An ecologist's guide for studying DNA methylation variation in wild vertebrates

The field of molecular biology is advancing fast with new powerful technologies, sequencing methods and analysis software being developed constantly. Commonly used tools originally developed for research on humans and model species are now regularly used in ecological and evolutionary research. There is also a growing interest in the causes and consequences of epigenetic variation in natural populations. Studying ecological epigenetics is currently challenging, especially for vertebrate systems, because of the required technical expertise, complications with analyses and interpretation, and limitations in acquiring sufficiently high sample sizes. Importantly, neglecting the limitations of the experimental setup, technology and analyses may affect the reliability and reproducibility, and the extent to which unbiased conclusions can be drawn from these studies. Here, we provide a practical guide for researchers aiming to study DNA methylation variation in wild vertebrates. We review the technical aspects of epigenetic research, concentrating on DNA methylation using bisulfite sequencing, discuss the limitations and possible pitfalls, and how to overcome them through rigid and reproducible data analysis. This review provides a solid foundation for the proper design of epigenetic studies, a clear roadmap on the best practices for correct data analysis and a realistic view on the limitations for studying ecological epigenetics in vertebrates. This review will help researchers studying the ecological and evolutionary implications of epigenetic variation in wild populations.     

Natural epigenetic variation in the female great roundleaf bat (Hipposideros armiger) populations

Epigenetic modifications are considered to have an important role in evolution. DNA methylation is one of the best studied epigenetic mechanisms and methylation variability is crucial for promoting phenotypic diversification of organisms in response to environmental variation. A critical first step in the assessment of the potential role of epigenetic variation in evolution is the identification of DNA methylation polymorphisms and their relationship with genetic variations in natural populations. However, empirical data is scant in animals, and particularly so in wild mammals. Bats are considered as bioindicators because of their sensitivity to environmental perturbations and they may present an opportunity to explore epigenetic variance in wild mammalian populations. Our study is the first to explore these questions in the female great roundleaf bat (Hipposideros armiger) populations using the methylation-sensitive amplified polymorphism (MSAP) technique. We obtained 868 MSAP sites using 18 primer combinations and found (1) a low genomic methylation level (21.3?% on average), but extensive DNA methylation polymorphism (90.2?%) at 5'-CCGG-3' sites; (2) epigenetic variation that is structured into distinct between- (29.8?%) and within- (71.2?%) population components, as does genetic variation; and (3) a significant correlation between epigenetic and genetic variations (P?相似文献   

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.     

Epigenetics for ecologists

There is now mounting evidence that heritable variation in ecologically relevant traits can be generated through a suite of epigenetic mechanisms, even in the absence of genetic variation. Moreover, recent studies indicate that epigenetic variation in natural populations can be independent from genetic variation, and that in some cases environmentally induced epigenetic changes may be inherited by future generations. These novel findings are potentially highly relevant to ecologists because they could significantly improve our understanding of the mechanisms underlying natural phenotypic variation and the responses of organisms to environmental change. To understand the full significance of epigenetic processes, however, it is imperative to study them in an ecological context. Ecologists should therefore start using a combination of experimental approaches borrowed from ecological genetics, novel techniques to analyse and manipulate epigenetic variation, and genomic tools, to investigate the extent and structure of epigenetic variation within and among natural populations, as well as the interrelations between epigenetic variation, phenotypic variation and ecological interactions.     

Interactions between natural and farmed fish populations: information from genetic markers

Studies on genetic changes in farmed fish populations are reviewed, and the potential interactions between wild and farm escapee, and between wild and stocked, fish populations are discussed. Examples of the application of genetic markers in studies concerning survival and reproduction of stocked fish, and genetic and ecological interactions between stocks, are given for brook trout, Salvelinus fontinalis , brown trout, Salmo trutta , rainbow trout, Salmo gairdneri , cod, Gadus morhua , Guadalupe bass, Micropterus treculi , walleye, Stizostedion vitreum vitreum and chum salmon, Oncorhynchus keta . The various studies produced different results. Evidence for successful reproduction and genetic interactions between released and wild stocks have been found in a few cases. Stocked genetic material sometimes had a lower reproductive success than wild material. In one case the transplanted genetic material failed to acclimatize, and was apparently lost from the genepool in two generations. Investigations on the genetic and ecological interactions between wild and farmed populations are of great importance to the preservation of wild populations and their genetic resources.     

Epigenetics in natural animal populations

Phenotypic plasticity is an important mechanism for populations to buffer themselves from environmental change. While it has long been appreciated that natural populations possess genetic variation in the extent of plasticity, a surge of recent evidence suggests that epigenetic variation could also play an important role in shaping phenotypic responses. Compared with genetic variation, epigenetic variation is more likely to have higher spontaneous rates of mutation and a more sensitive reaction to environmental inputs. In our review, we first provide an overview of recent studies on epigenetically encoded thermal plasticity in animals to illustrate environmentally‐mediated epigenetic effects within and across generations. Second, we discuss the role of epigenetic effects during adaptation by exploring population epigenetics in natural animal populations. Finally, we evaluate the evolutionary potential of epigenetic variation depending on its autonomy from genetic variation and its transgenerational stability. Although many of the causal links between epigenetic variation and phenotypic plasticity remain elusive, new data has explored the role of epigenetic variation in facilitating evolution in natural populations. This recent progress in ecological epigenetics will be helpful for generating predictive models of the capacity of organisms to adapt to changing climates.     

Genome‐scale sampling suggests cryptic epigenetic structuring and insular divergence in Canada lynx

Determining the molecular signatures of adaptive differentiation is a fundamental component of evolutionary biology. A key challenge is to identify such signatures in wild organisms, particularly between populations of highly mobile species that undergo substantial gene flow. The Canada lynx (Lynx canadensis) is one species where mainland populations appear largely undifferentiated at traditional genetic markers, despite inhabiting diverse environments and displaying phenotypic variation. Here, we used high‐throughput sequencing to investigate both neutral genetic structure and epigenetic differentiation across the distributional range of Canada lynx. Newfoundland lynx were identified as the most differentiated population at neutral genetic markers, with demographic modelling suggesting that divergence from the mainland occurred at the end of the last glaciation (20–33 KYA). In contrast, epigenetic structure revealed hidden levels of differentiation across the range coincident with environmental determinants including winter conditions, particularly in the peripheral Newfoundland and Alaskan populations. Several biological pathways related to morphology were differentially methylated between populations, suggesting that epigenetic modifications might explain morphological differences seen between geographically peripheral populations. Our results indicate that epigenetic modifications, specifically DNA methylation, are powerful markers to investigate population differentiation in wild and non‐model systems.     

中国野生大豆遗传资源搜集基本策略与方法

遗传资源搜集原则是通过种子采集追求样本具有最高程度的遗传多样性。为了合理而有效地搜集野生大豆资源,近年来通过野生大豆居群考察和遗传多样性分析,初步明确了野生大豆资源居群的遗传多样性分布动态:遗传多样性地理的和生态的区域性、生态系统内居群的遗传相关性及各种生境下居群遗传多样性差异,从理论上奠定了野生大豆资源合理有效搜集的依据。根据居群遗传多样性的分布规律,初步建立了居群野生大豆资源的搜集策略和方法。     

Epigenetic patterns newly established after interspecific hybridization in natural populations of Solanum

Interspecific hybridization is known for triggering genetic and epigenetic changes, such as modifications on DNA methylation patterns and impact on phenotypic plasticity and ecological adaptation. Wild potatoes (Solanum, section Petota) are adapted to multiple habitats along the Andes, and natural hybridizations have proven to be a common feature among species of this group. Solanum × rechei, a recently formed hybrid that grows sympatrically with the parental species S. kurtzianum and S. microdontum, represents an ideal model for studying the ecologically and evolutionary importance of hybridization in generating of epigenetic variability. Genetic and epigenetic variability and their correlation with morphological variation were investigated in wild and ex situ conserved populations of these three wild potato species using amplified fragment length polymorphism (AFLP) and methylation‐sensitive amplified polymorphism (MSAP) techniques. We observed that novel methylation patterns doubled the number of novel genetic patterns in the hybrid and that the morphological variability measured on 30 characters had a higher correlation with the epigenetic than with the genetic variability. Statistical comparison of methylation levels suggested that the interspecific hybridization induces genome demethylation in the hybrids. A Bayesian analysis of the genetic data reveled the hybrid nature of S. × rechei, with genotypes displaying high levels of admixture with the parental species, while the epigenetic information assigned S. × rechei to its own cluster with low admixture. These findings suggested that after the hybridization event, a novel epigenetic pattern was rapidly established, which might influence the phenotypic plasticity and adaptation of the hybrid to new environments.     

Epigenetic variation associated with responses to different habitats in the context of genetic divergence in Phragmites australis

The mechanisms underlying heritable phenotypic divergence associated with adaptation in response to environmental stresses may involve both genetic and epigenetic variations. Several prior studies have revealed even higher levels of epigenetic variation than genetic variation. However, few population‐level studies have explored the effects of epigenetic variation on species with high levels of genetic diversity distributed across different habitats. Using AFLP and methylation‐sensitive AFLP markers, we tested the hypothesis that epigenetic variation may contribute to differences in plants occupying different habitats when genetic variation alone cannot fully explain adaptation. As a cosmopolitan invasive species, Phragmites australis (common reed) together with high genetic diversity and remarkable adaptability has been suggested as a model for responses to global change and indicators of environmental fluctuations. We found high levels of genetic and epigenetic diversity and significant genetic/epigenetic structure within each of 12 studied populations sampled from four natural habitats of P. australis. Possible adaptive epigenetic variation was suggested by significant correlations between DNA methylation‐based epigenetic differentiation and adaptive genetic divergence in populations across the habitats. Meanwhile, various AMOVAs indicated that some epigenetic differences may respond to various local habitats. A partial Mantel test was used to tease out the correlations between genetic/epigenetic variation and habitat after controlling for the correlation between genetic and epigenetic variations. We found that epigenetic diversity was affected mostly by soil nutrient availability, suggesting that at least some epigenetic differentiation occurred independently of genetic variation. We also found stronger correlations between epigenetic variation and phenotypic traits than between genetic variation and such traits. Overall, our findings indicate that genetically based differentiation correlates with heterogeneous habitats, while epigenetic variation plays an important role in ecological differentiation in natural populations of P. australis. In addition, our results suggest that when assessing global change responses of plant species, intraspecific variation needs to be considered.     

Rooting Out Genetic Structure of Invasive Wild Pigs in Texas

Invasive wild pigs (Sus scrofa), also called feral swine or wild hogs, are recognized as among the most destructive invasive species in the world. Throughout the United States, invasive wild pigs have expanded rapidly over the past 40 years with populations now established in 38 states. Of the estimated 6.9 million wild pigs distributed throughout the United States, Texas supports approximately 40% of the population and similarly bears disproportionate ecological and economic costs. Genetic analyses are an effective tool for understanding invasion pathways and tracking dispersal of invasive species such as wild pigs and have been used recently in California and Florida, USA, which have similarly long-established populations and high densities of wild pigs. Our goals were to use molecular approaches to elucidate invasion and migration processes shaping wild pig populations throughout Texas, compare our results with patterns of genetic structure observed in California and Florida, and provide insights for effective management of this invasive species. We used a high-density single nucleotide polymorphism (SNP) array to evaluate population genetic structure. Genetic clusters of wild pigs throughout Texas demonstrate 2 distinct patterns: weakly resolved, spatially dispersed clusters and well-resolved, spatially localized clusters. The disparity in patterns of genetic structure suggests disparate processes are differentially shaping wild pig populations in various localities throughout the state. Our results differed from the patterns of genetic structure observed in California and Florida, which were characterized by localized genetic clusters. These differences suggest distinct biological and perhaps anthropogenic processes are shaping genetic structure in Texas. Further, these disparities demonstrate the need for location-specific management strategies for controlling wild pig populations and mitigating associated ecological and economic costs. © 2021 The Wildlife Society. This article has been contributed to by US Government employees and their work is in the public domain in the USA.     

Epigenetic and epigenomic variation in Arabidopsis thaliana

Arabidopsis thaliana (Arabidopsis) is ideally suited for studies of natural phenotypic variation. This species has also provided an unparalleled experimental system to explore the mechanistic link between genetic and epigenetic variation, especially with regard to cytosine methylation. Using high-throughput sequencing methods, genotype to epigenotype to phenotype observations can now be extended to plant populations. We review the evidence for induced and spontaneous epigenetic variants that have been identified in Arabidopsis in the laboratory and discuss how these experimental observations could explain existing variation in the wild.     

A truly ecological epigenetics study

Until a few years ago, epigenetics was a field of research that had nothing to do with ecology and that virtually no ecologist had ever heard of. This is now changing, as more and more ecologists learn about epigenetic processes and their potential ecological and evolutionary relevance, and a new research field of ecological epigenetics is beginning to take shape. One question that is particularly intriguing ecologists is to what extent epigenetic variation is an additional, and hitherto overlooked, source of natural variation in ecologically important traits. In this issue of Molecular Ecology, Herrera & Bazaga (2011) provide one of the first attempts to truly address this question in an ecological setting. They study variation of DNA methylation in a wild population of the rare, long-lived violet Viola cazorlensis, and they use these data to explore interrelations between environmental, genetic and epigenetic variation, and in particular the extent to which these factors are related to long-term differences in herbivore damage among plants. They find substantial epigenetic variation among plant individuals. Interestingly, this epigenetic variation is significantly correlated with long-term differences in herbivory, but only weakly with herbivory-related DNA sequence variation, which suggests that besides habitat, substrate and genetic variation, epigenetic variation may be an additional, and at least partly independent, factor influencing plant–herbivore interactions in the field. Although the study by Herrera & Bazaga (2011) raises at least as many new questions as it answers, it is a pioneering example of how epigenetics can be incorporated into ecological field studies, and it illustrates the value and potential novel insights to be gained from such efforts.     

Biological effects of chronic radiation exposure on plant populations

The findings from long-term field studies on biological effects in plant populations inhabiting radioactively contaminated territories contrast in levels and compositions of dose-forming radionuclides are presented. Plant populations developing under radioactive impact show enhanced frequencies of gene and chromosome mutations, and their reproductive potential is inferior to reference populations. Even relatively low levels of technogenic impact are able to increase genetic diversity and destroy regularities inherent for intact populations. Chronic radiation exposure from a certain level appears to be an ecological factor changing genetic structure of wild populations. Data presented indicate the presence of adaptation processes in plant populations in territories with technogenic impact. Under ecological stress, there are selection processes for resistance improvement in plant populations. But an appearance and rate of this process can essentially differ in dependence on radioecological conditions.     

Experimental alteration of DNA methylation affects the phenotypic plasticity of ecologically relevant traits in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Heritable phenotypic variation in plants can be caused not only by underlying genetic differences, but also by variation in epigenetic modifications such as DNA methylation. However, we still know very little about how relevant such epigenetic variation is to the ecology and evolution of natural populations. We conducted a greenhouse experiment in which we treated a set of natural genotypes of Arabidopsis thaliana with the demethylating agent 5-azacytidine and examined the consequences of this treatment for plant traits and their phenotypic plasticity. Experimental demethylation strongly reduced the growth and fitness of plants and delayed their flowering, but the degree of this response varied significantly among genotypes. Differences in genotypes’ responses to demethylation were only weakly related to their genetic relatedness, which is consistent with the idea that natural epigenetic variation is independent of genetic variation. Demethylation also altered patterns of phenotypic plasticity, as well as the amount of phenotypic variation observed among plant individuals and genotype means. We have demonstrated that epigenetic variation can have a dramatic impact on ecologically important plant traits and their variability, as well as on the fitness of plants and their ecological interactions. Epigenetic variation may thus be an overlooked factor in the evolutionary ecology of plant populations.     

Variation in DNA methylation transmissibility,genetic heterogeneity and fecundity‐related traits in natural populations of the perennial herb Helleborus foetidus

Inferences about the role of epigenetics in plant ecology and evolution are mostly based on studies of cultivated or model plants conducted in artificial environments. Insights from natural populations, however, are essential to evaluate the possible consequences of epigenetic processes in biologically realistic scenarios with genetically and phenotypically heterogeneous populations. Here, we explore associations across individuals between DNA methylation transmissibility (proportion of methylation‐sensitive loci whose methylation status persists unchanged after male gametogenesis), genetic characteristics (assessed with AFLP markers), seed size variability (within‐plant seed mass variance), and realized maternal fecundity (number of recently recruited seedlings), in three populations of the perennial herb Helleborus foetidus along a natural ecological gradient in southeastern Spain. Plants (sporophytes) differed in the fidelity with which DNA methylation was transmitted to descendant pollen (gametophytes). This variation in methylation transmissibility was associated with genetic differences. Four AFLP loci were significantly associated with transmissibility and accounted collectively for ~40% of its sample‐wide variance. Within‐plant variance in seed mass was inversely related to individual transmissibility. The number of seedlings recruited by individual plants was significantly associated with transmissibility. The sign of the relationship varied between populations, which points to environment‐specific, divergent phenotypic selection on epigenetic transmissibility. Results support the view that epigenetic transmissibility is itself a phenotypic trait whose evolution may be driven by natural selection, and suggest that in natural populations epigenetic and genetic variation are two intertwined, rather than independent, evolutionary factors.     

湖北星斗山台湾杉居群的遗传多样性研究

采用随机扩增多态性DNA(RAPD)方法对孑遗植物台湾杉分布于湖北星斗山的野生和栽培居群共42个个体进行了遗传多样性分析。从60个随机引物中筛选出13个引物,共扩增出155条清晰谱带,多态位点百分率为72.9%。野生居群和栽培居群的多态位点百分率分别为65.81%和59.35%;Nei’s基因多样性分别为0.2001和0.1911;Shannon’s信息指数分别为0.3120和0.2946。野生居群的遗传多样性高于栽培居群,表明这些原生古树的后代出现了一定程度的遗传衰退。用UPGMA方法对42个单株聚类可知,来自野生群体的个体和来自栽培群体的个体明显地各聚为一类。尽管孑遗物种台湾杉在湖北仅有一个野生群体,且为零星分布,总个体数40多株,但仍保持较高的遗传多样性水平,这说明遗传多样性不是导致其种群濒危的主要原因,导致台湾杉种群濒危的原因可能与台湾杉自然种群及群落所处生境的直接破坏及其本身生物学和生态学特性所导致的自然更新不良有着密切的联系。     

Loss of genetic diversity in an outbreeding species: small population effects in the African wild dog <Emphasis Type="Italic">(Lycaon pictus</Emphasis>)

Genetic studies on the endangered African wild dog (Lycaon pictus) have primarily focused on the few remaining large and viable populations. However, investigations on the many isolated small African wild dog populations might also be informative for species management because the majority of extant populations are small and may contain genetic variability that is important for population persistence and for species conservation. Small populations are at higher risk of extinction from stochastic and deterministic demographic processes than larger populations and this is often of more immediate conservation concern than loss of genetic diversity, particularly for species that exhibit out-breeding behaviour such as long distance dispersal which may maintain gene flow. However, the genetic advantages of out-breeding behaviour may be reduced if dispersal is compromised beyond reserve borders (edge effects), further weakening the integrity of small populations. Mitochondrial DNA and 11 microsatellite genetic markers were used to investigate population genetic structure in a small population of out-breeding African wild dogs in Zambia, which occupies an historical dispersal corridor for the species. Results indicated the Zambian population suffered from low allelic richness, and there was significant evidence of a recent population bottleneck. Concurrent ecological data suggests these results were due to habitat fragmentation and restricted dispersal which compromised natural out-breeding mechanisms. This study recommends conservation priorities and management units for the African wild dog that focus on conserving remaining levels of genetic diversity, which may also be applicable for a range of out-breeding species.     

Genome‐wide epigenetic isolation by environment in a widespread Anolis lizard

Epigenetic changes can provide a pathway for organisms to respond to local environmental conditions by influencing gene expression. However, we still know little about the spatial distribution of epigenetic variation in natural systems, how it relates to the distribution of genetic variation and the environmental structure of the landscape, and the processes that generate and maintain it. Studies examining spatial patterns of genetic and epigenetic variation can provide valuable insights into how ecological and population processes contribute to epigenetic divergence across heterogeneous landscapes. Here, we perform a comparative analysis of spatial genetic and epigenetic variation based on 8,459 single nucleotide polymorphisms (SNPs) and 8,580 single methylation variants (SMVs) from eight populations of the Puerto Rican crested anole, Anolis cristatellus, an abundant lizard in the adaptive radiations of anoles on the Greater Antilles that occupies a diverse range of habitats. Using generalized dissimilarity modelling and multiple matrix regression, we found that genome‐wide epigenetic differentiation is strongly correlated with environmental divergence, even after controlling for the underlying genetic structure. We also detected significant associations between key environmental variables and 96 SMVs, including 42 located in promoter regions or gene bodies. Our results suggest an environmental basis for population‐level epigenetic differentiation in this system and contribute to better understanding how environmental gradients structure epigenetic variation in nature.