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.

Clonality, genetic diversity and support for the diversifying selection hypothesis in natural populations of a flower-living yeast.

Vast amounts of effort have been devoted to investigate patterns of genetic diversity and structuring in plants and animals, but similar information is scarce for organisms of other kingdoms. The study of the genetic structure of natural populations of wild yeasts can provide insights into the ecological and genetic correlates of clonality, and into the generality of recent hypotheses postulating that microbial populations lack the potential for genetic divergence and allopatric speciation. Ninety-one isolates of the flower-living yeast Metschnikowia gruessii from southeastern Spain were DNA fingerprinted using amplified fragment length polymorphism (AFLP) markers. Genetic diversity and structuring was investigated with band-based methods and model- and nonmodel-based clustering. Linkage disequilibrium tests were used to assess reproduction mode. Microsite-dependent, diversifying selection was tested by comparing genetic characteristics of isolates from bumble bee vectors and different floral microsites. AFLP polymorphism (91%) and genotypic diversity were very high. Genetic diversity was spatially structured, as shown by amova (Φ(st) = 0.155) and clustering. The null hypothesis of random mating was rejected, clonality seeming the prevailing reproductive mode in the populations studied. Genetic diversity of isolates declined from bumble bee mouthparts to floral microsites, and frequency of five AFLP markers varied significantly across floral microsites, thus supporting the hypothesis of diversifying selection on clonal lineages. Wild populations of clonal fungal microbes can exhibit levels of genetic diversity and spatial structuring that are not singularly different from those shown by sexually reproducing plants or animals. Microsite-dependent, divergent selection can maintain high local and regional genetic diversity in microbial populations despite extensive clonality.

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.

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 x 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 x environment effects accounted for 38% of fecundity variance, which may help to explain the unexpectedly high levels of genetic variance for fecundity found.

Adding a third dimension to the edge of a species' range: altitude and genetic structuring in mountainous landscapes.

In addition to the topographical and ecological barriers, other landscape features may also subtly influence the patterns of gene flow and spatial genetic structuring at species' borders. This paper focuses on the role played by altitudinal gradients that characterize mountainous landscapes. We formulate and test the hypothesis that when the distribution boundaries of plant species intersect mountainous landscapes, altitudinal gradients in ecological conditions may considerably enhance population subdivision and genetic structuring at the regional level. Using amplified fragment length polymorphism markers, we studied genetic diversity and differentiation in a set of 21 peripheral populations of the evergreen shrub Lavandula latifolia Med. (Labiatae) at its southernmost distribution limit in the Betic mountain ranges of southern Spain. Population size and abundance, and within-population genetic diversity, varied predictably with altitude, being highest at middle elevations and declining steadily towards both the upper and lower altitudinal distribution margins. Genetic differentiation tended to follow the opposite trend. These altitudinal patterns result from variation with elevation in the relative influence of gene flow and drift on the distribution of genetic variation. Genetic drift prevails around the upper and lower altitudinal limits, whereas a situation closer to a drift-gene flow equilibrium exists at the center of the altitudinal distribution. Altitudinal variation in the relative influences of gene flow and drift appears as an essential element in the interpretation of regional genetic structuring of L. latifolia at its mountainous distribution edge, and a factor which may influence the evolutionary potential of peripheral populations and the likelihood of local adaptation.

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.