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Variation in nucleotide sequence (i.e. genuine genetic variation) is not the only information in chromosomes that is heritable across generations. Epigenetic information, which is based on variations in DNA methylation or chromatin states, often is also heritable during cell propagation, particularly in plants, where epigenetic states of genes that influence phenotypic traits can be inherited over generations (Henderson & Jacobsen, 2007; Jablonka & Raz, 2009; Verhoeven et al., 2010). In theory, heritable epigenetic variation could directly or indirectly influence the course of evolution in plants, as it can affect the processes of adaptation and divergence through selection of stable epigenetic variants without involvement of genetic variation, or through evolutionary change in which epigenetic modifications would guide the selection of correlated genetic variation (Kalisz & Purugganan, 2004; Rapp & Wendel, 2005; Jablonka & Raz, 2009). Nevertheless, before the hypothesized role of epigenetic variation in plant microevolution can be assessed, fundamental questions remain to be addressed in a real-world context about its magnitude, structuring within and among natural plant populations, possible correlations with genetic or phenotypic traits related to fitness, and degree of autonomy in relation to the genetic context (Kalisz & Purugganan, 2004; Rapp & Wendel, 2005; Richards, 2006). DNA methylation is the best-described epigenetic mechanism, and it is involved in nearly all well-documented instances of transgenerational epigenetic inheritance in plants (Akimoto et al., 2007; Henderson & Jacobsen, 2007; Jablonka & Raz, 2009; Verhoeven et al., 2010). Given also that relatively simple and cost-effective molecular methods are available to study patterns of genome-wide DNA methylation in nonmodel organisms lacking detailed genomic information (Reyna-López et al., 1997; Xiong et al., 1999), investigating natural patterns of DNA methylation in wild plant populations emerges as a first step towards assessing the potential significance of epigenetic variation in microevolution (Kalisz & Purugganan, 2004; Richards, 2006).
The molecular mechanisms and functions associated with DNA methylation are reasonably well understood for model plants (Finnegan et al., 1998b; Vanyushin, 2006; Zhang, 2008), and a number of studies have also documented intraspecific epigenetic variation in model and cultivated species (Xiong et al., 1999; Ashikawa, 2001; Cervera et al., 2002; Keyte et al., 2006; Vaughn et al., 2007; Salmon et al., 2008). Almost nothing is known, however, on the amount and structuring of standing methylation-based epigenetic variation in wild plant populations (but see Li et al., 2008). Of particular relevance from an evolutionary perspective is to ascertain whether species-level epigenetic variation is, like sequence-based genetic variation, structured into distinct between- and within-population components, and whether such epigenetic structuring can be interpreted in adaptive terms, that is, as maintained by divergent selection (Kalisz & Purugganan, 2004; Bossdorf et al., 2008). For example, under conditions of extensive gene flow connecting discrete populations of a species into a genetically coherent, panmictic unit, a significant correlation linking between-population epigenetic differentiation with adaptive genetic divergence would be suggestive of epigenetic differentiation being directly or indirectly driven by variable selection. In this paper we address these questions by studying the extent and pattern of cytosine methylation in a set of discrete populations of the southern Spanish endemic violet Viola cazorlensis (Violaceae), and explore the relationship to adaptive genetic divergence between populations.
We use the technique of methylation-sensitive amplified polymorphism (MSAP), a modification of the amplified fragment length polymorphism method (AFLP) which takes advantage of the differential sensitivity of a pair of isoschizomeric restriction enzymes to site-specific cytosine methylation (McClelland et al., 1994; Reyna-López et al., 1997). By allowing the determination of the methylation status of anonymous regions of the genome susceptible to methylation, the MSAP method enables the identification of methylation-based epiallelic markers in wild populations of nonmodel plants in absence of detailed genomic information. Populations of V. cazorlensis are ideally suited to a study of patterns and possible adaptive correlates of epigenetic variation. Genetic analyses of wild populations of this narrowly-distributed plant have shown that, despite their spatial discreteness, populations are interconnected by extensive gene flow, neutral genetic variation is not spatially structured across populations and there is a lack of regional drift–gene flow equilibrium because of an overwhelming predominance of gene flow over drift (Herrera & Bazaga, 2008). In the face of extensive gene flow, however, populations of V. cazorlensis exhibit distinctive signatures of adaptive genetic divergence (Herrera & Bazaga, 2008). These circumstances combine favourably to render V. cazorlensis an excellent study system to investigate possible adaptive epigenetic variation by searching for correlations between methylation-based epigenetic differentiation and adaptive genetic divergence between populations.
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The MSAP technique can underestimate genome-wide levels of DNA methylation, because it detects only methylation at CCGG sites and is unable to discriminate between methylation and fragment absence when the two cytosines are methylated on both strands, as neither HpaII nor MspI cleave such hypermethylated sites. Despite this limitation of the method (Cervera et al., 2002; Salmon et al., 2008), our study has revealed extensive levels of DNA methylation in the genome of wild-growing V. cazorlensis plants. Roughly half of the loci from the MSAP analysis were in a methylated state in a significant fraction of the individuals sampled. Ninety-five per cent of these methylation-susceptible loci were polymorphic, thus considerably higher than the 65% polymorphism exhibited by nonmethylated loci. Shannon’s index measurements of methylation-associated, epiallelic diversity occurring in the sample of individuals studied were greater than those corresponding to sequence-based genetic diversity. Variation in methylation levels among different organs or developmental stages has been observed for several plant species (Xiong et al., 1999; Portis et al., 2004), but such effects should be disregarded as important sources of the extensive individual variation in methylation found in this study, as all DNA samples analysed were obtained from the same organ and collected at identical developmental stage (newly expanded, fully-grown leaves). Polymorphism levels of methylation-susceptible and nonmethylated loci found here for V. cazorlensis are virtually identical to those obtained in a MSAP study of 30 populations and lines of wild and cultivated morphotypes of Brassica oleracea (96 vs 63%, respectively; Salmon et al., 2008). Similarly high methylation levels, and a greater polymorphism and/or diversity of methylation-susceptible loci relative to nonmethylated ones, have been also reported for other wild (Li et al., 2008; Marfil et al., 2009) and cultivated plants (Keyte et al., 2006; Fang et al., 2008). Together, these studies (see also Cervera et al., 2002; Riddle & Richards, 2002; Vaughn et al., 2007) suggest that extensive intraspecific epigenetic variation of the sort found here for V. cazorlensis is probably widespread in wild plant populations.
Recent studies have stressed the nearly complete absence of information on the organization of epigenetic variation in natural plant populations (Kalisz & Purugganan, 2004; Rapp & Wendel, 2005; Bossdorf et al., 2008). One important result of our study was therefore the discovery that naturally occurring, methylation-based epigenetic variation in V. cazorlensis was structured into distinct between- and within-population components, in a manner that could be considered analogous to the structuring of sequence-based, genuine genetic variation. There existed extensive within-population variance owing to individual epigenotypic variation (87% of total), a critical prerequisite for epigenetic variation to have some microevolutionary potential (Kalisz & Purugganan, 2004). In addition, there was significant epigenetic population differentiation at both single-locus and multilocus levels. About one-third of methylation-susceptible loci exhibited statistically significant variation across populations in the proportion of methylated and nonmethylated states, and the AMOVA revealed significant multilocus epigenetic differentiation among populations, the between-population component accounting for 13% of total epigenetic variance in the sample. Although no comparable quantitative data are available, epigenetic differences between populations have been also reported for another species (Li et al., 2008).
Viola cazorlensis plants are very long-lived and cannot be propagated vegetatively, so that direct verification of the transgenerational constancy of observed epigenetic variation between populations and individuals by means of controlled crosses or common garden experiments was not possible. Some constancy, however, can be safely assumed in view of the data showing that inheritance of DNA methylation-based epigenetic marks across generations seems to be the rule in plants (Henderson & Jacobsen, 2007; Jablonka & Raz, 2009; Verhoeven et al., 2010), and that intraspecific differences in DNA methylation patterns can be stably maintained for long periods, as inferred from differences between Arabidopsis thaliana ecotypes separated by relatively large evolutionary distances (Vaughn et al., 2007). Under that assumption, and provided that the populations of V. cazorlensis studied form a single panmictic unit connected by extensive gene flow (Herrera & Bazaga, 2008), the maintenance of a between-population component of epigenetic variation can be interpreted as reflecting stable population differences presumably maintained by variable selection, as discussed in later text.
The population genomic approach (Black et al., 2001) adopted in this study to examine adaptive genetic divergence between V. cazorlensis populations has proven useful for dissecting functionally important genetic variation in natural populations of nonmodel organisms (Vasemägi & Primmer, 2005; Foll & Gaggiotti, 2008). Scanning patterns of DNA polymorphisms at the genomic level through genotyping numerous random anonymous loci spread over the entire genome of individuals in several populations, it is possible to identify genomic regions that exhibit deviant patterns of variation relative to the rest of the genome, presumably because of direct or indirect (through linkage) selection (Black et al., 2001; Luikart et al., 2003; Foll & Gaggiotti, 2008). Amplified fragment length polymorphism genotyping allows for both a large number of markers and an accurate assessment of baseline levels of neutral genetic variation across the whole genome, and markers from methylation-insensitive EcoRI-MseI primer combinations are thus well suited to the population genomics analysis conducted in this study to detect loci showing signatures of divergent selection (Bonin et al., 2007; Meudt & Clarke, 2007; Foll & Gaggiotti, 2008). The Bayesian method used here revealed that c. 6% of conventional AFLP loci had a decisive posterior probability of being subject to selection (or being linked to selected loci). This proportion nearly triplicates the figure obtained in an earlier analysis of the same data set using a frequentist procedure (Herrera & Bazaga, 2008), although all the outlier loci revealed by the earlier study have been corroborated here. This finding agrees with results reported by Foll & Gaggiotti (2008) for another reanalysis of AFLP data using their Bayesian approach, further stressing the superiority of this method over frequentist ones in population genomic scans for detecting candidate loci subject to selection.
The present study has shown that, in the set of V. cazorlensis populations studied, methylation-based epigenetic differentiation of populations is associated with adaptive genetic divergence. By contrast, earlier studies also using the MSAP technique generally found weak or nonsignificant correlations between epigenetic and genetic variation across populations, ecotypes or accessions of the same species (Ashikawa, 2001; Cervera et al., 2002; Keyte et al., 2006; Li et al., 2008; Salmon et al., 2008). Differences in analytical approaches probably account for these contrasting results. In earlier studies, genetic characterization of the groups under comparison used multilocus approaches based on the whole set of nonmethylated AFLP fragments. As most of the latter presumably correspond to neutral, noncoding portions of the genome, their failure to find clear relationships between multilocus genetic and epigenetic differences is not surprising. By contrast, by focusing only on the small subset of adaptive, presumably nonneutral (or linked to nonneutral) AFLP loci, our analytical approach presumably enhanced the likelihood of detecting biologically significant relationships between genetic and epigenetic variation. At the between-population level, the dbRDA revealed that variation in allelic frequencies of 10 outlier loci accounted statistically for most epigenetic variance between populations. This strong statistical association between epigenetic and adaptive genetic divergence between population has a biological basis, as shown by dbRDA and association analyses at the individual plant level. These results provide compelling evidence for a close functional association in the genome of individual V. cazorlensis plants between the methylation state of some methylation-susceptible loci and the allelic condition of some nonneutral, adaptive AFLP loci. As outlined later in this text, this result has implications in relation to the evolutionary mechanisms underlying the adaptive differentiation of populations (Kalisz & Purugganan, 2004; Jablonka & Raz, 2009).
In plant genomes, DNA methylation controls gene expression levels through interfering with transcription and influencing the formation of transcriptionally silent heterochromatin (Finnegan et al., 1998b; Zilberman et al., 2007; Jablonka & Raz, 2009). When methylation affects large-effect genes, methylation changes can induce drastic discontinuous phenotypic alterations, including modification of floral symmetry, homeotic transformation of floral organs, and inhibition of fruit ripening (Finnegan et al., 1998a; Cubas et al., 1999; Manning et al., 2006; Marfil et al., 2009). However, methylation changes can also induce less dramatic phenotypic variation involving continuous traits such as flowering time, plant size, fecundity and resistance to pathogens or toxins (Finnegan et al., 1996, 1998a; Sha et al., 2005; Giménez et al., 2006; Akimoto et al., 2007; Jablonka & Raz, 2009), which suggests that genic methylation also affects suites of small-effect genes involved in the determination of complex quantitative traits (Johannes et al., 2009). Results of the present study on V. cazorlensis are compatible with a scenario in which natural phenotypic variation among individuals and populations in quantitative traits (Herrera, 1990, 1993) could be accounted for by a combination of adaptive genetic variation and genetically controlled epigenetic differences: first, the significant associations found across plants between the presence/absence of some adaptive AFLP loci and methylation state of some methylation-susceptible loci, is suggestive of functional genetic–epigenetic connections within individual genomes; second, the allelic frequencies of some of the adaptive AFLP loci found here to be related to epigenetic population differentiation (loci 220 and 246; Table 2) are also correlated with population differences in quantitative floral traits (Table 4 in Herrera & Bazaga, 2008); third, the broad phenotypic differences in average metric floral traits that characterize V. cazorlensis populations (Herrera, 1990) may be partly accounted for by the epigenetic population differentiation documented in this study.
Natural variation in gene methylation can be under complex genetic and epigenetic control (Kalisz & Purugganan, 2004; Zhang, 2008; Jablonka & Raz, 2009), and establishing the degree to which epigenetic variation is autonomous from genetic variation is central to evaluating the evolutionary relevance of the former as an additional, rather than redundant, inheritance system (Richards, 2006; Bossdorf et al., 2008). A key aspect thus remaining to be elucidated is the evolutionary mechanism(s) underlying the three-way relationship linking the adaptive genetic, epigenetic and phenotypic divergence of the populations of V. cazorlensis studied here and by Herrera & Bazaga (2008). Assuming that phenotypic and genetic differentiation of populations have been driven by variable selection acting on genetically determined phenotypic traits (Herrera & Bazaga, 2008), such selection could be responsible for shaping the distributions of genotypes, epigenotypes or both. The first situation would exemplify a classical scenario of local adaptation through divergent selection on genetically determined traits. The other two possibilities would correspond to unexplored, but theoretically possible scenarios where evolution could proceed through selection of epigenetic variants without involvement of genetic variation, or through simultaneous, correlated selection on functionally linked, causative epigenetic and genetic variation (Kalisz & Purugganan, 2004; Rapp & Wendel, 2005; Richards, 2006; Jablonka & Raz, 2009). The data currently available do not allow one to discriminate among these alternatives for the V. cazorlensis study system, but our results suggest that correlated selection on epigenetic and genetic variation is a plausible evolutionary pathway. This intriguing possibility would simultaneously account for the association between epigenetic and adaptive genetic differentiation of populations, and the within-genome association between presence/absence of adaptive loci and the methylation state of methylation-susceptible loci. As selection is a process occurring within populations, this hypothesis could be tested through studies dissecting the contributions to fitness differences of individual epigenetic variation, individual genetic variation and their interaction in natural plant populations. Such expanded genetic–epigenetic framework could provide some of the ‘many pieces missing from the epigenetic puzzle’ (Bossdorf et al., 2008).