Effects of Epigenetic Diversity

A. Epigenetic effects at the population level.

We cannot start to understand how epigenetic diversity affects populations without first talking about intraspecific diversity. A species is a group of living individuals that can exchange genes or interbreed. By definition, intraspecific diversity is the variation occurring between individuals belonging to the same species, and it is the main factor underlying diversity at the population level. While many studies on ecosystem functioning focus on the interspecific facet of diversity, diversity also includes an intraspecific facet that is defined by phenotypic, functional, and genetic diversity measured within a single species (Schaaf et al., 2003; Bolnick et al., 2011). Generally speaking, this intraspecific trait variability increases the functional diversity of plant communities, a key component of biodiversity with important implications for species coexistence and ecosystem functioning. Intraspecific genotypic and phenotypic diversity has been demonstrated to account for a big part of the total biodiversity measured in plants and animals, representing in some cases up to a quarter of the total variability measured in communities (Raffard et al. 2018, 2019). In the last couple of decades, it has also become clear that intraspecific variation exists at the epigenetic level, for example, DNA methylation.

Epigenetic variation can be under genetic control, but some parts are independent of it due to spontaneous epimutations (Becker et al., 2011; Van Der Graaf et al., 2015) or following environmental induction (Jiang et al., 2014; Quadrana & Colot, 2016). These independent epimutations hold the most potential for finding novel intraspecific phenotypic differences (Bossdorf et al., 2008; Richards et al., 2017). So far, in‐depth documentation of intraspecific epigenetic variation has been restricted to model plant species such as Arabidopsis thaliana, Oryza sativa, and Zea mays for which extensive genomic and epigenomic resources exist (Becker et al., 2011; Schmitz et al., 2013; Van Der Graaf et al., 2015; Kawakatsu et al., 2016). However, with high throughput sequencing techniques becoming more affordable and accurate every day, new plant species are being added to the pool for which genomic resources are readily available, increasing research on natural genetic and epigenetic variation in non-model species (Richards et al., 2017).

Some studies could show that variation in DNA methylation is common in natural populations exceeding DNA sequence variation when comparing populations from different ecological origins (Herrera & Bazaga, 2010; Richards et al., 2012; Schulz et al., 2014). It is, therefore, essential to consider how epigenetic diversity varies between populations. Take, for example, two populations that are separated in time and/or space and are subjected to different conditions or environmental cues. If environmental conditions can induce the occurrence of new epialleles, these two populations will, in theory, present different epigenetic marks that mirror their specific environmental conditions, and those marks will potentially be inherited by the offspring (Richards, 2006). In line with this expectation, several studies have confirmed that populations show epigenetic differentiation between habitats. For example, in 2012, Richards and colleagues found that variation in some epigenetic loci in Fallopia japonica plants might be associated with specific local conditions (Richards et al., 2012). Another study by Lira-Madeiros et al. (2010) showed that individuals from a single population had similar genetic but divergent epigenetic profiles characteristic for the population in a particular environment. The evidence in these studies suggests that epigenetic variation among populations could reflect local acclimation and thus can play a role in helping individuals to cope with different environments.

The most commonly documented ecological effects of epigenetic diversity on populations involve the productivity or fitness of the population studied, and they can occur through different mechanisms. In 2010, Bossdorf and colleagues found that experimental alteration of DNA methylation strongly affected growth, fitness, and phenology in A. thaliana individuals. In another study, Latzel et al. (2013) suggested that epigenetic variability might affect how different epiRILs ("Epigenetic Recombinant Inbred Lines" Johannes et al., 2009) respond to treatment with salicylic acid and jasmonate, which are the main hormones involved in plant defense responses. In a series of experiments, Fieldes and colleagues showed that demethylation agents affected the fitness and phenological traits of Linum usitatissimum (Fieldes 1994; Fieldes & Amyot 1999; Fieldes et al. 2005). Moreover, evidence is growing that shortfalls in genetic diversity can be balanced by epigenetic diversity and facilitate plant population success. In 2013, Rollins and colleagues used two herbaceous species (Arctotheca populifolia and Petrorhagia nanteuilii) that showed evidence of fast morphological changes to examine if high genetic diversity is necessary for invasion success. Interestingly, they found that these two species had successfully established and adapted to a new habitat range in Australia, despite having considerably low genetic diversity in their native ranges (Rollins et al., 2013). Their findings provide an example of successful colonization processes that do not depend on high genetic diversity and align with similar findings (Richards et al., 2012) and theoretical lines of thought (Geoghegan & Spencer, 2012).

Although most studies on epigenetic effects involve short-lived plant species, methylation differences can also affect morphological and physiological processes in tree species. For example, a study by Raj et al. (2011) suggests that there might be a possible epigenetic basis for differences in transcriptomic profiles between poplar trees growing in distinct environments. In addition, white mangrove trees can exhibit striking morphological differences that might be related to a higher epigenetic diversity (Lira-Madeiros et al., 2010). Furthermore, changes in epigenetic marks have been associated with a wide variety of processes, including aging, organ maturation, and bud set or bud burst (Fraga et al. 2002; Santamaria et al. 2009; Valledor et al. 2010; Carneros et al., 2017; Lu et al., 2019). In recent years, studies on tree species responses that are epigenetically regulated have become more common (reviewed in Bräutigam et al., 2013). However, due to the limitations of working with long-lived species, natural epigenetic variation in tree species remains under-explored (Heer et al., 2018b).

B. Epigenetic effects at the community level.

We have discussed how epigenetic diversity can affect intraspecific diversity and how intraspecific diversity, in turn, changes how populations respond to their environmental conditions. Populations, although, are part of ecological communities. Although several studies have explored the importance of genetic diversity at the community level (Downing et al., 2002; Helm et al., 2009; Taberlet et al., 2012, Lamy et al., 2016), given the limitations of working at large-scale levels, quite a number of these studies disregard intraspecific variation and focus instead on trait means and trait variation among species (Bolnick et al., 2011; Zeng, Durka & Fischer, 2017). Nevertheless, community phenotypes can also arise from intraspecific trait variation (Siefert et al., 2015; Des Roches, 2018; Bongers et al., 2020), and sometimes intra-and interspecific diversity can have opposing effects (Hanh et al., 2017). Little is known about the ecological implications of epigenetic diversity on plant natural populations (Herrera, 2017), and very few studies have tried to assess the contribution of epigenetic variation to ecosystem dynamics in plant communities (Balao, Paun & Alonso, 2017; Herrera, Medrano & Bazaga, 2017; Mounger et al., 2020). However, considering that genetic variation has been proven to affect community-level interactions, it is safe to assume that epigenetic variation can have similar effects on communities. This section will address the three primary biological interactions (competition, predation and symbiosis) occurring within a community and the implications of epigenetic diversity on them.

i. Herbivory

Epigenetic responses to plant herbivory have been discussed in a previous chapter, so we will not detail them here. We will instead focus on how genetic diversity and, consequently epigenetic diversity, might affect interspecific interactions at the community level. Numerous studies have found effects of plant diversity on arthropod species richness and abundance (Koricheva et al. 2000, Crutsinger et al. 2006; Haddad et al., 2009; Robinson et al., 2012) and consumptive interactions (Moreira & Mooney 2013, Abdala-Roberts et al., 2015), with the basis of such effects being variation in ecologically critical functional traits among plant species or genotypes within species (Elle & Hare 2002, Mooney & Singer, 2012; Fernandez-Conradi et al., 2017). The influence of host-plant intraspecific diversity on arthropod communities has been extensively investigated and is a perfect example of how genetic variance can affect community-level interactions (reviewed in Koricheva et al., 2018). Similarly, epigenetic mechanisms can influence how plants respond to herbivory by, for example, inducing plant defenses (Verhoeven et al, 2010, affecting within-plant herbivore distribution (Herrera et al., 2019), or changing phytohormone production and palatability (Latzel et al., 2020). Hence, epigenetic changes can directly affect arthropod communities through different processes.

In this latter example, the authors conducted a so-called Cafeteria test with Larvae from the Egyptian cotton leafworm (Spodoptera littoralis). This moth is a generalist herbivore that feeds on plants of at least 40 families. Due to this extreme polyphagy, it has been used in many bioassay experiments of leaf palatability. In this experiment, the Egyptian cotton leafworm was put on Trifolium repens plants that had been treated with various levels of 5-azacytidine application to induce various levels of methylation, as well as on normally methylated control plants. The caterpillars could then freely choose between the plants and preferred individuals that were intensively treated over control or moderately treated plants (F = 7.22, p < 0.001). To get an indication of the mechanism behind the varying preferences, Latzel et al. measured levels of phytohormones known to be involved in inducible defense production, such as jasmonic acid, which is considered the key hormone in establishing altered gene expression related to inducible defense.

Most plant diversity studies have focused exclusively on its effects within a single trophic level (herbivores), but plant diversity may also indirectly affect higher trophic levels, i.e., enemies and mutualists of herbivores (Haddad et al., 2011; Moreira & Mooney, 2013). There are two ways in which plant diversity can influence higher trophic levels. The first one is related to an increase in the number of herbivores present in a specific community. There is a positive correlation between plant species richness and the diversity of associated consumers. Approximately 90% of herbivorous insects present some degree of host specialization (Bernays & Graham, 1988). If plant species richness increases, so do the number of herbivore species, and if the number of herbivores increases, so will the abundance of mutualists and enemies of those herbivores. The second way is mediated by traits. In this case, plant diversity influences herbivore, mutualist, or enemy traits, such as herbivore susceptibility to enemies (Johnson et al., 2006; Moreira et al., 2012; Moreira & Mooney, 2013). If epigenetic diversity can increase (or decrease) plant species richness and plant diversity, it will also affect arthropod communities.

Changes in aboveground net primary productivity (ANPP) can also influence plant-herbivore interactions. Higher ANPP means more energy available for consumers, and therefore more herbivore species or a higher number of individuals per species will be able to use this energy. Consequently, a higher number of herbivores will result in a higher number of enemies or mutualists. There is evidence that epigenetic diversity positively affects biomass production in Arabidopsis (Latzel et al., 2013). Although further research is needed to assess if this positive effect also extends to other plant species, epigenetic diversity has the potential of influencing herbivore populations by increasing plant biomass production and ANPP.

ii. Competition

Competition is a long-term interaction between organisms in which both organisms suffer adverse effects. Several studies have shown that plasticity in relevant functional traits derived from genetic variation can contribute to the success of invasive plant species by increasing their fitness across a range of habitats (Richards et al., 2006; Muth & Pigliucci, 2007; Walls, 2010; Davidson et al. 2011). Invasive species can have significant effects on ecosystem functioning, and invasions can lead to a loss of plant diversity (Linders et al., 2019). As discussed above, changes in plant diversity can extend to other trophic or organizational levels. Hence the invasive capacity of certain plant species can have effects on plant populations and communities. Interestingly, many invasive plant species appear to perform well despite having low levels of genetic variation. Dlugosch & Parker (2008a,b) reported that even though several plant species had suffered substantial losses of genetic diversity when compared to source populations, only one showed a significant decline in phenotypic variance. Several authors have suggested that epigenetic diversity increases the chances of successful colonization events in invasive species (Richards et al., 2006; Loomis & Fishman, 2009; Douhovnikoff & Dodd, 2015; Slotkin, 2016; Mounger et al., 2020). Even if most of the research in this field is focused on understanding plant invasion dynamics, epigenetic diversity could help explain how pioneer plant species colonize new habitats in successional processes. A recent study by Venturelli and colleagues (2015) showed that some allelochemicals released by Triticum aestivum (and other plant species) inhibit histone deacetylases, a group of enzymes involved in chromatin modification. Even more recently, Puy et al. (2020) showed that differences in DNA methylation of parental individuals affected offspring phenotypes. In their study, offspring of plants under stronger competition presented resource-conservative phenotypes and developed faster, suggesting that transgenerational phenotypic plasticity influenced competitive plant-plant interactions (Puy et al., 2020).

iii. Symbiosis

Symbiotic relationships are long-term interactions between two organisms. The term "symbiosis" includes a broad range of biological interactions that can be classified according to the effects the relationship has on each organism (Relman, 2008). The three major types of symbiotic interactions are mutualism, commensalism, and parasitism. Mutualistic interactions have a positive effect on both organisms, commensalism has a positive effect on one individual while the other is neither benefited nor harmed and in parasitism, the parasite benefits from the host and the host is damaged in some way.

Plants harbor an extreme diversity of symbionts, and their responses to symbiotic microbes and fungal organisms are probably the most studied interactions. Substantial literature documents the range of phenotypic variants conferred by symbiotic organisms, and many examples of mutualist-induced changes that are genotype-dependent in plant traits have been reported (Vannier et al., 2015; Gilbert, Tozer & Westoby, 2019; Wen et al., 2020). Several mechanisms control these interactions, but the plant epigenome has emerged as a critical modulator of pathogenic and symbiotic interactions (Bazin et al., 2012; Yu et al., 2013; Espinas, Saze & Saijo, 2016; Zogli & Libault, 2017). Interestingly, some symbiotic interactions could be considered epigenetic phenomena because plant endophytes can alter gene expression and phenotype without causing changes in the underlying DNA structure (Rodriguez et al., 2008). A major part of the research on epigenetic control of plant-symbiont interactions focuses on mycorrhizal fungi due to the importance of these organisms for processes that can affect plant fitness (e.g., nitrogen-fixing reactions, nutrient uptake, and abiotic and biotic stress tolerance). Nevertheless, some studies have tried to determine the role of epigenetic changes in plant-pollinator interactions, another common type of mutualism, and plant-parasite interactions. For example, in 2009, Marfil and colleagues associated distinctive methylation profiles with aberrant flower phenotypes in Solanum ruiz-lealii plants. This can potentially influence pollination rates, as bumblebees, the only pollinator of Solanum plants, do not visit aberrant flowers (Marfil, Camado & Masueli, 2009). In another study by Kellenberger et al. (2016), demethylation of Brassica rapa plants resulted in reduced attractiveness of the plants to pollinator bees (Kellenberger, Schlüter and Schiestle, 2016). Quite recently, Samarth et al. (2020) have introduced the hypothesis of an "epigenetic summer memory" as a driver of mast flowering (mass synchronized flowering of perennial plants over a wide geographical area), an event that has major impacts on trophic interactions (Kelly, 1994).

Parasitic interactions have received a bit more attention, mainly due to the high economic losses plant parasites cause on crops, and recent studies have attempted to throw some light on the epigenetic regulation of plant-nematode interactions. For example, Hewezi et al. showed that cyst nematodes could induce changes in the root epigenome (Hewezi et al., 2017). In another study, Sahid et al. (2018) suggest that miRNA-mediated changes in the host gene expression might act in a way beneficial to the nematode, and Pratx and colleagues explored the role of the epigenetic machinery of the root-knot nematode Meloidogyne incognita (Pratx et al., 2018). However, despite the advances of the modern literature, to our knowledge, all the research so far has focused on understanding the epigenetic regulation of symbiotic interactions, and there are no studies on the potential effects of epigenetic diversity on community structure or dynamics.

C. Epigenetic effects at the ecosystem and landscape level

We have discussed how variation at the species level can extend into higher organizational levels. When communities are grouped with their abiotic surroundings, they are considered an ecosystem. Genetic diversity and epigenetic diversity shape the structure and dynamics of each level at the population and community levels. In contrast, landscape structure can have significant effects on the genetic diversity of populations (Münzbergová et al., 2013; González et al., 2020; Lehmair et al., 2020). The study of large-scale interactions is limited by the complexity of each system and the sheer number of different variables and processes encompassed in the system. The further up the organizational level we move, the more difficult it becomes to study possible epigenetic effects. From a traditional perspective, studies on genetic diversity at large-scale levels (i.e., over large areas and for many species) are still demanding, given the need for field sampling and the still more or less high costs of genetic analysis. (Taberlet et al., 2012).

Furthermore, plant functional traits can strongly influence ecosystem properties, acting in several contexts that include the effects of dominant or foundation species and keystone species or the interactions between the individuals of the ecosystem (Hooper et al., 2005). Regardless, several studies have shown that the genetic diversity of dominant plant species can affect essential ecosystem functions. Genotypic variation in trees of the Populus genus is a well-studied example of how genetic diversity can influence nutrient cycles and energy fluxes. Differences among several aspen (Populus tremula) genotypes can have substantial effects on litter decomposition and nutrient release (Madritch et al. 2007; Bandau et al., 2016; Hughes et al., 2018). Nutrient fluxes can also be affected by foliar secondary metabolites. For example, condensed tannins (CT) influence carbon and nitrogen cycles specifically by affecting the microbial communities that mediate these cycles. In other hybridizing cottonwoods (Populus fremontii, Populus angustifolia), the chemical composition of the leaf litter impacts the rate of decay and nutrient flux to a degree that is comparable with the effects of species diversity (Schweitzer et al. 2005). Since nutrient fluxes are ecosystem-wide processes, any change in these fluxes will affect every population or community within the ecosystem (Fischer et al., 2007).

Moreover, disparate groups of organisms demonstrate significant relationships between community composition and concentrations of CT in Populus that link above- and below-ground processes. For example, Schweitzer et al. (2008) showed that microbial community composition differs significantly in soils beneath Populus genotypes that varied in their expression of foliar CT. Thus, even when only a few traits vary between genotypes, microorganisms belonging to different functional groups can occur. In another study, Madritch and others (2007) found that the CT concentration of frass deposition of two herbivores affected below-ground respiration and extracellular enzyme activity of microbial communities (Schweitzer et al., 2008).

When including epigenetic diversity into large-scale studies, several layers of complexity are added to the mix. In contrast to genetic or genomic patterns, the strength, the effects of epigenetic diversity at a landscape level, and its evolutionary implications are poorly understood. Generally speaking, disentangling the effects of epigenetic variation from genetic variation is not a straightforward process. Some advances have been made in this regard by using clonally propagated species and common garden experiments (Bossdorf et al., 2008; Richards et al., 2017). Despite the limitations, many recent landscape-level studies have investigated the role of epigenetics in intraspecific trait variation and adaptation (Medrano et al., 2014; Dubin et al., 2015; Preite et al., 2015; Foust et al., 2016; Gugger et al., 2016; Herrera et al., 2016; Keller et al., 2016; Alakärpa et al., 2018; Gáspár, Bossdorf & Durka, 2018). These studies focus on the relationship between genetic and epigenetic variation at the landscape level, correlations between environmental variables and epigenetic marks, and correlations between epigenetic marks and plant phenotypic traits (Whipple & Holeski, 2016).