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.

Plant epigenetics: phenotypic and functional diversity beyond the DNA sequence.

Phenotypic variation determines the capacity of plants to adapt to changing environments and to colonize new habitats. Deciphering the mechanisms contributing to plant phenotypic variation and their effects on plant ecological interactions and evolutionary dynamics is thus central to all biological disciplines. In the past few decades, research on plant epigenetics is showing that (1) epigenetic variation is related to phenotypic variation and that some epigenetic marks drive major phenotypic changes in plants; (2) plant epigenomes are highly diverse, dynamic, and can respond rapidly to a variety of biotic and abiotic stimuli; (3) epigenetic variation can respond to selection and therefore play a role in adaptive evolution. Yet, current information in terms of species, geographic ranges, and ecological contexts analyzed so far is too limited to allow for generalizations about the relevance of epigenetic regulation in phenotypic innovation and plant adaptation across taxa. In this report, we contextualize the potential role of the epigenome in plant adaptation to the environment and describe the latest research in this field presented during the symposium "Plant epigenetics: phenotypic and functional diversity beyond the DNA sequence" held within the Botany 2020 conference framework in summer 2020.

Genetic structuring of the moss Pseudoscleropodium purum sampled at different distances from a pollution source.

In this study, we used amplified fragment length polymorphism analysis to investigate the genetic structure of the terrestrial moss Pseudoscleropodium purum (Hedw.) M. Fleish. naturally exposed to different levels of atmospheric deposition of heavy metals. We also determined the heavy metal concentrations in samples of this moss to evaluate whether there was a relationship between atmospheric pollution and population genetic diversity. A low level of genetic diversity and a limited gene flow among populations were observed which is in accordance to the prevalence of asexual reproduction in this species. In addition, no significant correlation was found between metal content and gene diversity in P. purum, probably because of the common history of the sampled populations and/or to the lack of a drastic reduction of the size of the population; nonetheless, a clear genetic structure was evident in relation to the existing pollution gradient. Thus, based on the results of the principal coordinate analysis and Bayesian analysis of the genotypes, the mixed structure of the second most polluted population would suggest an ongoing differentiation of metal-tolerant genotypes in the most polluted sites of the sampling area.

High Hg biomagnification in North Atlantic coast ecosystems and limits to the use of δ15N to estimate trophic magnification factors.

Mercury contamination is a global environmental problem. This pollutant is highly toxic and persistent which makes it extremely susceptible to biomagnify, i.e. increase its concentrations as it moves up the food chain, reaching levels that threaten wildlife and, ultimately, ecosystems' function and structure. Mercury monitoring is thus crucial to determine its potential to damage the environment. In this study, we assessed the temporal trends of the concentrations of Hg in two coastal animal species closely connected by a predator-prey interaction, and evaluated its potential transfer between trophic levels using the δ15N signatures of the two species. For this, we performed a multi-year survey of the concentrations of total Hg and the values of δ15N in the mussel Mytilus galloprovincialis (prey) and the dogwhelk Nucella lapillus (predator) sampled along ∼1500 km of the North Atlantic coast of Spain over a 30-year period (five surveys between 1990 and 2021). Concentrations of Hg decreased significantly between the first and the last survey in the two species studied. Except for the 1990 survey, the concentrations of Hg in mussels were amongst the lowest registered in the literature for the North East Atlantic Ocean (NEAO) and the Mediterranean Sea (MS) between 1985 and 2020. Nonetheless, we detected Hg biomagnification in almost all surveys. Worryingly, trophic magnification factors obtained here for total Hg were high and comparable to the found in the literature for methylmercury, the most toxic and readily biomagnified form of this element. The δ15N values were useful to detect Hg biomagnification under normal circumstances. However, we found that nitrogen pollution of coastal waters differentially affected the δ15N signatures of mussels and dogwhelks limiting the use of this parameter for this purpose. We conclude that Hg biomagnification could constitute an important environmental hazard even when found at very low concentrations in the lower trophic levels. Also, we warn that use of δ15N in biomagnification studies when there is some underlying nitrogen pollution problem might lead to misleading conclusions.

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.

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.

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.

Assessing the effects of heavy metal contamination on the proteome of the moss Pseudoscleropodium purum cross-transplanted between different areas.

Protein expression was assessed in samples of Pseudoscleropodium purum cross-transplanted between one unpolluted (UNP) and two polluted (POLL) sites. Firstly, the level of expression (LE) of 17 proteins differed between native mosses from both types of sites, but differences were only maintained throughout the experiment for 5 of them. The LE of these five proteins changed over time in mosses transplanted from UNP to POLL and vice versa, becoming similar to that in autotransplants. However, these changes occurred slower than changes in the heavy metal concentrations measured in the same samples, and therefore they were not related to atmospheric pollution. Although the proteins identified were associated with moss metabolism, the expected growth reduction in samples autotransplanted within POLL (as a result of the down-regulation of photosynthesis-related proteins), did not occur. This supports the hypothesis that mosses growing in polluted areas adapt to heavy metal pollution and are able to reduce/overcome their toxic effects (i.e., reduced growth). Nevertheless, further specific research must be carried out to identify the proteins involved in this type of response, as lack of information on the bryophyte genome precludes us from reaching further conclusions.

Assessing the tolerance of the terrestrial moss Pseudoscleropodium purum to high levels of atmospheric heavy metals: a reciprocal transplant study.

We measured the concentrations of Cd, Cu, Hg, Pb and Zn in samples of the terrestrial moss Pseudoscleropodium purum reciprocally transplanted between an unpolluted and two polluted sampling sites. At the beginning of the experiment, the concentrations of all these elements differed significantly between mosses from the unpolluted site and mosses from the polluted sites. In general, the concentrations of the heavy metals in mosses from both polluted sites transplanted to the unpolluted site decreased until they reached the same levels as in autotransplants at this site (after 480-840 days). However, the concentrations of all heavy metals in mosses transplanted from the unpolluted site to both polluted sites increased to higher levels than in the autotransplants (except for Cu, Hg and Pb at one of the sampling sites). These results led us to conclude that mosses that have been continuously exposed to high atmospheric deposition of heavy metals undergo an adaptive response (probably genotypic) to such conditions. We therefore recommend active rather than passive biomonitoring of air quality in industrial environments because atmospheric deposition could be underestimated, and also recommend further investigation into the mechanisms involved in this response.