Ricardo Pérez

INVISIBLE FLORAL LARCENIES: MICROBIAL COMMUNITIES DEGRADE FLORAL NECTAR OF BUMBLE BEE-POLLINATED PLANTS

The ecology of nectarivorous microbial communities remains virtually unknown, which precludes elucidating whether these organisms play some role in plant-pollinator mutualisms beyond minor commensalism. We simultaneously assessed microbial abundance and nectar composition at the individual nectary level in flowers of three southern Spanish bumble bee-pollinated plants (Helleborus foetidus, Aquilegia vulgaris, and Aquilegia pyrenaica cazorlensis). Yeasts were frequent and abundant in nectar of all species, and variation in yeast density was correlated with drastic changes in nectar sugar concentration and composition. Yeast communities built up in nectar from early to late floral stages, at which time all nectaries contained yeasts, often at densities between 10(4) and 10(5) cells/mm3. Total sugar concentration and percentage sucrose declined, and percentage fructose increased, with increasing density of yeast cells in nectar. Among-nectary variation in microbial density accounted for 65% (H. foetidus and A. vulgaris) and 35% (A. p. cazorlensis) of intraspecific variance in nectar sugar composition, and 60% (H. foetidus) and 38% (A. vulgaris) of variance in nectar concentration. Our results provide compelling evidence that nectar microbial communities can have detrimental effects on plants and/or pollinators via extensive nectar degradation and also call for a more careful interpretation of nectar traits in the future, if uncontrolled for yeasts.

Extreme intraplant variation in nectar sugar composition in an insect-pollinated perennial herb

Variation in nectar chemistry among plants, flowers, or individual nectaries of a given species has been only rarely explored, yet it is an essential aspect to our understanding of how pollinator-mediated selection might act on nectar traits. This paper describes variation in nectar sugar composition in a population of the perennial herb Helleborus foetidus (Ranunculaceae) and dissects it into components due to variation among plants, flowers of the same plant, and nectaries of the same flower. The proportions of sucrose, glucose, and fructose in single-nectary nectar samples collected at two times in the flowering season were determined using high performance liquid chromatography (HPLC). Sugar composition varied extensively among nectaries, and nearly all combinations of individual sugars were recorded. Population-wide variance was mainly accounted for by variation among flowers of the same plant (56% of total), nectaries of the same flower (30%), and only minimally by differences among plants (14%). In absolute terms, intraplant variation was similar to or greater than that ordinarily reported in interspecific comparisons. Results suggest that the prevailing notion of intraspecific constancy in nectar sugar composition may be unwarranted for some species and that more elaborate nectar sampling designs are required to detect and appropriately account for extensive within-plant variance. Within-plant variation in nectar sugar composition will limit the ability of pollinators to exert selection on nectar chemistry in H. foetidus and may be advantageous to plants by reducing the number of flowers visited per foraging bout by variance-sensitive, risk-averse pollinators.

Pollinator foraging modifies nectar sugar composition in Helleborus foetidus (Ranunculaceae):An experimental test

We experimentally tested the hypothesis that the extensive within-plant variation of nectar sugar composition in Helleborus foetidus (Ranunculaceae) and other species results from differences between flowers and nectaries in pollinator visitation history. Experiments were conducted to mimic single-nectary visits by wild-caught individuals of the main bee pollinators of H. foetidus, which were assayed for their capacity to modify the sugar composition of natural and artificial nectar. Experimental nectar probing with bee mouthparts induced extensive changes in proportional sugar composition 48 h after treatment, and bee taxa differed widely in their effects. Nectar probing by Andrena, medium-sized Anthophoridae, Apis mellifera, and Lasioglossum had no subsequent effects on nectar sugar composition, while probing by Bombus terrestris and B. pratorum induced an extensive reduction in percentage sucrose, a marked increase in percentage fructose, and a slight increase in percentage glucose. Results support the hypothesis that stochastic variations among flowers or nectaries in the taxonomic identity of recent visitors and their relative visitation frequencies may eventually generate very small-scale mosaics in nectar sugar composition. Changes in nectar sugar composition following bumblebee probing may be the consequence of nectar contamination with pollinator-borne nectarivorous yeasts.

Global DNA cytosine methylation as an evolving trait: phylogenetic signal and correlated evolution with genome size in angiosperms

DNA cytosine methylation is a widespread epigenetic mechanism in eukaryotes, and plant genomes commonly are densely methylated. Genomic methylation can be associated with functional consequences such as mutational events, genomic instability or altered gene expression, but little is known on interspecific variation in global cytosine methylation in plants. In this paper, we compare global cytosine methylation estimates obtained by HPLC and use a phylogenetically-informed analytical approach to test for significance of evolutionary signatures of this trait across 54 angiosperm species in 25 families. We evaluate whether interspecific variation in global cytosine methylation is statistically related to phylogenetic distance and also whether it is evolutionarily correlated with genome size (C-value). Global cytosine methylation varied widely between species, ranging between 5.3% (Arabidopsis) and 39.2% (Narcissus). Differences between species were related to their evolutionary trajectories, as denoted by the strong phylogenetic signal underlying interspecific variation. Global cytosine methylation and genome size were evolutionarily correlated, as revealed by the significant relationship between the corresponding phylogenetically independent contrasts. On average, a ten-fold increase in genome size entailed an increase of about 10% in global cytosine methylation. Results show that global cytosine methylation is an evolving trait in angiosperms whose evolutionary trajectory is significantly linked to changes in genome size, and suggest that the evolutionary implications of epigenetic mechanisms are likely to vary between plant lineages.

A concise synthesis of ortho-condensed oxane-oxene, oxepene, oxocene and oxonene ring systems

Abstract An efficient strategy for the synthesis of trans -fused oxabicyclic systems involving thioannulation followed by a Ramberg-Backlund olefination as the key step is described.

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.

Synthesis and biological studies of flexible brevetoxin/ciguatoxin models with marked conformational preference

Abstract A comparison of the more active polyether toxins which are selective activators of voltage-sensitive sodium channels (VSSC), indicate that these molecules are mostly flat, with a hinge part around the middle of the molecules and a large curvature at one of the ends. Assuming that the receptor is topographically complementary to the active molecules, from the result reported here we could conclude, that the specific requirements of the receptor region can be achieved by synthetic polyether models based on exclusive participation of oxane/oxepane moieties. A new convergent approach to give oxepene rings via double reduction of methyl diacetals is explored. In searching for biological models to further characterize Na+ channels, our studies show that different voltage-dependent Na+ channels are expressed in the rat uterus and activated by brevetoxin-B. However, selected compound models synthesized in this work, failed to inhibit or activate Na+ channel function.

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

UNLABELLEDn•nnnPREMISE OF THE STUDYnFew 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.•nnnMETHODSnWe 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.•nnnKEY RESULTSnAbout 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.•nnnCONCLUSIONSnThese 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.

GENDER DIMORPHISM AND ALTITUDINAL VARIATION OF SECONDARY COMPOUNDS IN LEAVES OF THE GYNODIOECIOUS SHRUB Daphne laureola

In this article, we analyzed the concentration of coumarins in leaves of female and hermaphrodite individuals of the gynodioecious shrub Daphne laureola, along an elevational gradient in southern Spain. Combining HPLC and NMR techniques, we identified three different glycosides of 7-methoxy-coumarin in leaves of this species. Total coumarin concentration averaged between 60 and 120 mg/g dry weight for mature summer leaves of D. laureola growing at six different populations. As predicted by optimal theory, females tended to have a higher concentration of coumarins than hermaphrodites, thus upholding the idea that male reproductive function is costly for hermaphrodites. Furthermore, concentrations in females but not hermaphrodites were positively correlated with increasing population altitude, and the magnitude of gender divergence in coumarin concentration varied among populations, suggesting that the cost of the male function may be context dependent. To our knowledge, this is the first evidence of gender differences in chemical defenses of a gynodioecious species in the field.

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.xa0broteri 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.