Genetic and epigenetic divergence between disturbed and undisturbed subpopulations of a Mediterranean shrub:a 20-year field experiment

Little is known on the potential of ecological disturbance to cause genetic andepigenetic changes in plant populations. We take advantage of a long-term fieldexperiment initiated in 1986 to study the demography of the shrub Lavandulalatifolia, and compare genetic and epigenetic characteristics of plants in twoadjacent subplots, one experimentally disturbed and one left undisturbed,20 years after disturbance. Experimental setup was comparable to an unrepli-cated ‘Before-After-Control-Impact’ (BACI) design where a single pair of per-turbed and control areas were compared. When sampled in 2005, plants in thetwo subplots had roughly similar ages, but they had established in contrastingenvironments: dense conspecific population (‘Undisturbed’ subpopulation) ver-sus open area with all conspecifics removed (‘Disturbed’ subpopulation). Plantswere characterized genetically and epigenetically using amplified fragmentlength polymorphism (AFLP) and two classes of methylation-sensitive AFLP(MSAP) markers. Subpopulations were similar in genetic diversity but differedin epigenetic diversity and multilocus genetic and epigenetic characteristics.Epigenetic divergence between subpopulations was statistically unrelated togenetic divergence. Bayesian clustering revealed an abrupt linear boundarybetween subpopulations closely coincident with the arbitrary demarcation linebetween subplots drawn 20 years back, which supports that genetic and epige-netic divergence between subpopulations was caused by artificial disturbance.There was significant fine-scale spatial structuring of MSAP markers in bothsubpopulations, which in the Undisturbed one was indistinguishable from thatof AFLP markers. Genetic differences between subpopulations could beexplained by divergent selection alone, while the concerted action of divergentselection and disturbance-driven appearance of new methylation variants in theDisturbed subpopulation is proposed to explain epigenetic differences. Thisstudy provides the first empirical evidence to date suggesting that relativelymild disturbances could leave genetic and epigenetic signatures on the nextadult generation of long-lived plants.Peer reviewe

Epigenetic variation predicts regional and local intraspecific functional diversity in a perennial herb

The ecological significance of epigenetic variation has been generally inferred from studies on model plants under artificial conditions, but the importance of epigenetic differences between individuals as a source of intraspecific diversity in natural plant populations remains essentially unknown. This study investigates the relationship between epigenetic variation and functional plant diversity by conducting epigenetic (methylation-sensitive amplified fragment length polymorphisms, MSAP) and genetic (amplified fragment length polymorphisms, AFLP) marker-trait association analyses for 20 whole-plant, leaf and regenerative functional traits in a large sample of wild-growing plants of the perennial herb Helleborus foetidus from ten sampling sites in south-eastern Spain. Plants differed widely in functional characteristics, and exhibited greater epigenetic than genetic diversity, as shown by per cent polymorphism of MSAP fragments (92%) or markers (69%) greatly exceeding that for AFLP ones (41%). After controlling for genetic structuring and possible cryptic relatedness, every functional trait considered exhibited a significant association with at least one AFLP or MSAP marker. A total of 27 MSAP (13.0% of total) and 12 AFLP (4.4%) markers were involved in significant associations, which explained on average 8.2% and 8.0% of trait variance, respectively. Individual MSAP markers were more likely to be associated with functional traits than AFLP markers. Between-site differences in multivariate functional diversity were directly related to variation in multilocus epigenetic diversity after multilocus genetic diversity was statistically accounted for. Results suggest that epigenetic variation can be an important source of intraspecific functional diversity in H. foetidus, possibly endowing this species with the capacity to exploit a broad range of ecological conditions despite its modest genetic diversity.Peer Reviewe

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.Peer Reviewe

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.Peer reviewe

Comparación de efectos de dos especies de levaduras florícolas Metschnikowia en el néctar

Nectar yeast communities in southern Spain are dominated by two closely-related species, Metschnikowia reukaufii Pitt & M.W. Mill. and M. gruessii Gim.-Jurado (Ascomycota, Saccharomycetales), although they tend to be distributed differentially across different host plants. We explore here the possibility that the two yeasts play different functional roles in floral nectar by differing in their impact on sugar concentration and composition of nectar. Experiments were undertaken under controlled conditions using bumblebees caught foraging on the flowers of two different host plants each of which is known to harbor predominantly one of the two yeasts. Bumblebees were used as sources of inocula to obtain two groups of samples from the nectar of Helleborus foetidus L. (Ranunculaceae): nectar samples inoculated with M. gruessii and samples inoculated with M. reukaufii. Metschnikowia gruessii was poorly represented in nectar samples, while M. reukaufii was by far the most common and had the highest cell density. Although the two yeasts caused relatively similar changes in nectar sugar composition, which involved increasing fructose and decreasing sucrose proportions, they marginally differed in their quantitative impact on total nectar sugar concentration. Results suggest that differential yeast occurrence across host plants may lead to yeast specialization and modify the outcomes of the plant-pollinator interface.Las comunidades de levaduras asociadas al néctar en el sur de España son dominadas por dos especies, Metschnikowia reukaufii Pitt & M.W. Mill. y M. gruessii Gim.-Jurado (Ascomycota, Saccharomycetales). Ambas levaduras son especies muy cercanas entre sí, pero se distribuyen diferencialmente entre las especies de plantas cuyo néctar las hospeda. En este trabajo se explora la posibilidad de que las levaduras tengan funciones distintas impactando diferencialmente la concentración y composición del néctar. Para esto se realizaron experimentos bajo condiciones controladas usando individuos de abejorros que fueron capturados cuando visitaban flores que albergan predominantemente una de las dos posibles levaduras mencionadas. Los abejorros fueron utilizados como agentes inoculantes naturales en el néctar de Helleborus foetidus L. para obtener dos grupos de muestras, unas con M. reukaufii y otras con M. gruessii. Los resultados mostraron que M. gruessii estuvo pobremente representada en las muestras; en contraste, M. reukaufii fue la más frecuente y de mayor densidad en el néctar. Aun cuando ambas levaduras tuvieron un efecto similar en la composición proporcional de los azúcares en el néctar, aumento de la fructosa y decremento de la sacarosa, las dos especies difieren marginalmente en su impacto en la concentración total de azúcares. Los resultados sugieren que la ocurrencia diferencial de las especies de levaduras entre sus plantas hospederas, puede conducir a una modificación en las interacciones entre las plantas y sus visitantes florales y también a una especialización de levaduras por hospedero

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 February 2013-31 March 2013

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. © 2013 John Wiley & Sons Ltd.Peer Reviewe

Desarrollo de herramientas metodológicas para incorporar contenidos de ciencia al aula y su valoración económica

La ciencia y la tecnología juegan un papel fundamental en nuestra sociedad. En este sentido, la ciudadanía parece necesitar unos conocimientos básicos que le permitan responder de manera autónoma a multitud de situaciones, tanto a nivel personal como social (Kolstø et al., 2006). Este requisito, conocido en la literatura como alfabetización científica, lleva siendo uno de los objetivos de la educación en las últimas décadas (Hodson, 2003; Feinstein, 2011). Sin embargo, parece que su consecución presenta algunos problemas. Una de las causas de estas dificultades parece ser la falta de conexión entre los contenidos que se trabajan en las clases y el día a día del ciudadano. Respecto a esto, una solución ampliamente citada en la literatura es contextualizar el aprendizaje, relacionando los contenidos trabajados en clase con escenarios cotidianos y familiares para el alumnado (Clegg & Kolodner, 2014). El proyecto pretendía que nuestros estudiantes llevasen a cabo un análisis de los contextos con contenidos científico-económicos y promovieran su utilización en el aula y en su futuro profesional. Es decir, formar a nuestros profesionales para que pudieran detectar e incorporar entre sus competencias la capacidad de conectar lo aprendido en las asignaturas con las necesidades sociales. En concreto, el proyecto tenía, en base a los problemas detectados en la percepción de la ciencia y en la formación de los ciudadanos, dos grandes tipos de objetivos: Objetivos relacionados con la percepción de los conocimientos científico-tecnológicos y su valoración económica (Ezquerra, Fernández-Sánchez, Cabezas, 2013; Ezquerra, Fernandez-Sanchez, 2014; Ezquerra, Fernandez-Sanchez, Magaña, 2015; 2016; Ezquerra, Fernandez-Sanchez, Magaña & Mingo, 2017): • Identificar en qué situaciones los ciudadanos, en su vida cotidiana, están en presencia de contenidos científicos o tecnológicos. • Determinar de qué modo llegan al ciudadano estos contenidos, con qué intensidad, cómo son propuestos, cómo son percibidos... • Analizar el modo en que podemos otorgar a estos contenidos una valoración económica. Objetivos educativos que se integran dentro de las asignaturas: • Analizar las correspondencias entre los currículos oficiales y los contenidos presentes en el entorno del ciudadano (Ezquerra, Fernandez-Sanchez, Magaña Ramos, 2015; Ezquerra y Magaña, 2016). • Introducir a los estudiantes en una propuesta de aprendizaje basada en la indagación (Rivero et al., 2013; Ezquerra, De Juanas, Martín del Pozo, 2015). • Valorar las estrategias y competencias de los futuros docentes para vincular sus conocimientos con la realidad circundante (De Juanas et al., 2012). • Analizar las actividades que los estudiantes propongan para identificar los contenidos de ciencia y estudiar su valoración económica

Epigenetic variation predicts regional and local intraspecific functional diversity in a perennial herb

The ecological significance of epigenetic variation has been generally inferred from studies on model plants under artificial conditions, but the importance of epigenetic differences between individuals as a source of intraspecific diversity in natural plant populations remains essentially unknown. This study investigates the relationship between epigenetic variation and functional plant diversity by conducting epigenetic (methylation-sensitive amplified fragment length polymorphisms, MSAP) and genetic (amplified fragment length polymorphisms, AFLP) marker-trait association analyses for 20 whole-plant, leaf and regenerative functional traits in a large sample of wild-growing plants of the perennial herb Helleborus foetidus from ten sampling sites in south-eastern Spain. Plants differed widely in functional characteristics, and exhibited greater epigenetic than genetic diversity, as shown by per cent polymorphism of MSAP fragments (92%) or markers (69%) greatly exceeding that for AFLP ones (41%). After controlling for genetic structuring and possible cryptic relatedness, every functional trait considered exhibited a significant association with at least one AFLP or MSAP marker. A total of 27 MSAP (13.0% of total) and 12 AFLP (4.4%) markers were involved in significant associations, which explained on average 8.2% and 8.0% of trait variance, respectively. Individual MSAP markers were more likely to be associated with functional traits than AFLP markers. Between-site differences in multivariate functional diversity were directly related to variation in multilocus epigenetic diversity after multilocus genetic diversity was statistically accounted for. Results suggest that epigenetic variation can be an important source of intraspecific functional diversity in H. foetidus, possibly endowing this species with the capacity to exploit a broad range of ecological conditions despite its modest genetic diversity.Peer Reviewe