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.

Physiology, gene expression, and epiphenotype of two Dianthus broteri polyploid cytotypes under temperature stress.

Increasing evidence supports a major role for abiotic stress response in the success of plant polyploids, which usually thrive in harsh environments. However, understanding the ecophysiology of polyploids is challenging due to interactions between genome doubling and natural selection. Here, we investigated physiological responses, gene expression, and the epiphenotype of two related Dianthus broteri cytotypes-with different genome duplications (4× and 12×) and evolutionary trajectories-to short extreme temperature events (42/28 °C and 9/5 °C). The 12× cytotype showed higher expression of stress-responsive genes (SWEET1, PP2C16, AI5L3, and ATHB7) and enhanced gas exchange compared with 4×. Under heat stress, both ploidies had greatly impaired physiological performance and altered gene expression, with reduced cytosine methylation. However, the 12× cytotype exhibited remarkable physiological tolerance (maintaining gas exchange and water status via greater photochemical integrity and probably enhanced water storage) while down-regulating PP2C16 expression. Conversely, 4× D. broteri was susceptible to thermal stress despite prioritizing water conservation, showing signs of non-stomatal photosynthetic limitations and irreversible photochemical damage. This cytotype also presented gene-specific expression patterns under heat, up-regulating ATHB7. These findings provide insights into divergent stress response strategies and physiological resistance resulting from polyploidy, highlighting its widespread influence on plant function.

Herbivory induced methylation changes in the Lombardy poplar: A comparison of results obtained by epiGBS and WGBS.

DNA cytosine methylation is an epigenetic mechanism involved in regulation of plant responses to biotic and abiotic stress and its ability to change can vary with the sequence context in which a cytosine appears (CpG, CHG, CHH, where H = Adenine, Thymine, Cytosine). Quantification of DNA methylation in model plant species is frequently addressed by Whole Genome Bisulfite Sequencing (WGBS), which requires a good-quality reference genome. Reduced Representation Bisulfite Sequencing (RRBS) is a cost-effective potential alternative for ecological research with limited genomic resources and large experimental designs. In this study, we provide for the first time a comprehensive comparison between the outputs of RRBS and WGBS to characterize DNA methylation changes in response to a given environmental factor. In particular, we used epiGBS (recently optimized RRBS) and WGBS to assess global and sequence-specific differential methylation after insect and artificial herbivory in clones of Populus nigra cv. 'italica'. We found that, after any of the two herbivory treatments, global methylation percentage increased in CHH, and the shift was detected as statistically significant only by epiGBS. As regards to loci-specific differential methylation induced by herbivory (cytosines in epiGBS and regions in WGBS), both techniques indicated the specificity of the response elicited by insect and artificial herbivory, together with higher frequency of hypo-methylation in CpG and hyper-methylation in CHH. Methylation changes were mainly found in gene bodies and intergenic regions when present at CpG and CHG and in transposable elements and intergenic regions at CHH context. Thus, epiGBS succeeded to characterize global, genome-wide methylation changes in response to herbivory in the Lombardy poplar. Our results support that epiGBS could be particularly useful in large experimental designs aimed to explore epigenetic changes of non-model plant species in response to multiple environmental factors.

Body mass decline in a Mediterranean community of solitary bees supports the size shrinking effect of climatic warming.

The long-known, widely documented inverse relationship between body size and environmental temperature ("temperature-size rule") has recently led to predictions of body size decline following current climatic warming ("size shrinking effect"). For keystone pollinators such as wild bees, body shrinking in response to warming can have significant effects on pollination processes but there is still little direct evidence of the phenomenon because adequate tests require controlling for confounding factors linked to climate change (e.g., habitat change). This paper assesses the shrinking effect in a community of solitary bees from well-preserved habitats in the core of a large nature reserve experiencing climatic warming without disturbances or habitat changes. Long-term variation in mean body mass was evaluated using data from 1704 individual bees (137 species, 27 genera, 6 families) sampled over 1990-2023. Climate warmed at a fast rate during this period, annual mean of daily maximum temperature increasing 0.069°C/year on average during 2000-2020. Changes in bee body mass verified expectations from the size shrinking effect. The mean individual body mass of the community of solitary bees declined significantly, irrespective of whether the analysis referred to the full species sample or only to the subset of species that were sampled in both the old (1990-1997) and recent (2022-2023) periods. On average, body mass declined ~0.7%·year-1 , or an estimated average cumulative reduction of ~20 mg per individual bee from 1990 to 2023. Proportional size reduction was greatest among large-bodied species, ranging from around -0.6%·year-1 for the smallest species to -0.9%·year-1 for the largest ones. Declining rate was steeper for cavity-nesting than ground-nesting species. The pollination and mating systems of bee-pollinated plants in the study region are probably undergoing significant alterations as a consequence of supra-annual decline in bee body mass.

Heavy metal tolerance in Scopelophila cataractae: Transcriptomic and epigenetic datasets.

Studying how different plant groups deal with heavy metal exposure is crucial to improve our understanding of the diversity of molecular mechanisms involved in plant stress response. Here, we used RNA sequencing (RNA-seq) and epigenotyping by sequencing (epiGBS) to assess gene expression and DNA methylation changes respectively in plants from four populations of the metallophyte moss Scopelophila cataractae treated with Cd or Cu in the laboratory. We built RNA-seq and epiGBS sequencing libraries from control and treated samples from each population and sequenced them using Illumina HiSeq 3000 (PE-150 bp) and Illumina HiSeq X-Ten System (PE-150 bp) respectively. For the RNA-seq data, we performed a read quality filter, mapped the reads to the de novo transcriptome created with Trinity, and estimated transcript abundance for each sample. For the epiGBS data, we used a custom pipeline (https://doi.org/10.5281/zenodo.7040291) to map the reads to a de novo reference genome and performed strand-specific nucleotide (single nucleotide polymorphisms, SNPs) and methylation (single cytosine methylation polymorphisms, SMPs) variant calling. We filtered out SNPs and SMPs with low coverage within (positions with <10 sequencing reads per sample) and across samples (positions with poor representation on the full set of samples). Finally, we performed pairwise comparisons between control and treated samples from each population and identified differentially expressed genes and differentially methylated cytosines associated to heavy metal exposure. We payed particular attention to the different responses of the more and the less tolerant populations of S. cataractae. These datasets could contribute to future comparative studies of abiotic stress response across plant groups.

Plant epigenetics: phenotypic and functional diversity beyond the DNA sequence.

Phenotypic variation determines the capacity of plants to adapt to changing environments and to colonize new habitats. Deciphering the mechanisms contributing to plant phenotypic variation and their effects on plant ecological interactions and evolutionary dynamics is thus central to all biological disciplines. In the past few decades, research on plant epigenetics is showing that (1) epigenetic variation is related to phenotypic variation and that some epigenetic marks drive major phenotypic changes in plants; (2) plant epigenomes are highly diverse, dynamic, and can respond rapidly to a variety of biotic and abiotic stimuli; (3) epigenetic variation can respond to selection and therefore play a role in adaptive evolution. Yet, current information in terms of species, geographic ranges, and ecological contexts analyzed so far is too limited to allow for generalizations about the relevance of epigenetic regulation in phenotypic innovation and plant adaptation across taxa. In this report, we contextualize the potential role of the epigenome in plant adaptation to the environment and describe the latest research in this field presented during the symposium "Plant epigenetics: phenotypic and functional diversity beyond the DNA sequence" held within the Botany 2020 conference framework in summer 2020.

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.

Comparative genetic and epigenetic diversity in pairs of sympatric, closely related plants with contrasting distribution ranges in south-eastern Iberian mountains.

Genetic diversity defines the evolutionary potential of a species, yet mounting evidence suggests that epigenetic diversity could also contribute to adaptation. Elucidating the complex interplay between genetic and epigenetic variation in wild populations remains a challenge for evolutionary biologists, and the intriguing possibility that epigenetic diversity could compensate for the loss of genetic diversity is one aspect that remains basically unexplored in wild plants. This hypothesis is addressed in this paper by comparing the extent and patterns of genetic and epigenetic diversity of phylogenetically closely related but ecologically disparate species. Seven pairs of congeneric species from Cazorla mountains in south-eastern Spain were studied, each pair consisting of one endemic, restricted-range species associated to stressful environments, and one widespread species occupying more favourable habitats. The prediction was tested that endemic species should have lower genetic diversity due to population fragmentation, and higher epigenetic diversity induced by environmental stress, than their widespread congeners. Genetic (DNA sequence variants) and epigenetic (DNA cytosine methylation variants) diversities and their possible co-variation were assessed in three populations of each focal species using amplified fragment length polymorphism (AFLP) and methylation-sensitive AFLP (MSAP). All species and populations exhibited moderate to high levels of genetic polymorphism irrespective of their ecological characteristics. Epigenetic diversity was greater than genetic diversity in all cases. Only in endemic species were the two variables positively related, but the difference between epigenetic and genetic diversity was greater at populations with low genetic polymorphism. Results revealed that the relationship between genetic and epigenetic diversity can be more complex than envisaged by the simple hypothesis addressed in this study, and highlight the need of additional research on the actual role of epigenetic variation as a source of phenotypic diversity before a realistic understanding of the evolutionary relevance of epigenetic phenomena in plant adaptation can be achieved.

Pollen on stigmas as proxies of pollinator competition and facilitation: complexities, caveats and future directions.

BACKGROUND: Pollen transfer via animals is necessary for reproduction by ~80 % of flowering plants, and most of these plants live in multispecies communities where they can share pollinators. While diffuse plant-pollinator interactions are increasingly recognized as the rule rather than the exception, their fitness consequences cannot be deduced from flower visitation alone, so other proxies, functionally closer to seed production and amenable for use in a broad variety of diverse communities, are necessary. SCOPE: We conceptually summarize how the study of pollen on stigmas of spent flowers can reflect key drivers and functional aspects of the plant-pollinator interaction (e.g. competition, facilitation or commensalism). We critically evaluate how variable visitation rates and other factors (pollinator pool and floral avoidance) can give rise to different relationships between heterospecific pollen and (1) conspecific pollen on the stigma and (2) conspecific tubes/grain in the style, revealing the complexity of potential interpretations. We advise on best practices for using these proxies, noting the assumptions and caveats involved in their use, and explicate what additional data are required to verify interpretation of given patterns. CONCLUSIONS: We conclude that characterizing pollen on stigmas of spent flowers provides an attainable indirect measure of pollination interactions, but given the complex processes of pollen transfer that generate patterns of conspecific-heterospecific pollen on stigmas these cannot alone determine whether competition or facilitation are the underlying drivers. Thus, functional tests are also needed to validate these hypotheses.

The role of alien species on plant-floral visitor network structure in invaded communities.

The interactions between pairs of native and alien plants via shared use of pollinators have been widely studied. Community level studies however, are necessary in order to fully understand the factors and mechanisms that facilitate successful plant invasion, but these are still scarce. Specifically, few community level studies have considered how differences in invasion level (alien flower abundance), and degree of floral trait similarity between native and invasive species, mediate effects on native plant-pollinator communities. Here, we evaluated the role of alien species on overall plant-floral visitor network structure, and on species-level network parameters, across nine invaded coastal communities distributed along 205 km in Yucatán, México that vary in alien species richness and flower abundance. We further assessed the potential the role of alien plant species on plant-floral visitor network structure and robustness via computational simulation of native and invasive plant extinction scenarios. We did not find significant differences between native and alien species in their functional floral phenotypes or in their visitation rate and pollinator community composition in these invaded sites. Variation in the proportion of alien plant species and flower abundance across sites did not influence plant-pollinator network structure. Species-level network parameters (i.e., normalized degree and nestedness contribution) did not differ between native and alien species. Furthermore, our simulation analyses revealed that alien species are functionally equivalent to native species and contribute equally to network structure and robustness. Overall, our results suggest that high levels of floral trait similarity and pollinator use overlap may help facilitate the integration of alien species into native plant-pollinator networks. As a result, alien species may also play a similar role than that of natives in the structure and stability of native plant and pollinator communities in the studied coastal sand dune ecosystem.

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.

The role of plant epigenetics in biotic interactions.

Contents Summary 731 I. Biotic interactions in the context of genetic, epigenetic and environmental diversity 731 II. Biotic interactions affect epigenetic configuration 732 III. Plant epigenetic configuration influences biotic interactions 733 IV. Epigenetic memory in the context of biotic interactions 734 V. Conclusions and future research 735 Acknowledgements 735 Author contributions 735 References 735 SUMMARY: Plants are hubs of a wide range of biotic interactions with mutualist and antagonist animals, microbes and neighboring plants. Because the quality and intensity of those relationships can change over time, a fast and reversible response to stress is required. Here, we review recent studies on the role of epigenetic factors such as DNA methylation and histone modifications in modulating plant biotic interactions, and discuss the state of knowledge regarding their potential role in memory and priming. Moreover, we provide an overview of strategies to investigate the contribution of epigenetics to environmentally induced phenotypic changes in an ecological context, highlighting possible transitions from whole-genome high-resolution analyses in plant model organisms to informative reduced representation analyses in genomically less accessible species.

Variation in sampling effort affects the observed richness of plant-plant interactions via heterospecific pollen transfer: implications for interpretation of pollen transfer networks.

PREMISE OF THE STUDY: There is growing interest in understanding plant-plant interactions via pollen transfer at the community level. Studies on the structure and spatial variability of pollen transfer networks have been valuable to this understanding. However, there is high variability in the intensity of sampling used to characterize pollen transfer interactions, which could influence network structure. To date, there is no knowledge of how sampling effort influences the richness of pollen on stigmas and thereby transfer interactions observed, nor how this may vary across species and study sites. METHODS: We use rarefaction curves on 16 species to characterize the relationship between sampling effort (number of stigmas analyzed) and the richness of pollen transfer interactions recorded. We further assess variability in this relationship among species, plant community types, and sites within a single plant community. KEY RESULTS: We show high among-species variation in the amount of sampling required to sufficiently characterize interspecific pollen transfer. We further reveal variability in the sampling effort-interaction richness relationship among different plant communities and even for the same species growing in different sites. CONCLUSIONS: The wide heterogeneity in the sampling effort required to accurately characterize pollen transfer interactions observed has the potential to influence the characterization of pollen transfer dynamics. Thus, sampling completeness should be considered in future studies to avoid overestimation of modularity and specialization in pollen transfer networks that may bias the predicted causes and expected consequences of such processes for plant-plant interactions.

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.

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.

Ecological plant epigenetics: Evidence from model and non-model species, and the way forward.

Growing evidence shows that epigenetic mechanisms contribute to complex traits, with implications across many fields of biology. In plant ecology, recent studies have attempted to merge ecological experiments with epigenetic analyses to elucidate the contribution of epigenetics to plant phenotypes, stress responses, adaptation to habitat, and range distributions. While there has been some progress in revealing the role of epigenetics in ecological processes, studies with non-model species have so far been limited to describing broad patterns based on anonymous markers of DNA methylation. In contrast, studies with model species have benefited from powerful genomic resources, which contribute to a more mechanistic understanding but have limited ecological realism. Understanding the significance of epigenetics for plant ecology requires increased transfer of knowledge and methods from model species research to genomes of evolutionarily divergent species, and examination of responses to complex natural environments at a more mechanistic level. This requires transforming genomics tools specifically for studying non-model species, which is challenging given the large and often polyploid genomes of plants. Collaboration among molecular geneticists, ecologists and bioinformaticians promises to enhance our understanding of the mutual links between genome function and ecological processes.

Epigenetic contribution to successful polyploidizations: variation in global cytosine methylation along an extensive ploidy series in Dianthus broteri (Caryophyllaceae).

Polyploidization is a significant evolutionary force in plants which involves major genomic and genetic changes, frequently regulated by epigenetic factors. We explored whether natural polyploidization in Dianthus broteri complex resulted in substantial changes in global DNA cytosine methylation associated to ploidy. Global cytosine methylation was estimated by high-performance liquid chromatography (HPLC) in 12 monocytotypic populations with different ploidies (2×, 4×, 6×, 12×) broadly distributed within D. broteri distribution range. The effects of ploidy level and local variation on methylation were assessed by generalized linear mixed models (GLMMs). Dianthus broteri exhibited a higher methylation percent (˜33%) than expected by its monoploid genome size and a large variation among study populations (range: 29.3-35.3%). Global methylation tended to increase with ploidy but did not significantly differ across levels due to increased variation within the highest-order polyploidy categories. Methylation varied more among hexaploid and dodecaploid populations, despite such cytotypes showing more restricted geographic location and increased genetic relatedness than diploids and tetraploids. In this study, we demonstrate the usefulness of an HPLC method in providing precise and genome reference-free global measure of DNA cytosine methylation, suitable to advance current knowledge of the roles of this epigenetic mechanism in polyploidization processes.

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.

Patterns of among- and within-species variation in heterospecific pollen receipt: The importance of ecological generalization.

PREMISE OF THE STUDY: Coflowering plants are at risk for receiving pollen from heterospecifics as well as conspecifics, yet evidence shows wide variation in the degree that heterospecific pollen transfer occurs. Evaluation of patterns and correlates of among- and within-species variation in heterospecific pollen (HP) receipt is key to understanding its importance for floral evolution and species coexistence; however, the rarity of deeply sampled multispecies comparisons has precluded such an evaluation. METHODS: We evaluated patterns of among- and within-species variation in HP load size and diversity in 19 species across three distinct plant communities. We assessed the importance of phenotypic specialization (floral phenotype), ecological specialization (contemporary visitor assemblage), and conspecific flower density as determinants of among-species variation. We present hypotheses for different accrual patterns of HP within species based on the evenness and quality of floral visitors and evaluated these by characterizing the relationship between conspecific pollen (CP) and HP receipt. KEY RESULTS: We found that within-species variation in HP receipt was greater than among-species and among-communities variation. Among species, ecological generalization emerged as the strongest driver of variation in HP receipt irrespective of phenotypic specialization. Within-species variation in HP load size and diversity was predicted most often from two CP-HP relationships (linear or exponentially decreasing), suggesting that two distinct types of plant-pollinator interactions prevail. CONCLUSIONS: Our results give important insights into the potential drivers of among- and within-species variation in HP receipt. They also highlight the value of explorations of patterns at the intraspecific level, which can ultimately shed light on plant-pollinator-mediated selection in diverse plant communities.