Pilar Bazaga

Epigenetic differentiation and relationship to adaptive genetic divergence in discrete populations of the violet Viola cazorlensis

*In plants, epigenetic variations based on DNA methylation are often heritable and could influence the course of evolution. Before this hypothesis can be assessed, fundamental questions about epigenetic variation remain to be addressed in a real-world context, including its magnitude, structuring within and among natural populations, and autonomy in relation to the genetic context. *Extent and patterns of cytosine methylation, and the relationship to adaptive genetic divergence between populations, were investigated for wild populations of the southern Spanish violet Viola cazorlensis (Violaceae) using the methylation-sensitive amplified polymorphism (MSAP) technique, a modification of the amplified fragment length polymorphism method (AFLP) based on the differential sensitivity of isoschizomeric restriction enzymes to site-specific cytosine methylation. *The genome of V. cazorlensis plants exhibited extensive levels of methylation, and methylation-based epigenetic variation was structured into distinct between- and within- population components. Epigenetic differentiation of populations was correlated with adaptive genetic divergence revealed by a Bayesian population-genomic analysis of AFLP data. Significant associations existed at the individual genome level between adaptive AFLP loci and the methylation state of methylation-susceptible MSAP loci. *Population-specific, divergent patterns of correlated selection on epigenetic and genetic individual variation could account for the coordinated epigenetic-genetic adaptive population differentiation revealed by this study.

Untangling individual variation in natural populations: ecological, genetic and epigenetic correlates of long-term inequality in herbivory

Individual variation in ecologically important features of organisms is a crucial element in ecology and evolution, yet disentangling its underlying causes is difficult in natural populations. We applied a genomic scan approach using amplified fragment length polymorphism (AFLP) markers to quantify the genetic basis of long‐term individual differences in herbivory by mammals at a wild population of the violet Viola cazorlensis monitored for two decades. In addition, methylation‐sensitive amplified polymorphism (MSAP) analyses were used to investigate the association between browsing damage and epigenetic characteristics of individuals, an aspect that has been not previously explored for any wild plant. Structural equation modelling was used to identify likely causal structures linking genotypes, epigenotypes and herbivory. Individuals of V. cazorlensis differed widely in the incidence of browsing mammals over the 20‐year study period. Six AFLP markers (1.6% of total) were significantly related to herbivory, accounting altogether for 44% of population‐wide variance in herbivory levels. MSAP analyses revealed considerable epigenetic variation among individuals, and differential browsing damage was significantly related to variation in multilocus epigenotypes. In addition, variation across plants in epigenetic characteristics was related to variation in several herbivory‐related AFLP markers. Statistical comparison of alternative causal models suggested that individual differences in herbivory are the outcome of a complex causal structure where genotypes and epigenotypes are interconnected and have direct and indirect effects on herbivory. Insofar as methylation states of MSAP markers influential on herbivory are transgenerationally heritable, herbivore‐driven evolutionary changes at the study population will involve correlated changes in genotypic and epigenotypic distributions.

Species Richness of Yeast Communities in Floral Nectar of Southern Spanish Plants

Floral nectar of insect-pollinated plants often contains dense yeast populations, yet little quantitative information exists on patterns and magnitude of species richness of nectar-dwelling yeasts in natural plant communities. This study evaluates yeast species richness at both the plant community and plant species levels in a montane forest area in southern Spain, and also explores possible correlations between the incidence of different yeast species in nectar and their reported tolerance to high sugar concentrations, and between yeast diversity and pollinator composition. Yeast species occurring in a total of 128 field-collected nectar samples from 24 plant species were identified by sequencing the D1/D2 domain of the large subunit rDNA, and rarefaction-based analyses were used to estimate yeast species richness at the plant community and plant species levels, using nectar drops as elemental sampling units. Individual nectar samples were generally characterized by very low species richness (1.2 yeast species/sample, on average), with the ascomycetous Metschnikowia reukaufii and Metschnikowia gruessii accounting altogether for 84.7% of the 216 isolates identified. Other yeasts recorded included species in the genera Aureobasidium, Rhodotorula, Cryptococcus, Sporobolomyces, and Lecythophora. The shapes and slopes of observed richness accumulation curves were quite similar for the nectar drop and plant species approaches, but the two approaches yielded different expected richness estimates. Expected richness was higher for plant species-based than for nectar drop-based analyses, showing that the coverage of nectar yeast species occurring in the region would be improved by sampling additional host plant species. A significant correlation was found between incidence of yeast species in nectar and their reported ability to grow in a medium containing 50% glucose. Neither diversity nor incidence of yeasts was correlated with pollinator composition across plant species.

Inhospitable sweetness: nectar filtering of pollinator-borne inocula leads to impoverished, phylogenetically clustered yeast communities

Identifying the rules and mechanisms that determine the composition and diversity of naturally co-occurring species assemblages is a central topic in community ecology. Although micro-organisms represent the ‘unseen majority’ of species, individuals and biomass in many ecosystems and play pivotal roles in community development and function, the study of the factors influencing the assembly of microbial communities has lagged behind that of plant and animal communities. In this paper, we investigate experimentally the mechanisms accounting for the low species richness of yeast communities inhabiting the nectar of the bumble-bee-pollinated Helleborus foetidus (Ranunculaceae), and explore the relationships between community assembly rules and phylogenetic relatedness. By comparing yeast communities on the glossae of foraging bumble-bees (the potential species pool) with those eventually establishing in virgin nectar probed with bee glossae (the realized community), we address the questions: (i) does nectar filter yeast inocula, so that the communities eventually established there are not random subsamples of species on bumble-bee glossae? and (ii) do yeast communities establishing in H. foetidus nectar exhibit some phylogenetic bias relative to the species pool on bumble-bee glossae? Results show that nectar filtering leads to species-poor, phylogenetically clustered yeast communities that are a predictable subset of pollinator-borne inocula. Such strong habitat filtering is probably due to H. foetidus nectar representing a harsh environment for most yeasts, where only a few phylogenetically related nectar specialists physiologically endowed to tolerate a combination of high osmotic pressure and fungicidal compounds are able to develop.

Jack of all nectars, master of most: DNA methylation and the epigenetic basis of niche width in a flower-living yeast

In addition to genetic differences between individuals as a result of nucleotide sequence variation, epigenetic changes that occur as a result of DNA methylation may also contribute to population niche width by enhancing phenotypic plasticity, although this intriguing possibility remains essentially untested. Using the nectar‐living yeast Metschnikowia reukaufii as study subject, we examine the hypothesis that changes in genome‐wide DNA methylation patterns underlie the ability of this fugitive species to exploit a broad resource range in its heterogeneous and patchy environment. Data on floral nectar characteristics and their use by M. reukaufii in the wild were combined with laboratory experiments and methylation‐sensitive amplified polymorphism (MSAP) analyses designed to detect epigenetic responses of single genotypes to variations in sugar environment that mimicked those occurring naturally in nectar. M. reukaufii exploited a broad range of resources, occurring in nectar of 48% of species and 52% of families surveyed, and its host plants exhibited broad intra‐ and interspecific variation in sugar‐related nectar features. Under experimental conditions, sugar composition, sugar concentration and their interaction significantly influenced the mean probability of MSAP markers experiencing a transition from unmethylated to methylated state. Alterations in methylation status were not random but predictably associated with certain markers. The methylation inhibitor 5‐azacytidine (5‐AzaC) had strong inhibitory effects on M. reukaufii proliferation in sugar‐containing media, and a direct relationship existed across sugar × concentration experimental levels linking inhibitory effect of 5‐AzaC and mean per‐marker probability of genome‐wide methylation. Environmentally induced DNA methylation polymorphisms allowed genotypes to grow successfully in extreme sugar environments, and the broad population niche width of M. reukaufii was largely made possible by epigenetic changes enabling genotype plasticity in resource use.

Population-genomic approach reveals adaptive floral divergence in discrete populations of a hawk moth- pollinated violet

Local adaptation to contrasting biotic or abiotic environments is an important evolutionary step that presumably precedes floral diversification at the species level, yet few studies have demonstrated the adaptive nature of intraspecific floral divergence in wild plant populations. We combine a population‐genomic approach with phenotypic information on floral traits to examine whether the differentiation in metric floral traits exhibited by 14 populations of the southern Spanish hawk moth‐pollinated violet Viola cazorlensis reflects adaptive divergence. Screening of many amplified fragment length polymorphism (AFLP) loci using a multiple‐marker‐based neutrality test identified nine outlier loci (2.6% of the total) that departed from neutral expectations and were potentially under selection. Generalized analysis of molecular variance revealed significant relationships between genetic distance and population divergence in three floral traits when genetic distance was based on outlier loci, but not when it was based on neutral ones. Population means of floral traits were closely correlated with population scores on the first principal coordinate axis of the genetic distance matrix using outlier loci, and with the allelic frequencies of four of the outlier loci. Results strongly support the adaptive nature of intraspecific floral divergence exhibited by V. cazorlensis and illustrate the potential of genome scans to identify instances of adaptive divergence when used in combination with phenotypic information.

Permanent Genetic Resources added to the Molecular Ecology Resources Database 1 February 2010-31 March 2010.

This article documents the addition of 142 microsatellite marker loci to the Molecular Ecology Resources database. Loci were developed for the following species: Agriophyllum squarrosum, Amazilia cyanocephala, Batillaria attramentaria, Fungal strain CTeY1 (Ascomycota), Gadopsis marmoratus, Juniperus phoenicea subsp. turbinata, Liriomyza sativae, Lupinus polyphyllus, Metschnikowia reukaufii, Puccinia striiformis and Xylocopa grisescens. These loci were cross‐tested on the following species: Amazilia beryllina, Amazilia candida, Amazilia rutila, Amazilia tzacatl, Amazilia violiceps, Amazilia yucatanensis, Campylopterus curvipennis, Cynanthus sordidus, Hylocharis leucotis, Juniperus brevifolia, Juniperus cedrus, Juniperus osteosperma, Juniperus oxycedrus, Juniperus thurifera, Liriomyza bryoniae, Liriomyza chinensis, Liriomyza huidobrensis and Liriomyza trifolii.

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.

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.

Quantifying the genetic component of phenotypic variation in unpedigreed wild plants: tailoring genomic scan for within-population use

The study of adaptive genetic variation in natural populations is central to evolutionary biology. Quantitative genetics methods, however, are hardly applicable to long‐lived organisms, and current knowledge on adaptive genetic variation in wild plants mostly refers to annuals and short‐lived perennials. Studies on long‐lived species are essential to explore possible life‐history correlates of genetic variation, selection, and trait heritability. In this paper, we propose a method based on molecular markers to quantify the genetic basis of individual phenotypic differences in wild plants under natural conditions. Rather than focusing on inferring individual relatedness to estimate the heritability of phenotypic traits, we directly estimate the proportion of observed phenotypic variance that is statistically accounted for by genotypic differences between individuals. This is achieved by (i) identifying loci that are correlated across individuals with the phenotypic trait of interest by means of an amplified fragment length polymorphism (AFLP)‐based explorative genomic scan, and (ii) fitting multiple regression and linear random effect models to estimate the effects of genotype, environment and genotype × environment on phenotypes. We apply this method to estimate genotypic and environmental effects on cumulative maternal fecundity in a wild population of the long‐lived Viola cazorlensis monitored for 20 years. Results show that between 56–63% (depending on estimation method) of phenotypic variance in fecundity is accounted for by genotypic differences in 11 AFLP loci that are significantly related to fecundity. Genotype × environment effects accounted for 38% of fecundity variance, which may help to explain the unexpectedly high levels of genetic variance for fecundity found.