Within-plant heterogeneity in fecundity and herbivory induced by localized DNA hypomethylation in the perennial herb Helleborus foetidus.

PREMISE: Phenotypic heterogeneity of reiterated, homologous structures produced by individual plants has ecological consequences for plants and their animal consumers. This paper examines experimentally the epigenetic mosaicism hypothesis, which postulates that within-plant variation in traits of reiterated structures may partly arise from different parts of the same genetic individual differing in patterns or extent of genomic DNA methylation. METHODS: Leaves of paired ramets borne by field-growing Helleborus foetidus plants were infiltrated periodically over the entire flowering period with either a water solution of the demethylating agent zebularine or just water as the control. The effects of the zebularine treatment were assessed by quantifying genome-wide DNA cytosine methylation in leaves and monitoring inflorescence growth and flower production, number of ovules per flower, pollination success, fruit set, seed set, seed size, and distribution of sap-feeding insects. RESULTS: Genomic DNA from leaves in zebularine-treated ramets was significantly less methylated than DNA from leaves in control ones. Inflorescences in treated ramets grew smaller and produced fewer flowers, with fewer ovules and lower follicle and seed set, but did not differ from inflorescences in untreated ramets in pollination success or seed size. The zebularine treatment influenced the within-plant distribution of sap-feeding insects. CONCLUSIONS: Experimental manipulation of genomic DNA methylation level in leaves of wild-growing H. foetidus plants induced considerable within-plant heterogeneity in phenotypic (inflorescences, flowers, fecundity) and ecologically relevant traits (herbivore distribution), which supports the hypothesis that epigenetic mosaicism may partly account for within-plant variation.

Comparative spatial genetics and epigenetics of plant populations: heuristic value and a proof of concept.

Despite the recent upsurge of interest on natural epigenetic variation of nonmodel organisms, factors conditioning the spatial structure of epigenetic diversity in wild plant populations remain virtually unexplored. We propose that information on processes shaping natural epigenetic variation can be gained using the spatial structure of genetic diversity as null model. Departures of epigenetic isolation-by-distance (IBD) patterns from genetic IBD patterns for the same sample, particularly differences in slope of similarity-distance regressions, will reflect the action of factors that operate specifically on epigenetic variation, including imperfect transgenerational inheritance and responsiveness to environmental factors of epigenetic marks. As a proof of concept, we provide a comparative analysis of spatial genetic and epigenetic structure of 200 mapped individuals of the perennial herb Helleborus foetidus. Plants were fingerprinted using nuclear microsatellites, amplified fragment length polymorphisms (AFLP) and methylation-sensitive AFLP markers. Expectations from individual-level IBD patterns were tested by means of kinship-distance regressions. Both genetic and epigenetic similarity between H. foetidus individuals conformed to theoretical expectations under individual-level IBD models. Irrespective of marker type, there were significant negative linear relationships between the kinship coefficient for plant pairs and their spatial separation. Regression slopes were significantly steeper for epigenetic markers. Epigenetic similarity between individuals was much greater than genetic similarity at shortest distances, such epigenetic 'kinship excess' tending to decrease as plant separation increased. Results suggest that moderate-to-high heritability and responsiveness to local environments are major drivers of epigenetic spatial structure in H. foetidus, and illustrate the heuristic value of comparing genetic and epigenetic spatial structure for formulating and testing hypotheses on forces shaping epigenetic diversity in wild plant populations.

MSAP markers and global cytosine methylation in plants: a literature survey and comparative analysis for a wild-growing species.

Methylation of DNA cytosines affects whether transposons are silenced and genes are expressed, and is a major epigenetic mechanism whereby plants respond to environmental change. Analyses of methylation-sensitive amplification polymorphism (MS-AFLP or MSAP) have been often used to assess methyl-cytosine changes in response to stress treatments and, more recently, in ecological studies of wild plant populations. MSAP technique does not require a sequenced reference genome and provides many anonymous loci randomly distributed over the genome for which the methylation status can be ascertained. Scoring of MSAP data, however, is not straightforward, and efforts are still required to standardize this step to make use of the potential to distinguish between methylation at different nucleotide contexts. Furthermore, it is not known how accurately MSAP infers genome-wide cytosine methylation levels in plants. Here, we analyse the relationship between MSAP results and the percentage of global cytosine methylation in genomic DNA obtained by HPLC analysis. A screening of literature revealed that methylation of cytosines at cleavage sites assayed by MSAP was greater than genome-wide estimates obtained by HPLC, and percentages of methylation at different nucleotide contexts varied within and across species. Concurrent HPLC and MSAP analyses of DNA from 200 individuals of the perennial herb Helleborus foetidus confirmed that methyl-cytosine was more frequent in CCGG contexts than in the genome as a whole. In this species, global methylation was unrelated to methylation at the inner CG site. We suggest that global HPLC and context-specific MSAP methylation estimates provide complementary information whose combination can improve our current understanding of methylation-based epigenetic processes in nonmodel plants.

Continuous within-plant variation as a source of intraspecific functional diversity: Patterns, magnitude, and genetic correlates of leaf variability in Helleborus foetidus (Ranunculaceae).

PREMISE OF THE STUDY: Continuous within-plant variation in quantitative traits of reiterated, homologous structures is a component of intraspecific variation, but its contribution to functional diversity remains largely unexplored. For the perennial Helleborus foetidus, we measured functional leaf traits to quantify the contribution of within-plant variation to intraspecific functional variance and evaluate whether within-plant variability itself deserves separate consideration. METHODS: Within-individual variation in eight leaf traits was quantified for 138 plants sampled from 10 widely spaced locations in the Sierra de Cazorla, southeastern Spain. An amplified fragment length polymorphism (AFLP) technique was used to look for associations between within-plant variability and specific AFLP markers. KEY RESULTS: Leaflets from basal positions in ramets were longer, heavier, had greater surface area and larger stomata, and lower specific area, stomatal index, and stomatal density than those from distal positions. Continuous variation between leaves from the same ramet was the main source of population-wide variance for most traits. Within-plant variability differed among populations. Individuals differed in within-plant variability, which was largely independent of trait means and associated with genetic characteristics. Up to four AFLP markers were associated with the within-plant variability level of a given leaf trait. CONCLUSIONS: Subindividual variability in continuous leaf traits was independent of plant means and related to genetic features. The within-individual component generally exceeded the between-individual component of intraspecific variance. Within-plant variation may broaden the ecological breadth and enhance stability and persistence of plant populations and communities and may provide novel insights when incorporated in trait-based community ecology models.

Epigenetic variation predicts regional and local intraspecific functional diversity in a perennial herb.

The ecological significance of epigenetic variation has been generally inferred from studies on model plants under artificial conditions, but the importance of epigenetic differences between individuals as a source of intraspecific diversity in natural plant populations remains essentially unknown. This study investigates the relationship between epigenetic variation and functional plant diversity by conducting epigenetic (methylation-sensitive amplified fragment length polymorphisms, MSAP) and genetic (amplified fragment length polymorphisms, AFLP) marker-trait association analyses for 20 whole-plant, leaf and regenerative functional traits in a large sample of wild-growing plants of the perennial herb Helleborus foetidus from ten sampling sites in south-eastern Spain. Plants differed widely in functional characteristics, and exhibited greater epigenetic than genetic diversity, as shown by per cent polymorphism of MSAP fragments (92%) or markers (69%) greatly exceeding that for AFLP ones (41%). After controlling for genetic structuring and possible cryptic relatedness, every functional trait considered exhibited a significant association with at least one AFLP or MSAP marker. A total of 27 MSAP (13.0% of total) and 12 AFLP (4.4%) markers were involved in significant associations, which explained on average 8.2% and 8.0% of trait variance, respectively. Individual MSAP markers were more likely to be associated with functional traits than AFLP markers. Between-site differences in multivariate functional diversity were directly related to variation in multilocus epigenetic diversity after multilocus genetic diversity was statistically accounted for. Results suggest that epigenetic variation can be an important source of intraspecific functional diversity in H. foetidus, possibly endowing this species with the capacity to exploit a broad range of ecological conditions despite its modest genetic diversity.

Individual variation in size and fecundity is correlated with differences in global DNA cytosine methylation in the perennial herb Helleborus foetidus (Ranunculaceae).

UNLABELLED: • PREMISE OF THE STUDY: Few studies have examined how epigenetic modifications of DNA may influence individual plant phenotypes and ecological processes in wild plant populations. We investigated natural variation in global DNA cytosine methylation and its phenotypic correlates in the perennial herb Helleborus foetidus.• METHODS: We focused specifically on individual differences in size- and fecundity-related traits and used HPLC to quantify percentage of total cytosines in the genome of young full-grown leaves that were methylated.• KEY RESULTS: About one third of all cytosines in H. foetidus genome were methylated. Methylation level differed significantly among individual plants (range = 26.4-36.6%; n = 60 plants), and this variation was significantly related to most size- and fecundity-related traits considered. Relatively hypomethylated plants bore more vegetative, reproductive, and total ramets, produced more flowers, larger inflorescences and more seed-bearing follicles, and their ramets remained vegetative for fewer years before reproducing sexually, than relatively hypermethylated ones. Taken together, results revealed that individual differences in size and reproductive output were inversely related to global cytosine methylation.• CONCLUSIONS: These results confirm, in a natural scenario, the association between DNA methylation and size- and fecundity-related traits that was previously found by experimental studies. Variations in global cytosine methylation were predictably related to individual differences in sexual reproduction through significant effects on flower and fruit production, which might ultimately influence patterns of selection and population dynamics in this species. This study provides novel insights on the potential ecological significance of epigenetic heterogeneity in wild plant populations.

Cytotoxic and genotoxic activity of some Helleborus species.

Despite their known toxic properties, various Helleborus species are used as medicaments in folk medicine to treat some diseases and health conditions. As the main mechanism of many cytostatic drugs is based on their cytotoxic activity, there is potential for the toxicity of hellebore to be used in anticancer therapy. This study tested the geno- and cytotoxic effects of extracts of three hellebore taxa (Helleborus odorus, Helleborus multifidus and Helleborus hercegovinus) on meristemic onion (Alliumcepa L.) cells and human lymphocytes. Treatments with Helleborus extracts induced cytotoxic and cytostatic effects in meristemic onion cells as well as in cultivated cytokinesis-blocked human lymphocytes. Cytokinesis-block micronucleus cytome assay indicated that treatments with hellebore extracts induce genotoxic effects in human lymphocytes, and that the significant mechanism of their antiproliferative activity is apoptosis induction.

Variation in DNA methylation transmissibility, genetic heterogeneity and fecundity-related traits in natural populations of the perennial herb Helleborus foetidus.

Inferences about the role of epigenetics in plant ecology and evolution are mostly based on studies of cultivated or model plants conducted in artificial environments. Insights from natural populations, however, are essential to evaluate the possible consequences of epigenetic processes in biologically realistic scenarios with genetically and phenotypically heterogeneous populations. Here, we explore associations across individuals between DNA methylation transmissibility (proportion of methylation-sensitive loci whose methylation status persists unchanged after male gametogenesis), genetic characteristics (assessed with AFLP markers), seed size variability (within-plant seed mass variance), and realized maternal fecundity (number of recently recruited seedlings), in three populations of the perennial herb Helleborus foetidus along a natural ecological gradient in southeastern Spain. Plants (sporophytes) differed in the fidelity with which DNA methylation was transmitted to descendant pollen (gametophytes). This variation in methylation transmissibility was associated with genetic differences. Four AFLP loci were significantly associated with transmissibility and accounted collectively for ~40% of its sample-wide variance. Within-plant variance in seed mass was inversely related to individual transmissibility. The number of seedlings recruited by individual plants was significantly associated with transmissibility. The sign of the relationship varied between populations, which points to environment-specific, divergent phenotypic selection on epigenetic transmissibility. Results support the view that epigenetic transmissibility is itself a phenotypic trait whose evolution may be driven by natural selection, and suggest that in natural populations epigenetic and genetic variation are two intertwined, rather than independent, evolutionary factors.

Epigenetic differentiation persists after male gametogenesis in natural populations of the perennial herb Helleborus foetidus (Ranunculaceae).

Despite the importance of assessing the stability of epigenetic variation in non-model organisms living in real-world scenarios, no studies have been conducted on the transgenerational persistence of epigenetic structure in wild plant populations. This gap in knowledge is hindering progress in the interpretation of natural epigenetic variation. By applying the methylation-sensitive amplified fragment length polymorphism (MSAP) technique to paired plant-pollen (i.e., sporophyte-male gametophyte) DNA samples, and then comparing methylation patterns and epigenetic population differentiation in sporophytes and their descendant gametophytes, we investigated transgenerational constancy of epigenetic structure in three populations of the perennial herb Helleborus foetidus (Ranunculaceae). Single-locus and multilocus analyses revealed extensive epigenetic differentiation between sporophyte populations. Locus-by-locus comparisons of methylation status in individual sporophytes and descendant gametophytes showed that ~75% of epigenetic markers persisted unchanged through gametogenesis. In spite of some epigenetic reorganization taking place during gametogenesis, multilocus epigenetic differentiation between sporophyte populations was preserved in the subsequent gametophyte stage. In addition to illustrating the efficacy of applying the MSAP technique to paired plant-pollen DNA samples to investigate epigenetic gametic inheritance in wild plants, this paper suggests that epigenetic differentiation between adult plant populations of H. foetidus is likely to persist across generations.

Micropropagation of Helleborus through axillary budding.

Helleborus genus, belonging to the Ranunculaceae family, has 20 species of herbaceous perennial flowering plants. The commercial exploitation of this plant is dependent on the selection and propagation of appropriate lines. High propagation rate could be accomplished by using a suitable tissue culture method enabling the rapid introduction of valuable selections in the market. However, in vitro cultivation of Helleborus is still very difficult. Thereby the development of reliable in vitro propagation procedures is crucial for future production systems. Axillary buds cultured on agar-solidified Murashige and Skoog medium supplemented with 1 mg/L benzyladenine, 0.1 mg/L ß-naphthoxyacetic acid, and 2 mg/L isopentenyl adenine develop shoots after 16 weeks of culture under 16 h light regime, 50-60 µmol/s/m(2), and 19 ± 1°C. The multiplication rate ranges from 1.4 to 2.1. However, the genotype and the number of subcultures affect the efficiency of the micropropagation process. The rooting of shoots is about 80% in solidified MS medium containing 1 mg/L 1-naphthaleneacetic acid and 3 mg/L indole-3-butyric acid. The described protocol provides information which can contribute to the commercial production of Helleborus plants.

Evaluation of reproductive barriers and realisation of interspecific hybridisations depending on genetic distances between species in the genus Helleborus.

The genus Helleborus comprises 22 species, which are allocated to six sections. H. x hybridus and H. niger, which belong to different Helleborus sections, are economically important ornamentals. Several other species with minor impact exhibit interesting features, e.g. flower size, flower colour, foliage, scent and disease resistance, which should be introgressed into H. x hybridus or H. niger through interspecific hybridisation. The aims of this study were to investigate whether and which kind of hybridisation barriers occur in crosses between Helleborus species and if they differ in their manifestations, depending on the genetic distance of the respective partners. In order to obtain interspecific hybrids despite crossing barriers, a method to overcome these barriers should be developed. Crossing barriers in Helleborus were localised as predominantly post-zygotic according to in situ pollen tube staining with aniline blue. For certain crosses, pre-zygotic barriers could also be assumed, but pollen tube growth was not totally inhibited. Therefore, embryo rescue techniques via ovule culture were established to overcome the post-zygotic barriers. Ovules were isolated from maternal plants 5-7 weeks after pollination in most cases and then cultured in vitro. Overall, 219 hybrids were successfully obtained, of which 16 were derived from inter-sectional crosses. Hybrids were verified by flow cytometry and/or by molecular DNA markers.

Quantitative high-resolution melting analysis for detecting adulterations.

Admixtures of different plant species are a common problem in raw materials for medicinal use. Two exemplary assays were developed to admixtures in Helleborus niger with high-resolution melting analysis. HRM proved to be a very sensitive tool in detecting admixtures, able to detect a ratio of 1:1000 with unknown species, and of 1:200,000 with Veratrum nigrum. The example proves the ability of HRM for quantification in multiplex PCR. The method is not limited to detecting adulterations. It can also be used to quantify a specific target by integrating a second amplicon in the assay as internal standard.

DNA-based identification of Helleborus niger by high-resolution melting analysis.

Hellbori nigri rhizoma is a drug that is difficult to distinguish from other species of the genus Helleborus. In this communication we present a DNA-based identification by high-resolution melting analysis (HRM) that is able to differentiate between Helleborus niger and other species of the genus. HRM is a very specific, time- and labour-saving method for identifying DNA sequence variations and is ideally suitable for routine PCR analysis. The HRM assay developed is specific for the genus Helleborus. This method not only detects the presence of the target species H. niger but also, to a certain extent, identifies other Helleborus species by their different melting curve shapes. Markers were developed based on the trnL-trnF intergenic spacer and on the matK sequence. For an unambiguous identification of Helleborus niger, melting curves of both markers should be used.

Conflicting selection on diaspore traits limits the evolutionary potential of seed dispersal by ants.

Conflicts of selection on diaspore traits throughout the dispersal cycle can limit the evolutionary consequences of seed dispersal. However, these conflicts have never been investigated in directed dispersal systems. We explored conflicts of selection through life stages of dispersal in the myrmecochorous herb Helleborus foetidus. Seeds are subject to two contrasting partial selective scenarios. Undispersed seeds are subject to positive directional selection on seed size characters, whereas seeds dispersed are subject to stabilizing selection for size. In both scenarios, seedling establishment determined the magnitude and direction of selection. This does not reflect ant preferences for seed size. However, total selection still depends largely on ant activity, as ants control the relative importance of each selective scenario. We advocate the use of analytical approaches combining multiplicative fitness and microenvironment-specific selection to more realistically estimate the realized selection on traits functional during several life stages. This approach may be extended to any organism dispersing offspring to different environments.