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DNA methylation can cause heritable phenotypic modifications in the absence of changes in DNA sequence. Environmental stresses can trigger methylation changes and this may have evolutionary consequences, even in the absence of sequence variation. However, it remains largely unknown to what extent environmentally induced methylation changes are transmitted to offspring, and whether observed methylation variation is truly independent or a downstream consequence of genetic variation between individuals. Genetically identical apomictic dandelion (Taraxacum officinale) plants were exposed to different ecological stresses, and apomictic offspring were raised in a common unstressed environment. We used methylation-sensitive amplified fragment length polymorphism markers to screen genome-wide methylation alterations triggered by stress treatments and to assess the heritability of induced changes. Various stresses, most notably chemical induction of herbivore and pathogen defenses, triggered considerable methylation variation throughout the genome. Many modifications were faithfully transmitted to offspring. Stresses caused some epigenetic divergence between treatment and controls, but also increased epigenetic variation among plants within treatments. These results show the following. First, stress-induced methylation changes are common and are mostly heritable. Second, sequence-independent, autonomous methylation variation is readily generated. This highlights the potential of epigenetic inheritance to play an independent role in evolutionary processes, which is superimposed on the system of genetic inheritance.

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.

• 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.

Ecological Epigenetics studies the relationship between epigenetic variation and ecologically relevant phenotypic variation. As molecular epigenetic mechanisms often control gene expression, even across generations, they may impact many evolutionary processes. Multiple molecular epigenetic mechanisms exist, but methylation of DNA so far has dominated the Ecological Epigenetic literature. There are several molecular techniques used to screen methylation of DNA; here, we focus on the most common technique, methylation-sensitive-AFLP (MS-AFLP), which is used to identify genome-wide methylation patterns. We review studies that used MS-AFLP to address ecological questions, that describe which taxa have been investigated, and that identify general trends in the field. We then discuss, noting the general themes, four studies across taxa that demonstrate characteristics that increase the inferences that can be made from MS-AFLP data; we suggest that future MS-AFLP studies should incorporate these methods and techniques. We then review the short-comings of MS-AFLP and suggest alternative techniques that might address some of these limitations. Finally, we make specific suggestions for future research on MS-AFLP and identify questions that are most compelling and tractable in the short term.

Background It has been pointed out that environmental factors or chemicals can cause diseases that are developmental in origin. To detect abnormal epigenetic alterations in DNA methylation, convenient and cost-effective methods are required for such research, in which multiple samples are processed simultaneously. We here present methylated site display (MSD), a unique technique for the preparation of DNA libraries. By combining it with amplified fragment length polymorphism (AFLP) analysis, we developed a new method, MSD-AFLP. Results Methylated site display libraries consist of only DNAs derived from DNA fragments that are CpG methylated at the 5′ end in the original genomic DNA sample. To test the effectiveness of this method, CpG methylation levels in liver, kidney, and hippocampal tissues of mice were compared to examine if MSD-AFLP can detect subtle differences in the levels of tissue-specific differentially methylated CpGs. As a result, many CpG sites suspected to be tissue-specific differentially methylated were detected. Nucleotide sequences adjacent to these methyl-CpG sites were identified and we determined the methylation level by methylation-sensitive restriction endonuclease (MSRE)-PCR analysis to confirm the accuracy of AFLP analysis. The differences of the methylation level among tissues were almost identical among these methods. By MSD-AFLP analysis, we detected many CpGs showing less than 5% statistically significant tissue-specific difference and less than 10% degree of variability. Additionally, MSD-AFLP analysis could be used to identify CpG methylation sites in other organisms including humans. Conclusion MSD-AFLP analysis can potentially be used to measure slight changes in CpG methylation level. Regarding the remarkable precision, sensitivity, and throughput of MSD-AFLP analysis studies, this method will be advantageous in a variety of epigenetics-based research.

• 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 resulrs: 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. Withinplant 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.

• Aroma variability in truffles has been attributed to maturation (Tuber borchii), linked to environmental factors (Tuber magnatum), but the involvement of genetic factors has been ignored. We investigated aroma variability in Tuber uncinatum, a species with wide distribution. Our aim was to assess aroma variability at different spatial scales (i.e. trees, countries) and to quantify how aroma was affected by genotype, fruiting body maturity, and geographical origin. • A volatile fingerprinting method was used to analyze the aroma of 223 T. uncinatum fruiting bodies from seven European countries. Maturity was estimated from spore melanization. Genotypic fingerprinting was performed by amplified fragment length polymorphism (AFLP). • Discriminant analysis revealed that, regardless of the geographical origin of the truffles, most of the aroma variability was caused by eight‐carbon‐containing volatiles (C8‐VOCs). In an orchard of T. uncinatum, truffles producing different concentrations of C8‐VOCs clustered around distinct host trees. This clustering was not associated with maturity, but was associated with fungal genotype. • These results indicate that the variation in C8‐VOCs in truffles is most likely under genetic control. They exemplify that understanding the factors behind aroma variability requires a holistic approach. Furthermore, they also raise new questions regarding the ecological role of 1‐octen‐3‐ol in truffles.

Hybridization is known to have a creative role in plant evolution. However, it can also have negative effects on parental species. Onopordum is a large genus whose species frequently hybridize. In the Southwest Iberian Peninsula, the rare O. hinojense co‐occurs with the widely distributed O. nervosum, and hybrids between these two taxa have been described as O. × onubense. In this study we determine the extinction risk in a hybrid zone, both for hybrids and parentals, using analyses of morphological and cytogenetic traits as well as genetic markers and demographic models. To investigate the introgression process we used amplified fragment length polymorphism (AFLP) markers, Bayesian analyses and genome scan methods. Morphology, genome size and molecular markers confirmed homoploid hybridization and also indicated unidirectional backcrossing of F₁hybrids with O. nervosum, which is likely to swamp O. hinojense, the parental with lower pollen size and a very low fruit set (8%). Genome scan methods revealed several loci significantly deviating from neutrality. Finally, our demographic modeling indicated that the higher fitness of O. nervosum threats the survival of O. hinojense by demographic swamping. Our study provides strong new evidence for a scenario of rapid extinction by unidirectional introgression and demographic swamping. The multifaceted approach used here sheds new light on the role of introgression in plant extinctions.

Sechium edule (Jacq.) Sw. (Cucurbitaceae) is a species native to Mexico and Central America. The collection, characterization, and evaluation of accessions maintained in genebanks is essential for the conservation of this species. However, there are no specific varietal descriptors that differ from those used in a phenetic approach and are adapted to international registration guidelines to help distinguish, improve, cluster, and protect intraspecific variants of common use and those obtained by breeding. Therefore, 65 morphological descriptors (qualitative and quantitative) were evaluated in 133 accessions obtained from Mexico, Guatemala, and Costa Rica located in the National Germplasm Bank of S. edule in Mexico. These characteristics were observed to be phenetically stable for five generations under the same agroclimatic conditions. In addition, an analysis of amplified fragment length polymorphism (AFLP) was applied to 133 samples from a set of 245 accessions. According to the multivariate analysis, 26 of the 65 descriptors evaluated (qualitative and quantitative) enabled differentiation of varieties of S. edule. The AFLP analysis showed a high level of polymorphism and genetic distance between cultivated accessions and their corresponding wild ancestor. The variations in S. edule suggest that the morphological characteristics have differentiated from an essentially derived initial edible variety (ancestral original variety), but unlike other cucurbits, there is no evidence of the ancestral edible for Sechium since the seed is unorthodox and there are no relicts.

The multispecies coalescent provides an elegant theoretical framework for estimating species trees and species demographics from genetic markers. However, practical applications of the multispecies coalescent model are limited by the need to integrate or sample over all gene trees possible for each genetic marker. Here we describe a polynomial-time algorithm that computes the likelihood of a species tree directly from the markers under a finite-sites model of mutation effectively integrating over all possible gene trees. The method applies to independent (unlinked) biallelic markers such as well-spaced single nucleotide polymorphisms, and we have implemented it in SNAPP, a Markov chain Monte Carlo sampler for inferring species trees, divergence dates, and population sizes. We report results from simulation experiments and from an analysis of 1997 amplified fragment length polymorphism loci in 69 individuals sampled from six species of Ourisia (New Zealand native foxglove).