Heavy metal tolerance in Scopelophila cataractae: Transcriptomic and epigenetic datasets.

Studying how different plant groups deal with heavy metal exposure is crucial to improve our understanding of the diversity of molecular mechanisms involved in plant stress response. Here, we used RNA sequencing (RNA-seq) and epigenotyping by sequencing (epiGBS) to assess gene expression and DNA methylation changes respectively in plants from four populations of the metallophyte moss Scopelophila cataractae treated with Cd or Cu in the laboratory. We built RNA-seq and epiGBS sequencing libraries from control and treated samples from each population and sequenced them using Illumina HiSeq 3000 (PE-150 bp) and Illumina HiSeq X-Ten System (PE-150 bp) respectively. For the RNA-seq data, we performed a read quality filter, mapped the reads to the de novo transcriptome created with Trinity, and estimated transcript abundance for each sample. For the epiGBS data, we used a custom pipeline (https://doi.org/10.5281/zenodo.7040291) to map the reads to a de novo reference genome and performed strand-specific nucleotide (single nucleotide polymorphisms, SNPs) and methylation (single cytosine methylation polymorphisms, SMPs) variant calling. We filtered out SNPs and SMPs with low coverage within (positions with <10 sequencing reads per sample) and across samples (positions with poor representation on the full set of samples). Finally, we performed pairwise comparisons between control and treated samples from each population and identified differentially expressed genes and differentially methylated cytosines associated to heavy metal exposure. We payed particular attention to the different responses of the more and the less tolerant populations of S. cataractae. These datasets could contribute to future comparative studies of abiotic stress response across plant groups.

Restoration of native saltmarshes can reverse arthropod assemblages and trophic interactions changed by a plant invasion.

Plant invasions profoundly impact both natural and managed ecosystems, and removal of the invasive plants addresses only part of the problem of restoring impacted areas. The rehabilitation of diverse communities and their ecosystem functions following removal of invasive plants is an important goal of ecological restoration. Arthropod assemblages and trophic interactions are important indicators of the success of restoration, but they have largely been overlooked in saltmarshes. We determined how arthropod assemblages and trophic interactions changed with the invasion of the exotic plant Spartina alterniflora and with the restoration of the native plant Phragmites australis following Spartina removal in a Chinese saltmarsh. We investigated multiple biotic and abiotic variables to gain insight into the factors underlying the changes in arthropod assemblages and trophic structure. We found that although Spartina invasion had changed arthropod diversity, community structure, feeding-guild composition, and the diets of arthropod natural enemies in the saltmarsh, these changes could be reversed by the restoration of native Phragmites vegetation following removal of the invader. The variation in arthropod assemblages and trophic structure were critically associated with four biotic and abiotic variables (aboveground biomass, plant density, leaf N, and soil salinity). Our findings demonstrate the positive effects of controlling invasive plants on biodiversity and nutrient cycling and provide a foundation for assessing the efficacy of ecological restoration projects in saltmarshes.

Repeated Genetic and Adaptive Phenotypic Divergence across Tidal Elevation in a Foundation Plant Species.

AbstractMicrogeographic genetic divergence can create fine-scale trait variation. When such divergence occurs within foundation species, then it might impact community structure and ecosystem function and cause other cascading ecological effects. We tested for parallel microgeographic trait and genetic divergence in Spartina alterniflora, a foundation species that dominates salt marshes of the US Atlantic and Gulf coasts. Spartina is characterized by tall-form (1-2 m) plants at lower tidal elevations and short-form (<0.5 m) plants at higher tidal elevations, yet whether this trait variation reflects plastic and/or genetically differentiated responses to these environmental conditions remains unclear. In the greenhouse, seedlings raised from tall-form plants grew taller than those from short-form plants, indicating a heritable difference in height. When we reciprocally transplanted seedlings back into the field for a growing season, composite fitness (survivorship and seed production) and key plant traits (plant height and biomass allocation) differed interactively across origin and transplant zones in a manner indicative of local adaptation. Further, a survey of single nucleotide polymorphisms revealed repeated, independent genetic differentiation between tall- and short-form Spartina at five of six tested marshes across the native range. The observed parallel, microgeographic genetic differentiation in Spartina likely underpins marsh health and functioning and provides an underappreciated mechanism that might increase capacity of marshes to adapt to rising sea levels.

Genetic structure in patchy populations of a candidate foundation plant: a case study of Leymus chinensis using genetic and clonal diversity.

PREMISE: The distribution of genetic diversity on the landscape has critical ecological and evolutionary implications. This may be especially the case on a local scale for foundation plant species because they create and define ecological communities, contributing disproportionately to ecosystem function. METHODS: We examined the distribution of genetic diversity and clones, which we defined first as unique multilocus genotypes (MLG), and then by grouping similar MLGs into multilocus lineages. We used 186 markers from inter-simple sequence repeats (ISSR) across 358 ramets from 13 patches of the foundation grass Leymus chinensis. We examined the relationship between genetic and clonal diversities, their variation with patch size, and the effect of the number of markers used to evaluate genetic diversity and structure in this species. RESULTS: Every ramet had a unique MLG. Almost all patches consisted of individuals belonging to a single multilocus lineages. We confirmed this with a clustering algorithm to group related genotypes. The predominance of a single lineage within each patch could be the result of the accumulation of somatic mutations, limited dispersal, some sexual reproduction with partners mainly restricted to the same patch, or a combination of all three. CONCLUSIONS: We found strong genetic structure among patches of L. chinensis. Consistent with previous work on the species, the clustering of similar genotypes within patches suggests that clonal reproduction combined with somatic mutation, limited dispersal, and some degree of sexual reproduction among neighbors causes individuals within a patch to be more closely related than among patches.

Inheritance of DNA methylation differences in the mangrove Rhizophora mangle.

The capacity to respond to environmental challenges ultimately relies on phenotypic variation which manifests from complex interactions of genetic and nongenetic mechanisms through development. While we know something about genetic variation and structure of many species of conservation importance, we know very little about the nongenetic contributions to variation. Rhizophora mangle is a foundation species that occurs in coastal estuarine habitats throughout the neotropics where it provides critical ecosystem functions and is potentially threatened by anthropogenic environmental changes. Several studies have documented landscape-level patterns of genetic variation in this species, but we know virtually nothing about the inheritance of nongenetic variation. To assess one type of nongenetic variation, we examined the patterns of DNA sequence and DNA methylation in maternal plants and offspring from natural populations of R. mangle from the Gulf Coast of Florida. We used a reduced representation bisulfite sequencing approach (epi-genotyping by sequencing; epiGBS) to address the following questions: (a) What are the levels of genetic and epigenetic diversity in natural populations of R. mangle? (b) How are genetic and epigenetic variation structured within and among populations? (c) How faithfully is epigenetic variation inherited? We found low genetic diversity but high epigenetic diversity from natural populations of maternal plants in the field. In addition, a large portion (up to ~25%) of epigenetic differences among offspring grown in common garden was explained by maternal family. Therefore, epigenetic variation could be an important source of response to challenging environments in the genetically depauperate populations of this foundation species.

Carbon and nitrogen pools and mobile fractions in surface soils across a mangrove saltmarsh ecotone.

In the subtropics, climate change is pushing woody mangrove forests into herbaceous saltmarshes, altering soil carbon (C) and nitrogen (N) pools, with implications for coastal wetland productivity and C and N exports. We quantified total C and N pools, and mobile fractions including extractable mineral N, extractable organic C and N, and active (aerobically mineralizable) C and N, in surface soils (top 7.6 cm) of adjacent mangrove (primarily Avicennia germinans) and saltmarsh (Juncus roemerianus) vegetation zones in tidal wetlands of west-central Florida (USA). We tested whether surface-soil accumulations of C, N, and their potentially mobile fractions are greater in mangrove than in saltmarsh owing to greater accumulations in the mangrove zone of soil organic matter (SOM) and fine mineral particles (C- and N-retaining soil constituents). Extractable organic fractions were 39-45% more concentrated in mangrove than in saltmarsh surface soil, and they scaled steeply and positively with SOM and fine mineral particle (silt + clay) concentrations, which themselves were likewise greater in mangrove soil. Elevation may drive this linkage. Mangrove locations were generally at lower elevations, which tended to have greater fine particle content in the surface soil. Active C and extractable mineral N were marginally (p < 0.1) greater in mangrove soil, while active N, total N, and total C showed no statistical differences between zones. Extractable organic C and N fractions composed greater shares of total C and N pools in mangrove than in saltmarsh surface soils, which is meaningful for ecosystem function, as persistent leaching of this fraction can perpetuate nutrient limitation. The active (mineralizable) C and N fractions we observed constituted a relatively small component of total C and N pools, suggesting that mangrove surface soils may export less C and N than would be expected from their large total C and N pools.

Epigenetic effects of parasites and pesticides on captive and wild nestling birds.

Anthropogenic changes to the environment challenge animal populations to adapt to new conditions and unique threats. While the study of adaptation has focused on genetic variation, epigenetic mechanisms may also be important. DNA methylation is sensitive to environmental stressors, such as parasites and pesticides, which may affect gene expression and phenotype. We studied the effects of an invasive ectoparasite, Philornis downsi, on DNA methylation of Galápagos mockingbirds (Mimus parvulus). We used the insecticide permethrin to manipulate P. downsi presence in nests of free-living mockingbirds and tested for effects of parasitism on nestling mockingbirds using epiGBS, a reduced-representation bisulfite sequencing (RRBS) approach. To distinguish the confounding effects of insecticide exposure, we conducted a matching experiment exposing captive nestling zebra finches (Taeniopygia guttata) to permethrin. We used zebra finches because they were the closest model organism to mockingbirds that we could breed in controlled conditions. We identified a limited number of differentially methylated cytosines (DMCs) in parasitized versus nonparasitized mockingbirds, but the number was not more than expected by chance. In contrast, we saw clear effects of permethrin on methylation in captive zebra finches. DMCs in zebra finches paralleled documented effects of permethrin exposure on vertebrate cellular signaling and endocrine function. Our results from captive birds indicate a role for epigenetic processes in mediating sublethal nontarget effects of pyrethroid exposure in vertebrates. Environmental conditions in the field were more variable than the laboratory, which may have made effects of both parasitism and permethrin harder to detect in mockingbirds. RRBS approaches such as epiGBS may be a cost-effective way to characterize genome-wide methylation profiles. However, our results indicate that ecological epigenetic studies in natural populations should consider the number of cytosines interrogated and the depth of sequencing in order to have adequate power to detect small and variable effects.

Epigenetics and the success of invasive plants.

Biological invasions impose ecological and economic problems on a global scale, but also provide extraordinary opportunities for studying contemporary evolution. It is critical to understand the evolutionary processes that underly invasion success in order to successfully manage existing invaders, and to prevent future invasions. As successful invasive species sometimes are suspected to rapidly adjust to their new environments in spite of very low genetic diversity, we are obliged to re-evaluate genomic-level processes that translate into phenotypic diversity. In this paper, we review work that supports the idea that trait variation, within and among invasive populations, can be created through epigenetic or other non-genetic processes, particularly in clonal invaders where somatic changes can persist indefinitely. We consider several processes that have been implicated as adaptive in invasion success, focusing on various forms of 'genomic shock' resulting from exposure to environmental stress, hybridization and whole-genome duplication (polyploidy), and leading to various patterns of gene expression re-programming and epigenetic changes that contribute to phenotypic variation or even novelty. These mechanisms can contribute to transgressive phenotypes, including hybrid vigour and novel traits, and may thus help to understand the huge successes of some plant invaders, especially those that are genetically impoverished. This article is part of the theme issue 'How does epigenetics influence the course of evolution?'

Artificial selection for host resistance to tumour growth and subsequent cancer cell adaptations: an evolutionary arms race.

BACKGROUND: Cancer progression is governed by evolutionary dynamics in both the tumour population and its host. Since cancers die with the host, each new population of cancer cells must reinvent strategies to overcome the host's heritable defences. In contrast, host species evolve defence strategies over generations if tumour development limits procreation. METHODS: We investigate this "evolutionary arms race" through intentional breeding of immunodeficient SCID and immunocompetent Black/6 mice to evolve increased tumour suppression. Over 10 generations, we injected Lewis lung mouse carcinoma cells [LL/2-Luc-M38] and selectively bred the two individuals with the slowest tumour growth at day 11. Their male progeny were hosts in the subsequent round. RESULTS: The evolved SCID mice suppressed tumour growth through biomechanical restriction from increased mesenchymal proliferation, and the evolved Black/6 mice suppressed tumour growth by increasing immune-mediated killing of cancer cells. However, transcriptomic changes of multicellular tissue organisation and function genes allowed LL/2-Luc-M38 cells to adapt through increased matrix remodelling in SCID mice, and reduced angiogenesis, increased energy utilisation and accelerated proliferation in Black/6 mice. CONCLUSION: Host species can rapidly evolve both immunologic and non-immunologic tumour defences. However, cancer cell plasticity allows effective phenotypic and population-based counter strategies.

Comparisons of Natural and Cultivated Populations of Corydalis yanhusuo Indicate Divergent Patterns of Genetic and Epigenetic Variation.

Epigenetic variation may contribute to traits that are important in domestication, but how patterns of genetic and epigenetic variation differ between cultivated and wild plants remains poorly understood. In particular, we know little about how selection may shape epigenetic variation in natural and cultivated populations. In this study, we investigated 11 natural populations and 6 major cultivated populations using amplified fragment length polymorphism (AFLP) and methylation-sensitive AFLP (MS-AFLP or MSAP) markers to identify patterns of genetic and epigenetic diversity among Corydalis yanhusuo populations. We further explored correlations among genetic, epigenetic, alkaloidal, and climatic factors in natural and cultivated C. yanhusuo. We found support for a single origin for all cultivated populations, from a natural population which was differentiated from the other natural populations. The magnitude of F ST based on AFLP was significantly correlated with that for MSAP in pairwise comparisons in both natural and cultivated populations, suggesting a relationship between genetic and epigenetic variation in C. yanhusuo. This relationship was further supported by dbRDA (distance-based redundancy analyses) where some of the epigenetic variation could be explained by genetic variation in natural and cultivated populations. Genetic variation was slightly higher in natural than cultivated populations, and exceeded epigenetic variation in both types of populations. However, epigenetic differentiation exceeded that of genetic differentiation among cultivated populations, while the reverse was observed among natural populations. The differences between wild and cultivated plants may be partly due to processes inherent to cultivation and in particular the differences in mode of reproduction. The importance of epigenetic compared to genetic modifications is thought to vary depending on reproductive strategies, and C. yanhusuo usually reproduces sexually in natural environments, while the cultivated C. yanhusuo are propagated clonally. In addition, alkaloid content of C. yanhusuo varied across cultivated populations, and alkaloid content was significantly correlated to climatic variation, but also to genetic (6.89%) and even more so to epigenetic (14.09%) variation in cultivated populations. Our study demonstrates that epigenetic variation could be important in cultivation of C. yanhusuo and serve as a source of variation for response to environmental conditions.

Epigenetic Approaches in Non-Model Plants.

Reduced representation bisulfite sequencing is an emerging methodology for evolutionary and ecological genomics and epigenomics research because it provides a cost-effective, high-resolution tool for exploration and comparative analysis of DNA methylation and genetic variation. Here we describe how digestion of genomic plant DNA with restriction enzymes, subsequent bisulfite conversion of unmethylated cytosines, and final DNA sequencing allow for the examination of genome-wide genetic and epigenetic variation in plants without the need for a reference genome. We explain how the use of several combinations of barcoded adapters for the creation of highly multiplexed libraries allows the inclusion of up to 144 different samples/individuals in only one sequencing lane.

Opportunities and limitations of reduced representation bisulfite sequencing in plant ecological epigenomics.

Contents Summary 738 I. Introduction 738 II. RRBS loci as genome-wide epigenetic markers 739 III. Exploiting functional annotation of RRBS loci 739 IV. Limitations of RRBS methods for nonmodel species 740 V. Maximising the impact of RRBS in plants 741 VI. Conclusions 741 Acknowledgements 741 SUMMARY: Investigating the features and implications of epigenetic mechanisms across the breadth of organisms and ecosystems is important for understanding the ecological relevance of epigenetics. Several cost-effective reduced representation bisulfite sequencing approaches (RRBS) have been recently developed and applied to different organisms that lack a well annotated reference genome. These new approaches improve the assessment of epigenetic diversity in ecological settings and may provide functional insights. We assess here the opportunities and limitations of RRBS in nonmodel plant species. Well thought out experimental designs that include complementary gene expression studies, and the improvement of genomics resources for the target group, promise to maximize the effect of future RRBS studies.

Quantitative epigenetics and evolution.

Epigenetics refers to chemical modifications of chromatin or transcribed DNA that can influence gene activity and expression without changes in DNA sequence. The last 20 years have yielded breakthroughs in our understanding of epigenetic processes that impact many fields of biology. In this review, we discuss how epigenetics relates to quantitative genetics and evolution. We argue that epigenetics is important for quantitative genetics because: (1) quantitative genetics is increasingly being combined with genomics, and therefore we should expand our thinking to include cellular-level mechanisms that can account for phenotypic variance and heritability besides just those that are hard-coded in the DNA sequence; and (2) epigenetic mechanisms change how phenotypic variance is partitioned, and can thereby change the heritability of traits and how those traits are inherited. To explicate these points, we show that epigenetics can influence all aspects of the phenotypic variance formula: VP (total phenotypic variance) = VG (genetic variance) + VE (environmental variance) + VGxE (genotype-by-environment interaction) + 2COVGE (the genotype-environment covariance) + Vɛ (residual variance), requiring new strategies to account for different potential sources of epigenetic effects on phenotypic variance. We also demonstrate how each of the components of phenotypic variance not only can be influenced by epigenetics, but can also have evolutionary consequences. We argue that no sources of epigenetic effects on phenotypic variance can be easily cast aside in a quantitative genetic research program that seeks to understand evolutionary processes.

Transcriptome response of the foundation plant Spartina alterniflora to the Deepwater Horizon oil spill.

Despite the severe impacts of the Deepwater Horizon oil spill, the foundation plant species Spartina alterniflora proved resilient to heavy oiling, providing an opportunity to identify mechanisms of response to the anthropogenic stress of crude oil exposure. We assessed plants from oil-affected and unaffected populations using a custom DNA microarray to identify genomewide transcription patterns and gene expression networks that respond to crude oil exposure. In addition, we used T-DNA insertion lines of the model grass Brachypodium distachyon to assess the contribution of four novel candidate genes to crude oil response. Responses in S. alterniflora to hydrocarbon exposure across the transcriptome as well as xenobiotic specific response pathways had little overlap with those previously identified in the model plant Arabidopsis thaliana. Among T-DNA insertion lines of B. distachyon, we found additional support for two candidate genes, one (ATTPS21) involved in volatile production, and the other (SUVH5) involved in epigenetic regulation of gene expression, that may be important in the response to crude oil. The architecture of crude oil response in S. alterniflora is unique from that of the model species A. thaliana, suggesting that xenobiotic response may be highly variable across plant species. In addition, further investigations of regulatory networks may benefit from more information about epigenetic response pathways.

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

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.

Adaptive plasticity and epigenetic variation in response to warming in an Alpine plant.

Environmentally induced phenotypic plasticity may be a critical component of response to changing environments. We examined local differentiation and adaptive phenotypic plasticity in response to elevated temperature in half-sib lines collected across an elevation gradient for the alpine herb, Wahlenbergia ceracea. Using Amplified Fragment Length Polymorphism (AFLP), we found low but significant genetic differentiation between low- and high-elevation seedlings, and seedlings originating from low elevations grew faster and showed stronger temperature responses (more plasticity) than those from medium and high elevations. Furthermore, plasticity was more often adaptive for plants of low-elevation origin and maladaptive for plants of high elevation. With methylation sensitive-AFLP (MS-AFLP), we revealed an increase in epigenetic variation in response to temperature in low-elevation seedlings. Although we did not find significant direct correlations between MS-AFLP loci and phenotypes, our results demonstrate that adaptive plasticity in temperature response to warming varies over fine spatial scales and suggest the involvement of epigenetic mechanisms in this response.

Ten years of transcriptomics in wild populations: what have we learned about their ecology and evolution?

Molecular ecology has moved beyond the use of a relatively small number of markers, often noncoding, and it is now possible to use whole-genome measures of gene expression with microarrays and RNAseq (i.e. transcriptomics) to capture molecular response to environmental challenges. While transcriptome studies are shedding light on the mechanistic basis of traits as complex as personality or physiological response to catastrophic events, these approaches are still challenging because of the required technical expertise, difficulties with analysis and cost. Still, we found that in the last 10 years, 575 studies used microarrays or RNAseq in ecology. These studies broadly address three questions that reflect the progression of the field: (i) How much variation in gene expression is there and how is it structured? (ii) How do environmental stimuli affect gene expression? (iii) How does gene expression affect phenotype? We discuss technical aspects of RNAseq and microarray technology, and a framework that leverages the advantages of both. Further, we highlight future directions of research, particularly related to moving beyond correlation and the development of additional annotation resources. Measuring gene expression across an array of taxa in ecological settings promises to enrich our understanding of ecology and genome function.

Ecological epigenetics.

Biologists have assumed that heritable variation due to DNA sequence differences (i.e., genetic variation) allows populations of organisms to be both robust and adaptable to extreme environmental conditions. Natural selection acts on the variation among different genotypes and ultimately changes the genetic composition of the population. While there is compelling evidence about the importance of genetic polymorphisms, evidence is accumulating that epigenetic mechanisms (e.g., chromatin modifications, DNA methylation) can affect ecologically important traits, even in the absence of genetic variation. In this chapter, we review this evidence and discuss the consequences of epigenetic variation in natural populations. We begin by defining the term epigenetics, providing a brief overview of various epigenetic mechanisms, and noting the potential importance of epigenetics in the study of ecology. We continue with a review of the ecological epigenetics literature to demonstrate what is currently known about the amount and distribution of epigenetic variation in natural populations. Then, we consider the various ecological contexts in which epigenetics has proven particularly insightful and discuss the potential evolutionary consequences of epigenetic variation. Finally, we conclude with suggestions for future directions of ecological epigenetics research.