Ecological plant epigenetics : Evidence from model and non-model species, and the way forward

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Ecological plant epigenetics : Evidence from model and non-model species, and the way forward Richards, Christina L.; Alonso, Conchita; Becker, Claude; Bossdorf, Oliver; Bucher, Etienne; Colome-Tatche, Maria; Durka, Walter; Engelhardt, Jan; Gaspar, Bence; GogolDoring, Andreas; Grosse, Ivo; van Gurp, Thomas P.; Heer, Katrin; Kronholm, Ilkka; Lampei, Christian; Latzel, Vit; Mirouze, Marie; Opgenoorth, Lars; Paun, Ovidiu; Prohaska, Sonja; Rensing, Stefan A.; Stadler, Peter; Trucchi, Emiliano; Ullrich, Kristian; Verhoeven, Koen J.F. Richards, C. L., Alonso, C., Becker, C., Bossdorf, O., Bucher, E., Colome-Tatche, M., Durka, W., Engelhardt, J., Gaspar, B., Gogol-Doring, A., Grosse, I., van Gurp, T. P., Heer, K., Kronholm, I., Lampei, C., Latzel, V., Mirouze, M., Opgenoorth, L., Paun, O., . . . Verhoeven, K. J. (2017). Ecological plant epigenetics : Evidence from model and non-model species, and the way forward. Ecology Letters, 20(12), 1576-1590. https://doi.org/10.1111/ele.12858 2017 REVIEW AND SYNTHESIS Ecological plant epigenetics: Evidence from model and non-model species, and the way forward Christina L. Richards, 1 * Conchita Alonso, 2 Claude Becker, 3 Oliver Bossdorf, 4 Etienne Bucher, 5 Maria Colom e-Tatch e, 6,7,8 Walter Durka, 9,10 Jan Engelhardt, 11 Bence Gaspar, 4 Andreas Gogol-D€ oring, 10,12 Ivo Grosse, 10,12 Thomas P. van Gurp, 13 Katrin Heer, 14 Ilkka Kronholm, 15 Christian Lampei, 16 V ıt Latzel, 17 Marie Mirouze, 18 Lars Opgenoorth, 19 Ovidiu Paun, 20 Sonja J. Prohaska, 11,23 Stefan A. Rensing, 21,22 Peter F. Stadler, 10,11,23,24 Emiliano Trucchi, 20 Kristian Ullrich 21 and Koen J. F. Verhoeven 13 Abstract Growing evidence shows that epigenetic mechanisms contribute to complex traits, with implications across many fields of biology. In plant ecology, recent studies have attempted to merge ecological experiments with epigenetic analyses to elucidate the contribution of epigenetics to plant phenotypes, stress responses, adaptation to habitat, and range distributions. While there has been some progress in revealing the role of epigenetics in ecological processes, studies with non-model species have so far been limited to describing broad patterns based on anonymous markers of DNA methylation. In contrast, studies with model species have benefited from powerful genomic resources,which contribute to a more mechanistic understanding but have limited ecological realism. Understanding the significance of epigenetics for plant ecology requires increased transfer of knowledge and methods from model species research to genomes of evolutionarily divergent species, and examination of responses to complex natural environments at a more mechanistic level. This requires transforming genomics tools specifically for studying non-model species, which is challenging given the large and often polyploid genomes of plants. Collaboration among molecular geneticists, ecologists and bioinformaticians promises to enhance our understanding of the mutual links between genome function and ecological processes. Keywords Bioinformatics, ecological epigenetics, genomics, phenotypic plasticity, response to environment. Ecology Letters (2017) 20: 1576–1590 INTRODUCTION The DNA of all higher organisms is subject to different chemical modifications that influence gene activity and expression, and that are summed up under the term ‘epigenetics’. One of these processes is DNA methylation, the addition of a methyl group to one of the four bases in the DNA molecule (usually cytosine). The idea that within-species variation in such epigenetic modifications may be important for the ecology and evolution of species has captivated biologists during recent years. 1 Department of Integrative Biology, University of South Florida, Tampa, FL 33620, USA 2 Estaci on Biol ogica de Do~ nana, CSIC, 41092 Sevilla, Spain 3 Gregor Mendel Institute of Molecular Plant Biology, 1030 Vienna, Austrian Academy of Sciences, Vienna Biocenter (VBC), Austria 4 Plant Evolutionary Ecology, University of T€ ubingen, 72076 T€ ubingen, Germany 5 Institut de Recherche en Horticulture et Semences, 49071 Beaucouz e Cedex, France 6 European Research Institute for the Biology of Ageing, University Medical Center Groningen, 9713 Groningen, The Netherlands 7 Institute of Computational Biology, Helmholtz Zentrum M€ unchen, 85764 Neuherberg, Germany 8 School of Life Sciences Weihenstephan, Technical University of Munich, 85354 Freising, Germany 9 Department of Community Ecology, Helmholtz Centre for Environmental Research –UFZ, 06120 Halle, Germany 10 German Centre for Integrative Biodiversity Research (iDiv) Halle-JenaLeipzig, 04103 Leipzig, Germany 11 Institut f€ ur Informatik, University of Leipzig, 04107 Leipzig, Germany 12 Institute of Computer Science, University of Halle, 06120 Halle, Germany 13 Netherlands Institute of Ecology (NIOO-KNAW), Wageningen, The Netherlands 14 Conservation Biology, Philipps-University of Marburg, 35037 Marburg, Germany 15 Department of Biological and Environmental Sciences, Center of Excellence in Biological Interactions, University of Jyv€ askyl€ a, 40014 Jyv€ askyl€ an yliopisto, Finland 16 Institute of Plant Breeding, Seed Science and Population Genetics, 70599 Stuttgart, Germany 17 Institute of Botany, The Czech Academy of Sciences, 25243 Pr uhonice, Czech Republic 18 Institut de Recherche pour le D eveloppement, Laboratoire G enome et D eveloppement des Plantes, 66860 Perpignan, France 19 Department of Ecology, Philipps-University Marburg, 35037 Marburg, Germany 20 Plant Ecological Genomics, University of Vienna, 1030 Vienna, Austria 21 Plant Cell Biology, Philipps-University Marburg, 35037 Marburg, Germany 22 BIOSS Centre for Biological Signaling Studies, University of Freiburg, 79098 Freiburg, Germany 23 The Santa Fe Institute, Santa Fe NM 87501, USA 24 Max Planck Institute for Mathematics in the Sciences, 04103 Leipzig, Germany *Correspondence: E-mail: [email protected] ©2017 The Authors. Ecology Letters published by CNRS and John Wiley & Sons Ltd This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Ecology Letters, (2017) 20: 1576–1590 doi: 10.1111/ele.12858 Earlier evidence of natural variation in DNA methylation, as well as of the inheritance and phenotypic effects of this epigenetic variation (e.g. Cubas et al. 1999), led to several conceptual papers that suggested its potential relevance to ecology and evolution (e.g. Richards 2006; Bossdorf et al. 2008; Jablonka & Raz 2009; Richards et al. 2010), and empirical work has been catching up slowly. Ecologists and evolutionary biologists are particularly interested in the unique contributions that epigenetic mechanisms might make. First, environment-sensitive epigenetic mechanisms could transmit responses to environmental changes across generation boundaries. Second, heritable epigenetic variants that arise stochastically, i.e. epimutations, that affect phenotypes may be under natural selection and might contribute to adaptation, independently from DNA sequence variation. More specifically, research in ecological and evolutionary epigenetics is concerned with (A) patterns of natural epigenetic variation, (B) the origins and drivers of this variation and (C) its ecological and evolutionary consequences (Bossdorf et al. 2008). Understanding these patterns, causes and consequences requires insight into a number of key questions that span research fields from molecular biology to ecology (Fig. 1): (A) What is the extent and structure of epigenetic variation in natural populations? (B1) What is the interplay between genetic variation and epigenetic variation? (B2) How frequently do spontaneous epimutations occur and how stable are they? (B3) To what extent can environmental changes induce heritable epigenetic changes? (C1) What is the relative importance of genetic vs. epigenetic variation in determining phenotypes? (C2) How important is epigenetic Figure 1 Research in ecological and evolutionary epigenetics is concerned with (A) patterns of natural epigenetic variation, (B) the origins and drivers of this variation and (C) its phenotypic, ecological and evolutionary consequences. Text outside of coloured boxes indicates additional contributions and down stream effects of these sources of variation that contribute to the seven key questions outlined in the text. Here, ‘Environmental change’ includes habitat reduction and fragmentation, which are primary causes of declines in population size, and can result in selection on phenotypic variation within populations. ©2017 The Authors. Ecology Letters published by CNRS and John Wiley & Sons Ltd Review and Synthesis Ecological plant epigenetics 1577 variation for biotic interactions, biodiversity and the structure and functioning of communities and ecosystems? (C3) Does epigenetic variation play a role in adaptation and the evolution of populations? For the most part these questions have been studied separately in two different research fields: molecular genetics and evolutionary ecology. In the plant sciences in particular, molecular genetics has made progress in understanding the mechanisms and dynamics of epigenetic variation by applying highresolution genomic analysis tools to model species like Arabidopsis thaliana,Oryza sativa and Zea mays. Evolutionary ecology on the other hand has started to explore epigenetic variation in a broad range of non-model species (that lack extensive genomic resources), and in natural settings. This adds the complexity of real environmental conditions to experimental investigations, and has uncovered correlations between epigenetic molecular markers and environmental variation. However, the lack of high-resolution genomic tools in most non-model plant species has made it difficult to firmly establish mechanistic links among genotype, epigenotype, phenotype and environment. To advance the study of ecological and evolutionary plant epigenetics, we need to better integrate the fields of molecular genetics and evolutionary ecology, by adding more ecological context and ecological questions to model species research (e.g. Latzel et al. 2013; Hagmann et al. 2015), and by adopting higher resolution tools in non-model species research (e.g. Platt et al. 2015; Xie et al. 2015; Gugger et al. 2016; van Gurp et al. 2016; Trucchi et al. 2016). In fact, a similar transition has recently been taking place in ecological and evolutionary genomics (Pavey et al. 2012; Narum et al. 2013; Alvarez et al. 2015), which has demonstrated the importance of testing hypotheses in natural environments. For example several studies have found that organismal responses under laboratory conditions may not reflect performance under natural conditions, and that non-adaptive processes may result in apparent genomic signatures of natural selection (Pavey et al. 2012; Alvarez et al. 2015). This review focuses on the progress in ecological plant epigenetics accomplished by molecular epigenetics as well as evolutionary ecology, following the series of seven key questions outlined above. Our review is largely restricted to studies of DNA methylation since DNA methylation is the most frequently studied and best-understood epigenetic process to date. However, it is important to note that histone modifications and small RNAs are involved in regulating epigenetic modifications (see e.g. Matzke & Mosher 2014; Kim et al. 2015), and the potential for interconnection among different epigenetic mechanisms is not yet fully understood (Becker et al. 2011). On the basis of our review of current progress, we identify next steps and discuss strategies and methodological challenges for future ecological epigenetics research. CURRENT PROGRESS IN ECOLOGICAL EPIGENETICS Patterns of natural epigenetic variation What is the extent and structure of epigenetic variation in natural populations? Across the genome, DNA methylation varies widely within and among plant species (4–40% of cytosines; Fig. 2; Alonso et al. 2014, 2015; Niederhuth et al. 2016). Whole-genome bisulfite sequencing (Box 1) provides information on the methylation of individual cytosines (Cokus et al. 2008), but this method has so far only been used in model plant species. Studies in A. thaliana (Schmitz et al. 2013; Kawakatsu et al. 2016) and rice (He et al. 2010; Chodavarapu et al. 2012; Li et al. 2012) found variable DNA methylation among different lines/genotypes, and that within genomes, DNA methylation depends on the sequence context (i.e. CG, CHG or CHH) and the type of genomic region (gene promoters, gene bodies, transposable elements [TEs]). While such detailed information on genomic context is lacking for non-model species, studies in wild plant populations have documented extensive natural intraspecific variation in DNA methylation, based on anonymous markers (Schrey et al. 2013), global DNA methylation estimates (Alonso et al. 2015) or analyses of specific genes (Xie et al. 2015; see Box 1). DNA methylation variation can result from genetic control, environmental induction and stochastic epimutations, and can in principle be shaped further by drift and natural selection. Consequently, the interpretation of patterns of natural epigenetic variation is not straightforward. Several ecological studies have searched for epigenetic variation that correlates with habitat or with population differentiation, and have found (1) that variation in DNA methylation usually exceeds variation in DNA sequence markers when populations from contrasting habitats are compared (e.g. Lira-Medeiros et al. 2010; Herrera & Bazaga 2010; Richards et al. 2012; Medrano et al. 2014; Schulz et al. 2014; but see Foust et al. 2016; Robertson et al. 2017), (2) that epigenetic differences are often correlated with ecological factors and (3) that some of these relationships are unrelated to patterns of genetic relatedness (Richards et al. Figure 2 Features and limitations of Arabidopsis thaliana as a model system for epigenetic studies. Rapid development, selfing ability and reduced chromosome number and genome size (C-value =0.30 pg) with relatively few repetitive and transposable elements, facilitate experimentation and simplify molecular analyses in A. thaliana. However, A. thaliana (black dot) is unusual within the range of variation in genome size and global cytosine methylation in study species, including some with a fully sequenced reference genome (filled dots) like rice (Oryza sativa), tomato (Solanum lycopersicum), maize (Zea mays) and wheat (Triticum aestivum). Redrawn from Alonso et al. (2015). ©2017 The Authors. Ecology Letters published by CNRS and John Wiley & Sons Ltd 1578 C. L. Richards et al. Review and Synthesis Box 1 Genome-wide screening for epigenetic variation in ecological epigenetics GLOBAL METHYLATION Global levels of DNA methylation can be assessed through HPLCand ELISA-based assays that estimate the proportion of methylated cytosines across the entire genome. These methods do not require any sequence knowledge, and they do not distinguish between different genomic locations or contexts (CG, CHG, CHH) of cytosine methylation. Nevertheless, they can help to clarify magnitudes of overall DNA methylation, its variation across species (Alonso et al. 2015), the structuring of natural intraspecific variation (Alonso et al. 2014) or the global response to environmental changes. METHYLATION-SENSITIVE MARKERS Genome-wide patterns of DNA methylation can be captured by molecular markers obtained with methylation-sensitive restriction enzymes. Methylation-sensitive AFLP (Reyna-Lopez et al. 1997; MS-AFLP/MSAP) follows a standard AFLP protocol but uses pairs of restriction enzymes, often HpaII and MspI, that have the same recognition sequence but differential sensitivity to DNA methylation. MS-AFLP typically evaluates a few hundred restriction sites. AFLP and MS-AFLP can be applied in parallel to compare genetic and epigenetic structures of populations, and their environmental correlates (e.g. Herrera & Bazaga 2010; Richards et al. 2012; Schulz et al. 2014; Foust et al. 2016; Robertson et al. 2017). Because of its easy application, the fact that no reference genome or advanced bioinformatics skills are required, and because of a lack of alternative methods for non-model organisms, the MS-AFLP method has been popular in ecological epigenetics. However, it is now gradually being replaced by bisulfite sequencing-based methods. For example EpiRADseq (Schield et al. 2015) combines methylation-sensitive restriction enzymes with next-generation sequencing. Similar to MS-AFLP, EpiRADseq detects methylation differences only in recognition sequences, but produces a much greater number of loci and thus characterises methylation patterns at much higher resolution. This approach also provides sequence information at the interrogated loci. BISULFITE SEQUENCING METHODS Bisulfite treatment converts unmethylated cytosines to uracil, allowing for the identification of methylated cytosines by comparing a treated sample to a reference sample (Cokus et al. 2008). Bisulfite sequencing is the gold standard of DNA methylation analysis, as it evaluates individual cytosines in a target sequence or for essentially all cytosines in a genome (i.e. whole-genome bisulfite sequencing or WGBS). While WGBS enables detailed analyses of DNA methylation variation, it is restricted to species with a highquality reference genome, and its costs may be prohibitive for large sample sizes and in species with large genomes, limiting its use for ecological studies. However, by restricting sequencing to specific subsets of the genome, bisulfite sequencing can be applied more broadly. If a high-quality reference transcriptome is available, bisulfite sequencing combined with exome capture permits methylation analyses of the expressed regions of the genome (and their flanking regions; Lee et al. 2011). Bisulfite sequencing can also be targeted to a selection of genomic fragments that are isolated with restriction enzymes (Reduced representation bisulfite sequencing,RRBS;Guet al. 2011), thus providing single-nucleotide resolution of DNA methylation within each of the fragments. Availability of both sequence and methylation variation from the same large set of loci allows direct comparison of genetic and epigenetic information, and the evaluation of the contributions of SNPs vs. DMPs to population divergence. RRBS has been adopted for plant population studies (Platt et al. 2015), and methods have recently been developed to incorporate bisulfite sequencing into popular reduced-representation sequencing approaches that can be applied to species for which no reference genomes are available (bsRADseq and epiGBS; Trucchi et al. 2016; van Gurp et al. 2016). Having precise sequence information for methylation polymorphisms contributes to functional analyses that can link DNA methylation variation to gene expression, which can then be linked to phenotypes that contribute to population divergence and local adaptation. CHIP-SEQUENCING Chromatin immunoprecipitation followed by NGS (ChIP-seq) determines the modification state of histone proteins (Park 2009). Specific antibodies bind to the histone modification of interest and immunoprecipitate fragments of DNA that are wrapped around the modified histones. These DNA fragments can be sequenced and mapped to the reference genome to determine specific regions where the modifications were present. ChIP-seq methods have not been used yet in ecological epigenetics. sRNA-SEQUENCING Diversity and abundance of small RNAs can be obtained by deep sequencing of small RNA molecules (Studholme 2012). Functional interpretations are aided by a reference genome or transcriptome, but it is possible to quantify differential sRNA abundance across populations, environments or treatments, even in the absence of such references (e.g. Morgado et al. 2017). ©2017 The Authors. Ecology Letters published by CNRS and John Wiley & Sons Ltd Review and Synthesis Ecological plant epigenetics 1579 2012; Schulz et al. 2014; Foust et al. 2016; Gugger et al. 2016). In summary, the extent and pattern of variation in DNA methylation in natural populations suggest that epigenetic variation might be relevant for ecological studies. Still, without whole-genome and -methylome data it is impossible to determine whether or not observed epigenetic patterns are under genetic control or make contributions independently from DNA sequence. Furthermore, in studies conducted in natural populations under field conditions, we cannot know whether observed differences in DNA methylation, and their correlations with environmental factors, reflect heritable (and thus potentially adaptive) variation or are merely repeatedly induced. Origins and drivers of epigenetic variation What is the interplay between genetic variation and epigenetic variation? Epigenetic variation may contribute to heritable trait variation, but if the epigenetic differences are entirely under genetic control then epigenetic variation simply reflects the underlying genetic variation (Richards 2006). Such genetic control of epigenetic variation has been shown in A. thaliana where sequence changes in genes related to the epigenetic machinery can have dramatic effects on the epigenome (Becker et al. 2011; Dubin et al. 2015; see also Box 2: Epigenetic mutants, and ‘What can be transferred from model to non-model species?’ below). Quantitative genetic studies in model plant species also suggest that many DNA methylation differences among individuals are associated with underlying genetic differences. For example the majority of differentially methylated regions (DMRs; Box 3) stably segregated with the local DNA sequence allele in maize (cis inheritance; Li et al. 2014). In contrast, only 35% of DMRs were associated with DNA sequence polymorphisms among 142 natural A. thaliana accessions (Schmitz et al. 2013). Although associations between DMRs and local genetic polymorphisms can indicate genetic control over DNA methylation, such associations can also arise in the absence of genetic control, when a spontaneous epimutation is stably inherited through epigenetic inheritance (Taudt et al. 2016). On the other hand, epigenetic changes can also influence genetic variation. Since DNA methylation is associated with the silencing of TEs, epigenetic change can induce novel genetic variation through regulating TE activity (Fig. 1). Reduction in DNA methylation can result in increased movement of TEs, thereby creating variation in DNA sequence. Such transposition may affect phenotypes where TEs insert near genes, attract DNA methylation and influence gene expression. In fact, many well-characterised epialleles are associated with TEs or repeats that can cause DNA methylation via RNA-directed DNA methylation (Paszkowski & Grossniklaus 2011; O’Malley & Ecker 2012; Matzke & Mosher 2014). Many previous epigenetic studies in non-model plants have attempted to control for genetic variation using species that have naturally low levels of genetic diversity such as asexually reproducing plants (e.g. Verhoeven et al. 2010; Richards et al. 2012; Verhoeven & Preite 2014). These studies cannot address the relationship between genetic and epigenetic variation. However, a few studies on outcrossing species have used statistical approaches to make inferences about the independence of genetic and epigenetic variation by evaluating how well overall similarities in DNA methylation profiles among individuals can be predicted from their DNA sequence similarities (Herrera & Bazaga 2010; Schulz et al. 2014; Foust et al. 2016). While these studies have frequently found that epigenetic variation appeared to be independent from genetic control, they usually used low-resolution molecular markers and thus could not rule out the possibility that critical genetic polymorphisms had gone unnoticed (Becker et al. 2011; Dubin et al. 2015). How often do spontaneous epimutations occur? Spontaneous epimutations have the potential to contribute to heritable trait variation in a way that is not predictable from DNA sequence variation, but their evolutionary potential is determined by the rate at which they appear and revert. Epimutation rates have been estimated in a study of the accumulation of mutations in A. thaliana, where DNA methylation polymorphisms accumulated at a much higher frequency (van der Graaf et al. 2015) than genetic mutations (Ossowski et al. 2010). Epimutations occurred more frequently in genic regions than in TEs. This pattern is consistent with the known RNA-directed repair mechanism, which limits the development of DNA methylation polymorphisms in TEs (Teixeira et al. 2009). Spontaneous epimutations also occurred at the level of DMRs, i.e. within particular regions rather than at specific single sites. These epimutations showed more functional relevance to gene expression, but they also occurred at much lower frequency, comparable to the rate at which genetic mutations arise (Becker et al. 2011). So far, there are virtually no data on epimutation rates in non-model plant species. A study on apomictic dandelions by Verhoeven et al. (2010) showed that even in a constant environment, appreciable DNA methylation differences developed among individual plants, and that most of these changes were passed on to offspring. However, further studies are needed that determine the rate and stability of epimutations across different species before we can draw any conclusions about their potential ecological and evolutionary significance. To what extent can environmental changes induce heritable epigenetic changes? Studies in A. thaliana have shown that the epigenome reacts to environmental changes such as abiotic and biotic stress (e.g. Dowen et al. 2012; Slaughter et al. 2012; Sani et al. 2013), and that these epigenetic changes are sometimes associated with changes in gene expression throughout the genome (Secco et al. 2015; Wibowo et al. 2016). Other studies have reported inheritance of stress-induced changes in DNA methylation (e.g. in rice: Kou et al. 2011; in A. thaliana: Bilichak et al. 2012). In particular, herbivore or pathogen effects can be passed on to offspring, and some of the best current evidence for epigenetically based inheritance of induced effects is in the context of such biotic interactions (Luna et al. 2012; Rasmann et al. 2012). However, several studies also found only limited inheritance of stress-induced DNA methylation ©2017 The Authors. Ecology Letters published by CNRS and John Wiley & Sons Ltd 1580 C. L. Richards et al. Review and Synthesis changes in A. thaliana and maize (Pecinka & Mittelsten Scheid 2012; Eichten & Springer 2015; Wibowo et al. 2016). Studies in non-model species have found that heritable changes in DNA methylation can occur in response to different environmental stresses, but that the strength of these effects depends on environmental conditions (Verhoeven et al. 2010; Alonso et al. 2016). For instance, Verhoeven et al. (2010) showed that plants with identical genotypes had a range of DNA methylation changes in response to different stresses, and that the degree to which these were inherited also varied. Richards et al. (2012) measured epigenetic differences in a dominant haplotype of the highly invasive clonal plant Box 2 Linking epigenetic variation to phenotype Establishing the causal links between epigenetic variation and phenotypes is one of the key challenges in ecological epigenetics, and a number of different approaches have been used to address this question. CANDIDATE GENES Detailed observational studies can reveal epigenetic polymorphisms at candidate loci that are associated with expression and phenotypic differences in a genetically uniform background (such as completely inbred lines, asexually propagated individuals or within the same individual, e.g. Cubas et al. 1999; Xie et al. 2015). Such observations also suggest autonomous epigenetic determinants of phenotypic variation. However, so far such studies have been restricted to a very limited number of phenotypes in model species. EPIGENETIC MUTANTS A specific tool in plant epigenetic research is epigenetic mutants. These are plant lines that carry mutations in genes required for proper functioning of the epigenetic machinery, e.g. those coding enzymes for initiating (de novo) or maintaining DNA methylation. Such mutations can cause genome-wide epigenetic alterations (in otherwise genetically uniform backgrounds), which can be exploited for proof-of-principle studies of whether certain phenotypes are influenced by epigenetic mechanisms. Epigenetic mutants have also been useful in experiments on environmental induction and epigenetic inheritance. Evidence for a role of epigenetics is provided if a certain environment cannot induce an expected phenotypic change in an epigenetic mutant (Luna et al. 2012; Rasmann et al. 2012), or the transmission of a phenotype is altered in the mutant (Crevillen et al. 2014). CHEMICAL MANIPULATION Mutant lines are often not available in non-model organisms, but there are chemicals that can be used to manipulate epigenetic variation. Examples are 5-azacytidine (Jones 1985) or zebularine (Cheng et al. 2003) that both inhibit DNA methylation. There are also chemicals that target different epigenetic mechanisms, such as inhibition of histone demethylation (Kruidenier et al. 2012). However, many of these chemicals have cytotoxic or other off-target effects, and they can be biased to specific loci (Hagemann et al. 2011). In ecological studies, DNA methylation inhibitors have been used to demonstrate the importance of DNA methylation for plant responses to environmental conditions, and genotypic variation therein (Bossdorf et al. 2010; Herrera et al. 2012), for maintaining the effects of inbreeding (Vergeer & Ouborg 2012), flowering time differences (Wilschut et al. 2016) and inheritance of induced phenotypes (Herman et al. 2016). EPIGENETIC ASSOCIATION AND QTL-MAPPING Ecologists are most interested in natural variation, and in principle the same methods of quantitative genetics that are applicable for the study of natural genetic variation can be used to investigate natural epigenetic variation. Epigenetic variation can then be treated in two distinct ways: First, it can be treated as a phenotype, and then one can screen for genetic markers that contribute to differences in DNA methylation patterns (e.g. Dubin et al. 2015), which helps to reveal genetically controlled vs. autonomous methylation variation. Second, DMPs or DMRs that are stably inherited across generations can be treated the same way as conventional genetic markers and used in mapping approaches to explain phenotypic variation. Long et al. (2011) and Li et al. (2014) found that many methylation polymorphisms segregated in a normal Mendelian fashion, and could be used for epi-QTL-mapping in Brassica napus and maize respectively. Epigenetic markers have also been used for QTL analysis in plants in the absence of DNA sequence polymorphisms (i.e. A. thaliana epiRILs; Cortijo et al. 2014). We are not aware of any studies that use such markers for association mapping in non-model species other than in some low-resolution methylation sensitive AFLP studies with plants. Such marker-based mapping studies can shed light on the epigenetic contribution to phenotypic variation. However, a statistical epigenetic marker-trait association does not necessarily imply a true epigenetic contribution to the observed trait variation, as phenotypic effects may generally also be caused by a tightly linked genetic polymorphism. Follow-up studies are thus required to functionally characterise suspected epi-QTLs and evaluate them in manipulative studies. ©2017 The Authors. Ecology Letters published by CNRS and John Wiley & Sons Ltd Review and Synthesis Ecological plant epigenetics 1581 Japanese knotweed (Fallopia japonica) collected from different habitats, but grown in a common environment. They found that part of the observed DNA methylation variation correlated with habitat of origin, suggesting environment-specific DNA methylation changes that persisted through clonal propagation in a common environment. Similarly, after growing the invasive plant Ageratina adenophora under controlled conditions, Xie et al. (2015) found that stable inheritance of demethylation at the promoter region of a specific gene was correlated with variation in cold tolerance. All of these studies thus indicate a correlation between environment and stable epigenetic variation. However, heritable epigenetic differences between populations may also be the result of neutral processes. Further, the changes that are potentially advantageous can result not only from environmental induction and subsequent inheritance, but may also reflect natural selection acting on variation created by spontaneous epimutations. The studies above usually cannot discriminate between these two possible explanations. Environmental responses that are mediated by epigenetic mechanisms could be particularly relevant for asexually reproducing plants (Verhoeven & Preite 2014; Douhovnikoff & Dodd 2015; Rendina Gonz alez et al. 2016; Spens & Douhovnikoff 2016). While some DNA methylation is stable through sexual reproduction, epigenetic changes that are environmentally induced often show limited meiotic stability, or epigenetic resetting during meiosis (Wibowo et al. 2016). Asexual plants use vegetative (clonal) reproduction, so meiotic epigenetic reset does not occur. The mitotic stability of epigenetic responses might allow different plant parts to respond to different microenvironments, and transmit environmentally induced DNA methylation changes. Therefore, different ramets of the same clonal individual may show different epigenetic profiles in an otherwise uniform genetic background (Rendina Gonz alez et al. 2016; Spens & Douhovnikoff 2016). For instance environmental effects on DNA methylation in CHH contexts tend to occur at TE loci (Dubin et al. 2015), but the epigenetic silencing of such loci can be reinforced via small RNAs during sexual reproduction (Mart ınez et al. 2016). By circumventing this process, stable TE-associated epialleles may arise in clonal plants, as observed in clonally propagated oil palm (Ong-Abdullah et al. 2015). The Consequences of Epigenetic Variation What is the contribution of DNA methylation to phenotypes? So far, only a few natural epialleles have been functionally characterised using various approaches (Box 2; Cubas et al. 1999; Manning et al. 2006; Paszkowski & Grossniklaus 2011; Xie et al. 2015). Model plants offer powerful tools to isolate epigenetic effects from genetic effects on phenotype. Of particular note are two collections of A. thaliana lines that were derived from crosses between the Columbia wild type, and mutants in the Columbia background that have decreased DNA methylation genome-wide (ddm1 and met1 mutants; Johannes et al. 2009; Reinders et al. 2009; see below in ‘What can be transferred from model to non-model species?’). These epigenetic recombinant inbred lines (epiRILs) are therefore nearly identical in DNA sequence genome-wide. However, individual lines from the epiRIL collections differ from each other in DNA methylation because their genomes are mosaics of the wild type methylation patterns, and the methylation patterns of the parent with decreased DNA methylation (Johannes et al. 2009). Such populations allow for an assessment of the phenotypic consequences of epigenetic variation independent of variation in DNA sequence. By specifically isolating epigenetic from genetic information, the epiRILs allow for the study of the dynamics and phenotypic consequences of DNA methylation in the almost complete absence of DNA sequence variation. Both met1 and ddm1 epiRIL populations displayed significant phenotypic variation (Zhang et al. 2013; Cortijo et al. 2014), and linkage mapping in the ddm1 epiRILs identified DMRs that explained heritable phenotypic effects for root length and flowering time (QTL epi ; Cortijo et al. 2014). Interestingly, a significant fraction of the DMRs created in the epiRILs are also variable in natural A. thaliana populations, and it is possible that they are also functionally important in the wild. In non-model species, several studies have found correlations between anonymous methylation-sensitive AFLP markers and leaf traits (Herrera & Bazaga 2013), flower morphology (Herrera & Bazaga 2010) and fitness-related traits (Medrano et al. 2014) in natural populations. While it is tempting to interpret these correlations in terms of contributions to adaptation, they may be simply the result of genetic differences. Experiments will be required to identify which loci are involved, and confirm causal relationships between epigenetic variation and phenotypes. In quantitative and ecological genetics, phenotypic variation is described with the classic equation V P =V G +V E +V G9E where V P is the total variance in phenotype, and V G ,V E and V G9E are the fractions of V P that can be ascribed to genetic variation, environmental influences or the interaction between the two. If epigenetically based heritable phenotypes exist not only in epiRILs but also in nature, then the equation might be improved to V P =V G +V EPI +V E +V G9EPI +V G9E + V EPI9E +V G9EPI9E (see also Gorelick 2005). So far, nearly all evidence for this is correlative and therefore inconclusive, but with increasing possibilities for whole-genome and -methylome sequencing, we should now be able to begin to explore this equation with empirical data. What are the ecological consequences of epigenetic variation? By mediating phenotypic plasticity, DNA methylation can facilitate response to various biotic and abiotic conditions, and persistence in different environments. To test this hypothesis, Herrera et al. (2012) investigated the relationship between epigenetic variation and environmental conditions in an easily manipulated yeast. Using in vivo demethylation by the methylation inhibitor azacytidine, they showed that the exploitation of nectar of varying sugar concentrations in flowers depended on DNA methylation. This type of experiment is ideal for isolating phenotypic plasticity, but more difficult to achieve for many long-lived plants. Nevertheless, a handful of studies have provided evidence that DNA methylation may contribute to the response to environmental factors under natural conditions. For example Herrera & Bazaga (2011) found both DNA sequence and methylation polymorphisms in Viola ©2017 The Authors. Ecology Letters published by CNRS and John Wiley & Sons Ltd 1582 C. L. Richards et al. Review and Synthesis cazorlensis were correlated with herbivory damage and habitat. In addition, variation in genomic DNA methylation has been correlated with shifts in species range (Richards et al. 2012; Xie et al. 2015), and differentiation of populations (Platt et al. 2015; Foust et al. 2016; but see Robertson et al. 2017). Interactions between plants and biotic or abiotic stressors can prime plants for a more rapid or vigorous response upon a second exposure in the future (Conrath et al. 2002), and additional epigenetic mechanisms such as histone modifications may be responsible for such sustained stress memory (e.g. Jaskiewicz et al. 2011). In the model plant A. thaliana, there has been some evidence suggesting that the epigenetic contribution to response to ecologically important factors like nutrient availability varied among natural accessions (Bossdorf et al. 2010). Experiments with epiRILs demonstrated that epigenetic variation among lines led to functional diversity that had very similar effects on population and ecosystem functioning as found for genetic and species diversity effects: higher epigenetic variation created variation in phenotypes that translated into increased productivity and resistance of experimental populations (Latzel et al. 2013). The combination of studies in natural accessions and epiRILs suggests that epigenetic diversity may be an important component of functional biodiversity, and that epigenetic variation can be indirectly involved in evolution by modifying natural selection at the community level. What are the evolutionary consequences of epigenetic variation? Many studies have investigated the role of environmentally induced and spontaneous epigenetic modifications in evolutionary theory (Jablonka & Raz 2009; Slatkin 2009; Day & Bonduriansky 2011; Geoghegan & Spencer 2012; Klironomos et al. 2013; Charlesworth & Jain 2014; Furrow 2014; Wang & Fan 2014; Kronholm & Collins 2016). Ultimately, the impact of spontaneous epigenetic variation in evolution will depend on the rates and stability of epigenetic changes, and the distribution of their phenotypic effects (Kronholm & Collins 2016). Modelling studies show that if spontaneous epigenetic changes occur at faster rates than genetic changes, this could lead to evolutionary dynamics where phenotypic changes are first driven by epigenetic changes, and become genetically encoded later (Klironomos et al. 2013; Kronholm & Collins 2016). These modelling studies have also shown that environmentally induced epigenetic changes that are inherited across generations could be adaptive in rapidly changing environments (Robertson & Richards 2015). However, the significance of such environmentally induced variation for adaptation will strongly depend on its persistence. While theoretical models show that epigenetic variation has the potential to change evolutionary dynamics, more data are needed to clarify the role of epigenetics. For instance detailed analyses in A. thaliana have revealed little evidence for environment-induced epigenetic variation that persists for several generations (Hagmann et al. 2015). We also have little insight into what determines transmissibility of epigenetic variants, and how this varies between species or contexts. In Helleborus foetidus, there was natural variation in transmission of DNA methylation to pollen, and this was correlated with seed size and seedling recruitment success (Herrera et al. 2014). This suggests that transmission fidelity may be a selectable trait. Long-term data on epigenetic response to environmental conditions in different species and ecological contexts would provide critical insight about when epigenetic mechanisms are potentially important for evolutionary processes. WHAT CAN BE TRANSFERRED FROM MODEL TO NONMODEL SPECIES? The transfer of information from model systems combined with advances in sequencing and bioinformatics approaches has initiated a powerful next step for ecological epigenetics due to more precise insights into function, and an increase in genome coverage. Some of the detailed information on epigenetic mechanisms that we have learned from model species is already useful in non-model systems. Gene annotations in model species provide information on genes that code for conserved components of the epigenetic machinery or genes that are epigenetically regulated. Studies of how DNA methylation is directed to specific genomic regions via small RNAs (RNA directed DNA methylation [RdDM]; Khraiwesh et al. 2010), methyltransferases (Noy-Malka et al. 2014), and other epigenetic modifications like histone modifications demonstrate that epigenetic mechanisms are controlled by evolutionarily conserved machinery (Rensing et al. 2008; Widiez et al. 2014). This information can be used to identify homologs in non-model species, which can then be targets for knock-outs or transformation to validate function in future studies (Kobayashi et al. 2013; Alvarez et al. 2015; Xie et al. 2015). Mutations occurring in genes related to the epigenetic machinery can have strong effects on epigenetic variation. In a study of the accumulation of mutations in replicate A. thaliana lines, one line showed a single spontaneous mutation in a methyltransferase, MEE57. This mutation appears to have led to an increased rate of epimutation at CG sites, resulting in 40% more methylation differences after 30 generations in this line compared to the other lines (Becker et al. 2011). Similarly, in natural A. thaliana populations, Dubin et al. (2015) found a link between alternative alleles of the DNA methyltransferase CMT2 (responsible for CHG and CHH methylation of certain classes of TEs) and the epigenome’s capacity to respond to temperature changes. In contrast, the CMT3 homolog double mutants in maize are not viable (Li et al. 2014), indicating that loss of methylation can have more drastic effects in some species. Studies in A. thaliana have exposed how DNA methylation effects are often associated with TE silencing. The vast majority of small RNAs (more than 60%) are complementary to TE sequences, and are involved in guiding DNA methylation to TEs through RdDM (Matzke & Mosher 2014). Small RNAs target DNA methylation to long terminal repeats (LTRs) of retrotransposons, and inhibit TE transcription. Reduction in DNA methylation at that location can be associated with the upregulation of TE expression, potentially creating new genetic variants, and phenotypes. Studies in RdDM deficient A. thaliana have shown that TEs get activated and introduce new copies of themselves in the genome (Mirouze et al. 2009; Ito et al. 2011). Thus, RdDM could be an important mechanism that protects the genome from TE ©2017 The Authors. Ecology Letters published by CNRS and John Wiley & Sons Ltd Review and Synthesis Ecological plant epigenetics 1583 Ragunathan, K., Jih, G. & Moazed, D. (2014). Epigenetic inheritance uncoupled from sequence-specific recruitment. 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