Plant Phenotypes as Distributions: Johannsen's Beans Revisited.

AbstractIn the early twentieth century, Wilhelm Johannsen's breeding experiments on pure lines of beans provided empirical support for his groundbreaking distinction between phenotype and genotype, the foundation stone of classical genetics. In contrast with the controversial history of the genotype concept, the notion of phenotype has remained essentially unrevised since then. The application of the Johannsenian concept of phenotype to modularly built, nonunitary plants, however, needs reexamination. In the first part of this article it is shown that Johannsen's appealing solution for dealing with the multiplicity of nonidentical organs produced by plant individuals (representing individual plant phenotypes by arithmetic means), which has persisted to this day, reflected his intellectual commitment to nineteenth-century typological thinking. Revisitation of Johannsen's results using current statistical tools upholds his major conclusion about the nature of heredity but at the same time falsifies two important ancillary conclusions of his experiments-namely, the alleged homogeneity of pure lines (genotypes) regarding seed weight variability and the lack of transgenerational effects of within-line (within-genotype) seed weight variation. The canonical notion of individual plant phenotypes as arithmetic means should therefore be superseded by a concept of phenotype as a dual property, consisting of central tendency and variability components of organ trait distribution. Phenotype duality offers a unifying framework applicable to all nonunitary organisms.

Comparative epigenetic and genetic spatial structure in Mediterranean mountain plants: a multispecies study.

Changes in epigenetic states can allow individuals to cope with environmental changes. If such changes are heritable, this may lead to epigenetic adaptation. Thus, it is likely that in sessile organisms such as plants, part of the spatial epigenetic variation found across individuals will reflect the environmental heterogeneity within populations. The departure of the spatial epigenetic structure from the baseline genetic variation can help in understanding the value of epigenetic regulation in species with different breadth of optimal environmental requirements. Here, we hypothesise that in plants with narrow environmental requirements, epigenetic variability should be less structured in space given the lower variability in suitable environmental conditions. We performed a multispecies study that considered seven pairs of congeneric plant species, each encompassing a narrow endemic with habitat specialisation and a widespread species. In three populations per species we used AFLP and methylation-sensitive AFLP markers to characterise the spatial genetic and epigenetic structures. Narrow endemics showed a significantly lower epigenetic than genetic differentiation between populations. Within populations, epigenetic variation was less spatially structured than genetic variation, mainly in narrow endemics. In these species, structural equation models revealed that such pattern was associated to a lack of correlation between epigenetic and genetic information. Altogether, these results show a greater decoupling of the spatial epigenetic variation from the baseline spatial genetic pattern in endemic species. These findings highlight the value of studying genetic and epigenetic spatial variation to better understand habitat specialisation in plants.

Sugar Concentration, Nitrogen Availability, and Phylogenetic Factors Determine the Ability of Acinetobacter spp. and Rosenbergiella spp. to Grow in Floral Nectar.

The floral nectar of angiosperms harbors a variety of microorganisms that depend predominantly on animal visitors for their dispersal. Although some members of the genus Acinetobacter and all currently known species of Rosenbergiella are thought to be adapted to thrive in nectar, there is limited information about the response of these bacteria to variation in the chemical characteristics of floral nectar. We investigated the growth performance of a diverse collection of Acinetobacter (n = 43) and Rosenbergiella (n = 45) isolates obtained from floral nectar and the digestive tract of flower-visiting bees in a set of 12 artificial nectars differing in sugar content (15% w/v or 50% w/v), nitrogen content (3.48/1.67 ppm or 348/167 ppm of total nitrogen/amino nitrogen), and sugar composition (only sucrose, 1/3 sucrose + 1/3 glucose + 1/3 fructose, or 1/2 glucose + 1/2 fructose). Growth was only observed in four of the 12 artificial nectars. Those containing elevated sugar concentration (50% w/v) and low nitrogen content (3.48/1.67 ppm) were limiting for bacterial growth. Furthermore, phylogenetic analyses revealed that the ability of the bacteria to grow in different types of nectar is highly conserved between closely related isolates and genotypes, but this conservatism rapidly vanishes deeper in phylogeny. Overall, these results demonstrate that the ability of Acinetobacter spp. and Rosenbergiella spp. to grow in floral nectar largely depends on nectar chemistry and bacterial phylogeny.

Contrasting effects of nectar yeasts on the reproduction of Mediterranean plant species.

PREMISE: Yeasts are often present in floral nectar and can influence plant fitness directly (independently of pollinators) or indirectly by influencing pollinator visitation and behavior. However, few studies have assessed the effect of nectar yeasts on plant reproductive success or compared effects across different plant species, limiting our understanding of the relative impact of direct vs. indirect effects. METHODS: We inoculated the nectar of six plant species in the field with the cosmopolitan yeast Metschnikowia reukaufii to analyze the direct and indirect effects on female reproductive success over 2 years. The pollinator assemblage for each species was recorded during both flowering years. RESULTS: Direct yeast effects on female fecundity were statistically nonsignificant for all plant species. There were significant indirect, pollinator-mediated effects on fruit production and seed mass for the two species pollinated almost exclusively by bumblebees or hawkmoths, with the direction of the effects differing for the quantity- and quality-related fitness components. There were no consistent effects of the yeast on maternal fecundity for any of the species with diverse pollinator assemblages. CONCLUSIONS: Effects of M. reukaufii on plant reproduction ranged from negative to neutral or positive depending on the plant species. The among-species variation in the indirect effects of nectar yeasts on plant pollination could reflect variation in the pollinator community, the specific microbes colonizing the nectar, and the order of microbial infection (priority effects), determining potential species interactions. Elucidating the nature of these multitrophic plant-pollinator-microbe interactions is important to understand complex processes underlying plant pollination.

Lifetime genealogical divergence within plants leads to epigenetic mosaicism in the shrub Lavandula latifolia (Lamiaceae).

Epigenetic mosaicism is a possible source of within-plant phenotypic heterogeneity, yet its frequency and developmental origin remain unexplored. This study examines whether extant epigenetic heterogeneity within Lavandula latifolia (Lamiaceae) shrubs reflects recent epigenetic modifications experienced independently by different plant parts or, alternatively, it is the cumulative outcome of a steady lifetime process. Leaf samples from different architectural modules (branch tips) were collected from three L. latifolia plants and characterized epigenetically by global DNA cytosine methylation and methylation state of methylation-sensitive amplified fragment-length polymorphism (MS-AFLP) markers. Epigenetic characteristics of modules were then assembled with information on the branching history of plants. Methods borrowed from phylogenetic research were used to assess genealogical signal of extant epigenetic variation and reconstruct within-plant genealogical trajectory of epigenetic traits. Plants were epigenetically heterogeneous, as shown by differences among modules in global DNA methylation and variation in the methylation states of 6 to 8% of MS-AFLP markers. All epigenetic features exhibited significant genealogical signal within plants. Events of epigenetic divergence occurred throughout the lifespan of individuals and were subsequently propagated by branch divisions. Internal epigenetic diversification of L. latifolia individuals took place steadily during their development, a process which eventually led to persistent epigenetic mosaicism.

Nitrogen Assimilation Varies Among Clades of Nectar- and Insect-Associated Acinetobacters.

Floral nectar is commonly colonized by yeasts and bacteria, whose growth largely depends on their capacity to assimilate nutrient resources, withstand high osmotic pressures, and cope with unbalanced carbon-to-nitrogen ratios. Although the basis of the ecological success of these microbes in the harsh environment of nectar is still poorly understood, it is reasonable to assume that they are efficient nitrogen scavengers that can consume a wide range of nitrogen sources in nectar. Furthermore, it can be hypothesized that phylogenetically closely related strains have more similar phenotypic characteristics than distant relatives. We tested these hypotheses by investigating the growth performance on different nitrogen-rich substrates of a collection of 82 acinetobacters isolated from nectar and honeybees, representing members of five species (Acinetobacter nectaris, A. boissieri, A. apis, and the recently described taxa A. bareti and A. pollinis). We also analyzed possible links between growth performance and phylogenetic affiliation of the isolates, while taking into account their geographical origin. Results demonstrated that the studied isolates could utilize a wide variety of nitrogen sources, including common metabolic by-products of yeasts (e.g., ammonium and urea), and that phylogenetic relatedness was associated with the variation in nitrogen assimilation among the studied acinetobacters. Finally, nutrient source and the origin (sample type and country) of isolates also predicted the ability of the acinetobacters to assimilate nitrogen-rich compounds. Overall, these results demonstrate inter-clade variation in the potential of the acinetobacters as nitrogen scavengers and suggest that nutritional dependences might influence interactions between bacteria and yeasts in floral nectar.

Gradual replacement of wild bees by honeybees in flowers of the Mediterranean Basin over the last 50 years.

Evidence for pollinator declines largely originates from mid-latitude regions in North America and Europe. Geographical heterogeneity in pollinator trends combined with geographical biases in pollinator studies can produce distorted extrapolations and limit understanding of pollinator responses to environmental changes. In contrast with the declines experienced in some well-investigated European and North American regions, honeybees seem to have increased recently in some areas of the Mediterranean Basin. Because honeybees can have negative impacts on wild bees, it was hypothesized that a biome-wide alteration in bee pollinator assemblages may be underway in the Mediterranean Basin involving a reduction in the relative number of wild bees. This hypothesis was tested using published quantitative data on bee pollinators of wild and cultivated plants from studies conducted between 1963 and 2017 in 13 countries from the European, African and Asian shores of the Mediterranean Sea. The density of honeybee colonies increased exponentially and wild bees were gradually replaced by honeybees in flowers of wild and cultivated plants. The proportion of wild bees at flowers was four times greater than that of honeybees at the beginning of the period, the proportions of both groups becoming roughly similar 50 years later. The Mediterranean Basin is a world biodiversity hotspot for wild bees and wild bee-pollinated plants, and the ubiquitous rise of honeybees to dominance as pollinators could in the long run undermine the diversity of plants and wild bees in the region.

Interspecific variation across angiosperms in global DNA methylation: phylogeny, ecology and plant features in tropical and Mediterranean communities.

The interspecific range of epigenetic variation and the degree to which differences between angiosperm species are related to geography, evolutionary history, ecological settings or species-specific traits, remain essentially unexplored. Genome-wide global DNA cytosine methylation is a tractable 'epiphenotypic' feature suitable for exploring these relationships. Global cytosine methylation was estimated in 279 species from two distant, ecologically disparate geographical regions: Mediterranean Spain and tropical México. At each region, four distinct plant communities were analyzed. Global methylation spanned a 10-fold range among species (4.8-42.2%). Interspecific differences were related to evolutionary trajectories, as denoted by a strong phylogenetic signal. Genomes of tropical species were on average less methylated than those of Mediterranean ones. Woody plants have genomes with lower methylation than perennial herbs, and genomes of widespread species were less methylated than those of species with restricted geographical distribution. The eight communities studied exhibited broad and overlapping interspecific variances in global cytosine methylation and only two of them differed in average methylation. Altogether, our broad taxonomic survey supported global methylation as a plant 'epiphenotypic' trait largely associated with species evolutionary history, genome size, range size and woodiness. Additional studies are required for better understanding the environmental components underlying local and geographical variation.

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.

Within-plant variation in seed size and inflorescence fecundity is associated with epigenetic mosaicism in the shrub Lavandula latifolia (Lamiaceae).

Background and Aims: Sub-individual variation in traits of homologous structures has multiple ecological consequences for individuals and populations. Assessing the evolutionary significance of such effects requires an improved knowledge of the mechanisms underlying within-plant phenotypic heterogeneity. The hypothesis that continuous within-plant variation in some phenotypic traits can be associated with epigenetic mosaicism was examined. Methods: Fifteen individuals of the long-lived, evergreen Mediterranean shrub Lavandula latifolia were studied. Five widely spaced 'modules', each consisting of a single inflorescence plus all its subtending basal leaves, were collected from each shrub. Genomic DNA was extracted from leaf samples and genome-wide cytosine methylation determined by reversed phase high-performance liquid chromatography (HPLC) with spectrofluorimetric detection. The number and mean mass of seeds produced were determined for each inflorescence. An assessment was made of whether (1) leaves from different modules in the same plant differed significantly in global DNA cytosine methylation, and (2) mosaicism in cytosine methylation contributed to explain variation across modules in number and size of seeds. Key Results: Leaves from different modules in the same plant differed in global DNA cytosine methylation. The magnitude of epigenetic mosaicism was substantial, as the variance in DNA methylation among modules of the same shrub was greater than the variance between individuals. Number and mean mass of seeds produced by individual inflorescences varied within plants and were quadratically related to cytosine methylation of subtending leaves, with an optimum at an intermediate methylation level (approx. 25 %). Conclusions: The results support a causal link between global cytosine methylation of leaves in a module and the size and numbers of seeds produced by the associated inflorescence. It is proposed that variation in global DNA methylation within L. latifolia shrubs may result from the concerted action of plant sectoriality and differential exposure of different plant parts to some environmental factor(s) with a capacity to induce durable epigenetic changes.

Transgenerational epigenetics: Inheritance of global cytosine methylation and methylation-related epigenetic markers in the shrub Lavandula latifolia.

PREMISE OF THE STUDY: The ecological and evolutionary significance of natural epigenetic variation (i.e., not based on DNA sequence variants) variation will depend critically on whether epigenetic states are transmitted from parents to offspring, but little is known on epigenetic inheritance in nonmodel plants. METHODS: We present a quantitative analysis of transgenerational transmission of global DNA cytosine methylation (= proportion of all genomic cytosines that are methylated) and individual epigenetic markers (= methylation status of anonymous MSAP markers) in the shrub Lavandula latifolia. Methods based on parent-offspring correlations and parental variance component estimation were applied to epigenetic features of field-growing plants ('maternal parents') and greenhouse-grown progenies. Transmission of genetic markers (AFLP) was also assessed for reference. KEY RESULTS: Maternal parents differed significantly in global DNA cytosine methylation (range = 21.7-36.7%). Greenhouse-grown maternal families differed significantly in global methylation, and their differences were significantly related to maternal origin. Methylation-sensitive amplified polymorphism (MSAP) markers exhibited significant transgenerational transmission, as denoted by significant maternal variance component of marker scores in greenhouse families and significant mother-offspring correlations of marker scores. CONCLUSIONS: Although transmission-related measurements for global methylation and MSAP markers were quantitatively lower than those for AFLP markers taken as reference, this study has revealed extensive transgenerational transmission of genome-wide global cytosine methylation and anonymous epigenetic markers in L. latifolia. Similarity of results for global cytosine methylation and epigenetic markers lends robustness to this conclusion, and stresses the value of considering both types of information in epigenetic studies of nonmodel plants.

Flowers as a reservoir of yeast diversity: description of Wickerhamiella nectarea f.a. sp. nov., and Wickerhamiella natalensis f.a. sp. nov. from South African flowers and pollinators, and transfer of related Candida species to the genus Wickerhamiella as new combinations.

Flowers offer favourable microenvironments for yeast growth, and are increasingly recognised as a rich source of novel yeast species. Independent surveys of yeasts associated with flowers and pollinators in South Africa led to the discovery of 38 strains of two new species. Physiological profiles and analysis of the internal transcribed spacer and the D1/D2 domains of the large subunit rRNA gene showed that they represent two novel species that belong to the Wickerhamiella clade. We describe the species as Wickerhamiella nectarea f.a. sp. nov. (type strain EBDCdVSA11-1T, CBS 14162T, NRRL Y-63791T) and W. natalensis f.a. sp. nov. (type strain EBDCdVSA7-1T, CBS 14161T, NRRL Y-63790T). We extend the known range of flower-associated Wickerhamiella species to South Africa and discuss the ecology and phylogenetic relationships of the clade in relation to its host species and biogeography. Examination of growth characteristics supports that the Wickerhamiella clade exhibits a high degree of evolutionary lability, and that specialisation to different niches may occur rapidly. We review the current status of floral yeast biodiversity and nectar as a reservoir of species diversity, and the importance of pollinators and biogeography. In addition, 18 species formerly assigned to the genus Candida are reassigned formally to the genus Wickerhamiella.

Comparative epigenetic and genetic spatial structure of the perennial herb Helleborus foetidus: Isolation by environment, isolation by distance, and functional trait divergence.

PREMISE OF THE STUDY: Epigenetic variation can play a role in local adaptation; thus, there should be associations among epigenetic variation, environmental variation, and functional trait variation across populations. This study examines these relationships in the perennial herb Helleborus foetidus (Ranunculaceae). METHODS: Plants from 10 subpopulations were characterized genetically (AFLP, SSR markers), epigenetically (MSAP markers), and phenotypically (20 functional traits). Habitats were characterized using six environmental variables. Isolation-by-distance (IBD) and isolation-by-environment (IBE) patterns of genetic and epigenetic divergence were assessed, as was the comparative explanatory value of geographical and environmental distance as predictors of epigenetic, genetic, and functional differentiation. KEY RESULTS: Subpopulations were differentiated genetically, epigenetically, and phenotypically. Genetic differentiation was best explained by geographical distance, while epigenetic differentiation was best explained by environmental distance. Divergence in functional traits was correlated with environmental and epigenetic distances, but not with geographical and genetic distances. CONCLUSIONS: Results are compatible with the hypothesis that epigenetic IBE and functional divergence reflected responses to environmental variation. Spatial analyses simultaneously considering epigenetic, genetic, phenotypic and environmental information provide a useful tool to evaluate the role of environmental features as drivers of natural epigenetic variation between populations.

Species coexistence in simple microbial communities: unravelling the phenotypic landscape of co-occurring Metschnikowia species in floral nectar.

Identifying the ecological processes that underlie the distribution and abundance of species in microbial communities is a central issue in microbial ecology and evolution. Classical trade-off based niche theories of resource competition predict that co-occurrence in microbial communities is more likely when the residing species show trait divergence and complementary resource use. We tested the prediction that niche differentiation explained the co-occurrence of two yeast species (Metschnikowia reukaufii and M. gruessii) in floral nectar. Assessment of the phenotypic landscape showed that both species displayed a significantly different physiological profile. Comparison of utilization profiles in single versus mixed cultures indicated that these two species did not compete for most carbon and nitrogen sources. In mixed cultures, M. reukaufii grew better in sucrose solutions and in the presence of the antimicrobial compound digitonin than when grown as pure culture. M. gruessii, on the other hand, grew better in mixed cultures in glucose and fructose solutions. Overall, these results provide clear evidence that M. reukaufii and M. gruessii frequently co-occur in nectar and that they differ in their phenotypic response to variation in environmental conditions, suggesting that niche differentiation and resource partitioning are important mechanisms contributing to species co-occurrence in nectar yeast communities.

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.

Nectar yeasts of the Metschnikowia clade are highly susceptible to azole antifungals widely used in medicine and agriculture.

The widespread use of azole antifungals in medicine and agriculture and the resulting long-persistent residues could potentially affect beneficial fungi. However, there is very little information on the tolerance of non-target environmental fungi to azoles. In this study, we assessed the susceptibility of diverse plant- and insect-associated yeasts from the Metschnikowia clade, including several ecologically important species, to widely used medical and agricultural azoles (epoxiconazole, imazalil, ketoconazole and voriconazole). A total of 120 strains from six species were tested. Minimum inhibitory concentrations (MICs) were determined by the EUCAST broth microdilution procedure after some necessary modifications were made. The majority of species tested were highly susceptible to epoxiconazole, ketoconazole and voriconazole (>95% of strains showed MICs ≤ 0.125 mg l(-1)). Most strains were also very susceptible to imazalil, although MIC values were generally higher than for the other azoles. Furthermore, certain Metschnikowia reukaufii strains displayed a 'trailing' phenotype (i.e. showed reduced but persistent growth at antifungal concentrations above the MIC), but this characteristic was dependent on test conditions. It was concluded that exposure to azoles may pose a risk for ecologically relevant yeasts from the Metschnikowia clade, and thus could potentially impinge on the tripartite interaction linking these fungi with plants and their insect pollinators.

To be or not to be better pollinated: Differences between sex morphs in marginal gynodioecious populations.

PREMISE OF THE STUDY: Changes in the pollinator communities of marginal plant populations can affect their pollination quantity or quality. Geographic variation in pollination success can alter the reproductive advantage that female plants require to persist within gynodioecious populations. Particularly valuable is determining the pollination success at the prezygotic stage in self-compatible gynodioecious species whose females do not exhibit enhanced seed production. METHODS: In core and marginal populations of Daphne laureola, we analyzed the differences between hermaphrodites and females in the proportion of flowers visited, the stigma pollen loads, and the quantity of pollen tubes in styles. We also examined the relationship between the number of pollen tubes in styles vs. the number of pollen grains on stigmas using piecewise regression and binomial generalized linear mixed models. KEY RESULTS: Pollinators deposited larger pollen loads on flowers in marginal populations. In marginal populations, female flowers received more pollinator visits and more pollen grains on their stigmas, and they had more pollen tubes in their styles than did female flowers in core populations. Both piecewise regression and binomial GLMM analyses showed that females in marginal populations had a lower proportion of grains that developed tubes than females in the core populations, which suggests decreased pollination quality. CONCLUSIONS: More efficient pollination services in marginal populations decreased the overall differences in the prezygotic pollination success between the sex morphs. Our results also suggest that pollination quality is lower in females of marginal populations, which could be counteracting the increased pollination in females in marginal populations.

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.