruderalis and carried out whole-genome (PacBio) sequencing for one individual each of subsp. montivagans and subsp. polymorpha as well as Illumina resequencing of additional genomes for all three subspecies. The three subspecies were compared against M. paleacea as outgroup. Our analyses revealed separation of the three taxa, but all three possible topologies were richly represented across the genomes, and the underlying divergence order less obvious. This uncertainty could be the result of the divergence of the three subspecies close in time, or that introgression has been frequent since divergence. In particular, we found that pseudo-chromosome 2 in subsp. montivagans was much more diverged than other parts of the genomes. This could either be explained by specific capture of chromosome 2 from an unknown related species through hybridization or by conservation of chromosome 2 despite intermittent or ongoing introgression affecting more permeable parts of the genomes. A higher degree of chromosomal rearrangements on pseudo-chromosome 2 support the second hypothesis. Species tree analyses recovered an overall topology where subsp. montivagans diverged first and subsp. ruderalis and subsp. polymorpha appeared as sister lineages. Each subspecies was associated with its own chloroplast and mitochondrial haplotype group. Our data suggest introgression but refute a previous hypothesis that subsp. ruderalis is a new stabilized hybrid between the other two subspecies.

The effective size of bryophyte populations.

Bryophytes with their dominant haploid stage conform poorly to the life cycles generally treated in population genetical models. Here we make a detailed analysis of what effective sizes bryophyte model populations have as a function of their breeding system. It is found that the effective size is rarely much smaller than the scored number of haploid gametophytic individuals, even when the limited number of diploids (sporophytes) formed is taken into account. The most severe decrease in effective size occurs when unisexual gametophytic females produce only a small number of fertile diploid sporophytes in male biased populations; this effect is due to the restricted sampling of male gametophytic individuals that then occurs. It is shown that the harmonic mean of diploid sporophytes formed per haploid gametophytic individuals is the relevant measure in these calculations and not the standard (and generally larger) arithmetic mean.

Recombination and introgression of nuclear and chloroplast genomes between the peat mosses, Sphagnum capillifolium and Sphagnum quinquefarium.

Haploid hybrid gametophytes are often present at low frequencies in sympatric populations of Sphagnum capillifolium and Sphagnum quinquefarium. We used intersimple sequence repeat (ISSR) markers and polymerase chain reaction-restriction fragment length polymorphism of the trnL(UAA) intron of the chloroplast genome to reveal the nuclear and chloroplast composition of mature hybrid gametophytes from natural populations and of gametophytes derived from spores of hybrid sporophytes collected in nature. Asymmetrical nuclear inheritance was found in the progeny of the hybrid sporophytes, indicating that only spores with a low level of recombination of parental genomes were viable. A similarly skewed nuclear composition was found among the naturally occurring hybrid gametophytes. All hybrid genomes contained a larger proportion of S. capillifolium ISSR markers, combined with only two to five S. quinquefarium markers together with a chloroplast haplotype derived from S. quinquefarium. In this way, a pattern resembling introgression is created within a single generation. Some individuals possessed nuclear genomes typical for S. capillifolium in combination with the chloroplast haplotype of S. quinquefarium, possibly indicating backcrossing. Our results indicate that hybridization between S. capillifolium and S. quinquefarium is relatively common, but the resistance of large parts of the genome against heterospecific genes maintains the genetic distinctness of the species. Further evolutionary and phylogenetic consequences of restricted interspecific gene exchange are discussed.

Factors influencing reproductive success in the clonal moss, Hylocomium splendens.

Female reproductive success in the unisexual perennial clonal moss Hylocomium splendens was examined by recording, if the segment was reproductive [produced sporophyte(s)] or not, together with several distance-to-male and male density variables, and segment size. This was done for every female segment in a population over a 5 year study period. A high fraction of the population could be sexed because we monitored the population in situ for 5 years, and thereafter harvested the population for electrophoretic analysis from which the clonal identity and expressed sex could be deduced. Fertilization distances in H. splendens were short, indicated by the fact that as many as 85% of the female segments with sporophytes were situated within a distance of 5.0 cm from the nearest male. The longest distance measured between a sporophytic female and the closest male was 11.6 cm. However, analysed within a generalized linear modelling (GLM) framework, the year was the best single predictor for the presence of H. splendens sporophyte although female-segment size and distance to the closest situated male were also strongly significant. The two latter factors explained larger fractions of variation in sporophyte presence in a GLM model with three predictors than in single-predictor models. This is because (i) the large variation in sporophyte production among years partly obscures the strong general increase in sporophyte production with increasing female-segment size and vitality, and (ii) the between-year variation and the size obscure the effect of the distance to the most proximate male. To our knowledge, this study is the first to incorporate into one model the relative importance of several factors for bryophyte reproductive success. Our results demonstrate the value of multiple-predictor approaches in studies of reproductive success.

Microarthropods mediate sperm transfer in mosses.

Among flowering plants, animals commonly act as pollinators. We showed that fertile moss shoots attract springtails and mites, which in turn carry moss sperm, thereby enhancing the fertilization process. Previously, fertilization of mosses was thought to depend on the capacity of individual sperm to swim through a continuous water layer. The role of microarthropods in moss fertilization resembles the role of animals as pollinators of flowering plants but may be evolutionarily much older because of the antiquity of the organism groups involved.