Early developmental carry-over effects on exploratory behaviour and DNA methylation in wild great tits (Parus major).

Adverse, postnatal conditions experienced during development are known to induce lingering effects on morphology, behaviour, reproduction and survival. Despite the importance of early developmental stress for shaping the adult phenotype, it is largely unknown which molecular mechanisms allow for the induction and maintenance of such phenotypic effects once the early environmental conditions are released. Here we aimed to investigate whether lasting early developmental phenotypic changes are associated with post-developmental DNA methylation changes. We used a cross-foster and brood size experiment in great tit (Parus major) nestlings, which induced post-fledging effects on biometric measures and exploratory behaviour, a validated personality trait. We investigated whether these post-fledging effects are associated with DNA methylation levels of CpG sites in erythrocyte DNA. Individuals raised in enlarged broods caught up on their developmental delay after reaching independence and became more explorative as days since fledging passed, while the exploratory scores of individuals that were raised in reduced broods remained stable. Although we previously found that brood enlargement hardly affected the pre-fledging methylation levels, we found 420 CpG sites that were differentially methylated between fledged individuals that were raised in small versus large sized broods. A considerable number of the affected CpG sites were located in or near genes involved in metabolism, growth, behaviour and cognition. Since the biological functions of these genes line up with the observed post-fledging phenotypic effects of brood size, our results suggest that DNA methylation provides organisms the opportunity to modulate their condition once the environmental conditions allow it. In conclusion, this study shows that nutritional stress imposed by enlarged brood size during early development associates with variation in DNA methylation later in life. We propose that treatment-associated DNA methylation differences may arise in relation to pre- or post-fledging phenotypic changes, rather than that they are directly induced by the environment during early development.

Herbivory induced methylation changes in the Lombardy poplar: A comparison of results obtained by epiGBS and WGBS.

DNA cytosine methylation is an epigenetic mechanism involved in regulation of plant responses to biotic and abiotic stress and its ability to change can vary with the sequence context in which a cytosine appears (CpG, CHG, CHH, where H = Adenine, Thymine, Cytosine). Quantification of DNA methylation in model plant species is frequently addressed by Whole Genome Bisulfite Sequencing (WGBS), which requires a good-quality reference genome. Reduced Representation Bisulfite Sequencing (RRBS) is a cost-effective potential alternative for ecological research with limited genomic resources and large experimental designs. In this study, we provide for the first time a comprehensive comparison between the outputs of RRBS and WGBS to characterize DNA methylation changes in response to a given environmental factor. In particular, we used epiGBS (recently optimized RRBS) and WGBS to assess global and sequence-specific differential methylation after insect and artificial herbivory in clones of Populus nigra cv. 'italica'. We found that, after any of the two herbivory treatments, global methylation percentage increased in CHH, and the shift was detected as statistically significant only by epiGBS. As regards to loci-specific differential methylation induced by herbivory (cytosines in epiGBS and regions in WGBS), both techniques indicated the specificity of the response elicited by insect and artificial herbivory, together with higher frequency of hypo-methylation in CpG and hyper-methylation in CHH. Methylation changes were mainly found in gene bodies and intergenic regions when present at CpG and CHG and in transposable elements and intergenic regions at CHH context. Thus, epiGBS succeeded to characterize global, genome-wide methylation changes in response to herbivory in the Lombardy poplar. Our results support that epiGBS could be particularly useful in large experimental designs aimed to explore epigenetic changes of non-model plant species in response to multiple environmental factors.

Phylogenomic analysis provides insights into MADS-box and TCP gene diversification and floral development of the Asteraceae, supported by de novo genome and transcriptome sequences from dandelion (Taraxacum officinale).

The Asteraceae is the largest angiosperm family with more than 25,000 species. Individual studies have shown that MADS-box and TCP transcription factors are regulators of the development and symmetry of flowers, contributing to their iconic flower-head (capitulum) and floret. However, a systematic study of MADS-box and TCP genes across the Asteraceae is lacking. We performed a comparative analysis of genome sequences of 33 angiosperm species including our de novo assembly of diploid sexual dandelion (Taraxacum officinale) and 11 other Asteraceae to investigate the lineage-specific evolution of MADS-box and TCP genes in the Asteraceae. We compared the phylogenomic results of MADS-box and TCP genes with their expression in T. officinale floral tissues at different developmental stages to demonstrate the regulation of genes with Asteraceae-specific attributes. Here, we show that MADS-box MIKC c and TCP-CYCLOIDEA (CYC) genes have expanded in the Asteraceae. The phylogenomic analysis identified AGAMOUS-like (AG-like: SEEDSTICK [STK]-like), SEPALATA-like (SEP3-like), and TCP-PROLIFERATING CELL FACTOR (PCF)-like copies with lineage-specific genomic contexts in the Asteraceae, Cichorioideae, or dandelion. Different expression patterns of some of these gene copies suggest functional divergence. We also confirm the presence and revisit the evolutionary history of previously named "Asteraceae-Specific MADS-box genes (AS-MADS)." Specifically, we identify non-Asteraceae homologs, indicating a more ancient origin of this gene clade. Syntenic relationships support that AS-MADS is paralogous to FLOWERING LOCUS C (FLC) as demonstrated by the shared ancient duplication of FLC and SEP3.

Developmental stress does not induce genome-wide DNA methylation changes in wild great tit (Parus major) nestlings.

The environment experienced during early life is a crucial factor in the life of many organisms. This early life environment has been shown to have profound effects on morphology, physiology and fitness. However, the molecular mechanisms that mediate these effects are largely unknown, even though they are essential for our understanding of the processes that induce phenotypic variation in natural populations. DNA methylation is an epigenetic mechanism that has been suggested to explain such environmentally induced phenotypic changes early in life. To investigate whether DNA methylation changes are associated with experimentally induced early developmental effects, we cross-fostered great tit (Parus major) nestlings and manipulated their brood sizes in a natural study population. We assessed experimental brood size effects on pre-fledging biometry and behaviour. We linked this to genome-wide DNA methylation levels of CpG sites in erythrocyte DNA, using 122 individuals and an improved epiGBS2 laboratory protocol. Brood enlargement caused developmental stress and negatively affected nestling condition, predominantly during the second half of the breeding season, when conditions are harsher. Brood enlargement, however, affected nestling DNA methylation in only one CpG site and only if the hatch date was taken into account. In conclusion, this study shows that nutritional stress in enlarged broods does not associate with direct effects on genome-wide DNA methylation. Future studies should assess whether genome-wide DNA methylation variation may arise later in life as a consequence of phenotypic changes during early development.

Adaptive plasticity and fitness costs of endangered, nonendangered, and invasive plants in response to variation in nitrogen and phosphorus availabilities.

Global change drivers such as eutrophication and plant invasions will create novel environments for many plant species. Through adaptive trait plasticity plants may maintain their performance under these novel conditions and may outcompete those showing low-adaptive trait plasticity. In a greenhouse study, we determined if plasticity in traits is adaptive or maladaptive in endangered, nonendangered, and invasive plant species in response to variation of nitrogen (N) and phosphorus (P) availability (N:P ratios 1.7, 15, and 135) and whether plastic trait responses are adaptive and/or costly for fitness (i.e., biomass). Species choice comprised 17 species from three functional groups (legumes, nonlegume forbs, and grasses), either classified as endangered, nonendangered, or invasive. After 2 months, plants were harvested and nine traits related to carbon assimilation and nutrient uptake were measured (leaf area, SLA, LDMC, SPAD, RMR, root length, SRL, root surface area, and PME activity). We found more traits responding plastically to variation in P than in N. Plasticity only created costs when P was varied. Plasticity in traits was mostly adaptively neutral toward fitness, with plasticity in three traits being similarly adaptive across all species groups: SPAD (as a measure of chlorophyll content, adaptive to N and P limitation), leaf area, and root surface area (adaptive to P limitation). We found little differences in trait plasticity between endangered, nonendangered, and invasive species. Synthesis. Along a gradient from N limitation, balanced N:P supply, and P limitation, we found that the type of fluctuating nutrient (i.e., if N or P is varied) is decisive for the adaptive value of a trait. Variation in P availability (from balanced supply to P limitation) created both a stronger reduction in fitness as well as created plasticity costs in more traits than variation in N availability (from balanced supply to N limitation). However, the patterns observed in our study may change if nutrient availability is altered, either by nutrient inputs or by a shift in nutrient availabilities, for example, by decreasing N input as foreseen by European Legislation, but without simultaneously decreasing P input.

Variation in DNA Methylation in Avian Nestlings Is Largely Determined by Genetic Effects.

As environmental fluctuations are becoming more common, organisms need to rapidly adapt to anthropogenic, climatic, and ecological changes. Epigenetic modifications and DNA methylation in particular provide organisms with a mechanism to shape their phenotypic responses during development. Studies suggest that environmentally induced DNA methylation might allow for adaptive phenotypic plasticity that could last throughout an organism's lifetime. Despite a number of studies demonstrating environmentally induced DNA methylation changes, we know relatively little about what proportion of the epigenome is affected by environmental factors, rather than being a consequence of genetic variation. In the current study, we use a partial cross-foster design in a natural great tit (Parus major) population to disentangle the effects of common origin from common rearing environment on DNA methylation. We found that variance in DNA methylation in 8,315 CpG sites was explained by a common origin and only in 101 by a common rearing environment. Subsequently, we mapped quantitative trait loci for the brood of origin CpG sites and detected 754 cis and 4,202 trans methylation quantitative trait loci, involving 24% of the CpG sites. Our results indicate that the scope for environmentally induced methylation marks independent of the genotype is limited and that the majority of variation in DNA methylation early in life is determined by genetic factors instead. These findings suggest that there may be little opportunity for selection to act on variation in DNA methylation. This implies that most DNA methylation variation likely does not evolve independently of genomic changes.

Environmental and genealogical effects on DNA methylation in a widespread apomictic dandelion lineage.

DNA methylation in plant genomes occurs in different sequences and genomic contexts that have very different properties. DNA methylation that occurs in CG (mCG) sequence context shows transgenerational stability and high epimutation rate, and can thus provide genealogical information at short time scales. However, due to meta-stability and because mCG variants may arise due to other factors than epimutation, such as environmental stress exposure, it is not clear how well mCG captures genealogical information at micro-evolutionary time scales. Here, we analysed DNA methylation variation between accessions from a geographically widespread, apomictic common dandelion (Taraxacum officinale) lineage when grown experimentally under different light conditions. Using a reduced-representation bisulphite sequencing approach, we show that the light treatment induced differentially methylated cytosines (DMCs) in all sequence contexts, with a bias towards transposable elements. Accession differences were associated mainly with DMCs in CG context. Hierarchical clustering of samples based on total mCG profiles revealed a perfect clustering of samples by accession identity, irrespective of light conditions. Using microsatellite information as a benchmark of genetic divergence within the clonal lineage, we show that genetic divergence between accessions correlates strongly with overall mCG profiles. However, our results suggest that environmental effects that do occur in CG context may produce a heritable signal that partly dilutes the genealogical signal. Our study shows that methylation information in plants can be used to reconstruct micro-evolutionary genealogy, providing a useful tool in systems that lack genetic variation such as clonal and vegetatively propagated plants.

Exploring the Interspecific Interactions and the Metabolome of the Soil Isolate Hylemonella gracilis.

Microbial community analysis of aquatic environments showed that an important component of its microbial diversity consists of bacteria with cell sizes of ~0.1 µm. Such small bacteria can show genomic reductions and metabolic dependencies with other bacteria. However, so far, no study has investigated if such bacteria exist in terrestrial environments like soil. Here, we isolated soil bacteria that passed through a 0.1-µm filter. The complete genome of one of the isolates was sequenced and the bacterium was identified as Hylemonella gracilis. A set of coculture assays with phylogenetically distant soil bacteria with different cell and genome sizes was performed. The coculture assays revealed that H. gracilis grows better when interacting with other soil bacteria like Paenibacillus sp. AD87 and Serratia plymuthica. Transcriptomics and metabolomics showed that H. gracilis was able to change gene expression, behavior, and biochemistry of the interacting bacteria without direct cell-cell contact. Our study indicates that in soil there are bacteria that can pass through a 0.1-µm filter. These bacteria may have been overlooked in previous research on soil microbial communities. Such small bacteria, exemplified here by H. gracilis, can induce transcriptional and metabolomic changes in other bacteria upon their interactions in soil. In vitro, the studied interspecific interactions allowed utilization of growth substrates that could not be utilized by monocultures, suggesting that biochemical interactions between substantially different sized soil bacteria may contribute to the symbiosis of soil bacterial communities. IMPORTANCE Analysis of aquatic microbial communities revealed that parts of its diversity consist of bacteria with cell sizes of ~0.1 µm. Such bacteria can show genomic reductions and metabolic dependencies with other bacteria. So far, no study investigated if such bacteria exist in terrestrial environments such as soil. Here, we show that such bacteria also exist in soil. The isolated bacteria were identified as Hylemonella gracilis. Coculture assays with phylogenetically different soil bacteria revealed that H. gracilis grows better when cocultured with other soil bacteria. Transcriptomics and metabolomics showed that H. gracilis was able to change gene expression, behavior, and biochemistry of the interacting bacteria without direct contact. Our study revealed that bacteria are present in soil that can pass through 0.1-µm filters. Such bacteria may have been overlooked in previous research on soil microbial communities and may contribute to the symbiosis of soil bacterial communities.

Changes in the rearing environment cause reorganization of molecular networks associated with DNA methylation.

Disentangling the interaction between the genetic basis and environmental context underlying phenotypic variation is critical for understanding organismal evolution. Environmental change, such as increased rates of urbanization, can induce shifts in phenotypic plasticity with some individuals adapting to city life while others are displaced. A key trait that can facilitate adaptation is the degree at which animals respond to stressors. This stress response, which includes elevation of baseline circulating concentrations of glucocorticoids, has a heritable component and exhibits intra- and inter-individual variation. However, the mechanisms behind this variability and whether they might be responsible for adaptation to different environments are not known. Variation in DNA methylation can be a potential mechanism that mediates environmental effects on the stress response, as early-life stressors increase glucocorticoid concentrations and change adult phenotype. We used an inter- and intra-environmental cross-foster experiment to analyse the contribution of DNA methylation to early-life phenotypic variation. We found that at hatching, urban house wren (Troglodytes aedon) offspring had higher methylation frequencies compared with their rural counterparts. We also observed age-related patterns in offspring methylation, indicating the developmental effects of the rearing environment on methylation. At fledgling, differential methylation analyses showed that cellular respiration genes were differentially methylated in broods of different origins and behavioural and metabolism genes were differentially methylated in broods of different rearing environments. Lastly, hyper-methylation of a single gene (CNTNAP2) is associated with decreased glucocorticoid levels and the rearing environment. These differential methylation patterns linked to a specific physiological phenotype suggest that DNA methylation may be a mechanism by which individuals adjust to novel environments during their lifespan. Characterizing genetic and environmental influences on methylation is critical for understanding the role of epigenetic mechanisms in evolutionary adaptation.

DNA methylation in clonal duckweed (Lemna minor L.) lineages reflects current and historical environmental exposures.

Environmentally induced DNA methylation variants may mediate gene expression responses to environmental changes. If such induced variants are transgenerationally stable, there is potential for expression responses to persist over multiple generations. Our current knowledge in plants, however, is almost exclusively based on studies conducted in sexually reproducing species where the majority of DNA methylation changes are subject to resetting in germlines, limiting the potential for transgenerational epigenetics stress memory. Asexual reproduction circumvents germlines, and may therefore be more conducive to long-term inheritance of epigenetic marks. Taking advantage of the rapid clonal reproduction of the common duckweed Lemna minor, we hypothesize that long-term, transgenerational stress memory from exposure to high temperature can be detected in DNA methylation profiles. Using a reduced representation bisulphite sequencing approach (epiGBS), we show that temperature stress induces DNA hypermethylation at many CG and CHG cytosine contexts but not CHH. Additionally, differential methylation in CHG context that was observed was still detected in a subset of cytosines, even after 3-12 generations of culturing in a common environment. This demonstrates a memory effect of stress reflected in the methylome and that persists over multiple clonal generations. Structural annotation revealed that this memory effect in CHG methylation was enriched in transposable elements. The observed epigenetic stress memory is probably caused by stable transgenerational persistence of temperature-induced DNA methylation variants across clonal generations. To the extent that such epigenetic memory has functional consequences for gene expression and phenotypes, this result suggests potential for long-term modulation of stress responses in asexual plants.

Adapting to climate with limited genetic diversity: Nucleotide, DNA methylation and microbiome variation among populations of the social spider Stegodyphus dumicola.

Understanding the role of genetic and nongenetic variants in modulating phenotypes is central to our knowledge of adaptive responses to local conditions and environmental change, particularly in species with such low population genetic diversity that it is likely to limit their evolutionary potential. A first step towards uncovering the molecular mechanisms underlying population-specific responses to the environment is to carry out environmental association studies. We associated climatic variation with genetic, epigenetic and microbiome variation in populations of a social spider with extremely low standing genetic diversity. We identified genetic variants that are associated strongly with environmental variation, particularly with average temperature, a pattern consistent with local adaptation. Variation in DNA methylation in many genes was strongly correlated with a wide set of climate parameters, thereby revealing a different pattern of associations than that of genetic variants, which show strong correlations to a more restricted range of climate parameters. DNA methylation levels were largely independent of cis-genetic variation and of overall genetic population structure, suggesting that DNA methylation can work as an independent mechanism. Microbiome composition also correlated with environmental variation, but most strong associations were with precipitation-related climatic factors. Our results suggest a role for both genetic and nongenetic mechanisms in shaping phenotypic responses to local environments.

epiGBS2: Improvements and evaluation of highly multiplexed, epiGBS-based reduced representation bisulfite sequencing.

Several reduced-representation bisulfite sequencing methods have been developed in recent years to determine cytosine methylation de novo in nonmodel species. Here, we present epiGBS2, a laboratory protocol based on epiGBS with a revised and user-friendly bioinformatics pipeline for a wide range of species with or without a reference genome. epiGBS2 is cost- and time-efficient and the computational workflow is designed in a user-friendly and reproducible manner. The library protocol allows a flexible choice of restriction enzymes and a double digest. The bioinformatics pipeline was integrated in the Snakemake workflow management system, which makes the pipeline easy to execute and modular, and parameter settings for important computational steps flexible. We implemented bismark for alignment and methylation analysis and we preprocessed alignment files by double masking to enable single nucleotide polymorphism calling with Freebayes (epiFreebayes). The performance of several critical steps in epiGBS2 was evaluated against baseline data sets from Arabidopsis thaliana and great tit (Parus major), which confirmed its overall good performance. We provide a detailed description of the laboratory protocol and an extensive manual of the bioinformatics pipeline, which is publicly accessible on github (https://github.com/nioo-knaw/epiGBS2) and zenodo (https://doi.org/10.5281/zenodo.4764652).

The methylome of Biomphalaria glabrata and other mollusks: enduring modification of epigenetic landscape and phenotypic traits by a new DNA methylation inhibitor.

BACKGROUND: 5-Methylcytosine (5mC) is an important epigenetic mark in eukaryotes. Little information about its role exists for invertebrates. To investigate the contribution of 5mC to phenotypic variation in invertebrates, alteration of methylation patterns needs to be produced. Here, we apply new non-nucleoside DNA methyltransferase inhibitors (DNMTi) to introduce aleatory changes into the methylome of mollusk species. RESULTS: Flavanone inhibitor Flv1 was efficient in reducing 5mC in the freshwater snails Biomphalaria glabrata and Physa acuta, and to a lesser degree, probably due to lower stability in sea water, in the oyster Crassostrea gigas. Flv1 has no toxic effects and significantly decreased the 5mC level in the treated B. glabrata and in its offspring. Drug treatment triggers significant variation in the shell height in both generations. A reduced representation bisulfite-sequencing method called epiGBS corroborates hypomethylation effect of Flv1 in both B. glabrata generations and identifies seven Differential Methylated Regions (DMR) out of 32 found both in Flv1-exposed snails and its progeny, from which 5 were hypomethylated, demonstrating a multigenerational effect. By targeted bisulfite sequencing, we confirmed hypomethylation in a locus and show that it is associated with reduced gene expression. CONCLUSIONS: Flv1 is a new and efficient DNMTi that can be used to induce transient and heritable modifications of the epigenetic landscape and phenotypic traits in mollusks, a phylum of the invertebrates in which epigenetics is understudied.

Gene body DNA methylation in seagrasses: inter- and intraspecific differences and interaction with transcriptome plasticity under heat stress.

The role of DNA methylation and its interaction with gene expression and transcriptome plasticity is poorly understood, and current insight comes mainly from studies in very few model plant species. Here, we study gene body DNA methylation (gbM) and gene expression patterns in ecotypes from contrasting thermal environments of two marine plants with contrasting life history strategies in order to explore the potential role epigenetic mechanisms could play in gene plasticity and responsiveness to heat stress. In silico transcriptome analysis of CpGO/E ratios suggested that the bulk of Posidonia oceanica and Cymodocea nodosa genes possess high levels of intragenic methylation. We also observed a correlation between gbM and gene expression flexibility: genes with low DNA methylation tend to show flexible gene expression and plasticity under changing conditions. Furthermore, the empirical determination of global DNA methylation (5-mC) showed patterns of intra and inter-specific divergence that suggests a link between methylation level and the plants' latitude of origin and life history. Although we cannot discern whether gbM regulates gene expression or vice versa, or if other molecular mechanisms play a role in facilitating transcriptome responsiveness, our findings point to the existence of a relationship between gene responsiveness and gbM patterns in marine plants.

Epigenetics of Animal Personality: DNA Methylation Cannot Explain the Heritability of Exploratory Behavior in a Songbird.

The search for the hereditary mechanisms underlying quantitative traits traditionally focused on the identification of underlying genomic polymorphisms such as single-nucleotide polymorphisms. It has now become clear that epigenetic mechanisms, such as DNA methylation, can consistently alter gene expression over multiple generations. It is unclear, however, if and how DNA methylation can stably be transferred from one generation to the next and can thereby be a component of the heritable variation of a trait. In this study, we explore whether DNA methylation responds to phenotypic selection using whole-genome and genome-wide bisulfite approaches. We assessed differential erythrocyte DNA methylation patterns between extreme personality types in the Great Tit (Parus major). For this, we used individuals from a four-generation artificial bi-directional selection experiment and siblings from eight F2 inter-cross families. We find no differentially methylated sites when comparing the selected personality lines, providing no evidence for the so-called epialleles associated with exploratory behavior. Using a pair-wise sibling design in the F2 intercrosses, we show that the genome-wide DNA methylation profiles of individuals are mainly explained by family structure, indicating that the majority of variation in DNA methylation in CpG sites between individuals can be explained by genetic differences. Although we found some candidates explaining behavioral differences between F2 siblings, we could not confirm this with a whole-genome approach, thereby confirming the absence of epialleles in these F2 intercrosses. We conclude that while epigenetic variation may underlie phenotypic variation in behavioral traits, we were not able to find evidence that DNA methylation can explain heritable variation in personality traits in Great Tits.

AlphaBeta: computational inference of epimutation rates and spectra from high-throughput DNA methylation data in plants.

Stochastic changes in DNA methylation (i.e., spontaneous epimutations) contribute to methylome diversity in plants. Here, we describe AlphaBeta, a computational method for estimating the precise rate of such stochastic events using pedigree-based DNA methylation data as input. We demonstrate how AlphaBeta can be employed to study transgenerationally heritable epimutations in clonal or sexually derived mutation accumulation lines, as well as somatic epimutations in long-lived perennials. Application of our method to published and new data reveals that spontaneous epimutations accumulate neutrally at the genome-wide scale, originate mainly during somatic development and that they can be used as a molecular clock for age-dating trees.

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.

Transgenerational Effects and Epigenetic Memory in the Clonal Plant Trifolium repens.

Transgenerational effects (TGE) can modify phenotypes of offspring generations playing thus a potentially important role in ecology and evolution of many plant species. These effects have been studied mostly across generations of sexually reproducing species. A substantial proportion of plant species are however reproducing asexually, for instance via clonal growth. TGE are thought to be enabled by heritable epigenetic modification of DNA, although unambiguous evidence is still scarce. On the clonal herb white clover (Trifolium repens), we tested the generality of clonal TGE across five genotypes and five parental environments including soil contamination and above-ground competition. Moreover, by genome wide-methylation variation analysis we explored the role of drought, one of the parental environments that triggered the strongest TGE. We tested the induction of epigenetic changes in offspring generations using several intensities and durations of drought stress. We found that TGE of different environments were highly genotype specific and all tested environments triggered TGE at least in some genotypes. In addition, parental drought stresses triggered epigenetic change in T. repens and most of the induced epigenetic change was maintained across several clonal offspring generations. We conclude that TGE are common and genotype specific in clonal plant T. repens and potentially under epigenetic control.

Cross-species interference of gene expression.

Microbes can contribute to protection of animals and plants against diseases. A recent study reveals a mechanism by which a bacterium controls fungal infection in wheat, involving secretion of a metabolite that affects histone acetyltransferase activity of a plant pathogenic fungus.

LAESI mass spectrometry imaging as a tool to differentiate the root metabolome of native and range-expanding plant species.

MAIN CONCLUSION: LAESI-MSI, an innovative high-throughput technique holds a unique potential for untargeted detection, profiling and spatial localization of metabolites from intact plant samples without need for extraction or extensive sample preparation. Our understanding of chemical diversity in biological samples has greatly improved through recent advances in mass spectrometry (MS). MS-based-imaging (MSI) techniques have further enhanced this by providing spatial information on the distribution of metabolites and their relative abundance. This study aims to employ laser-ablation electrospray ionization (LAESI) MSI as a tool to profile and compare the root metabolome of two pairs of native and range-expanding plant species. It has been proposed that successful range-expanding plant species, like introduced exotic invaders, have a novel, or a more diverse secondary chemistry. Although some tests have been made using aboveground plant materials, tests using root materials are rare. We tested the hypothesis that range-expanding plants possess more diverse root chemistries than native plant species. To examine the root chemistry of the selected plant species, LAESI-MSI was performed in positive ion mode and data were acquired in a mass range of m/z 50-1200 with a spatial resolution of 100 µm. The acquired data were analyzed using in-house scripts, and differences in the spatial profiles were studied for discriminatory mass features. The results revealed clear differences in the metabolite profiles amongst and within both pairs of congeneric plant species, in the form of distinct metabolic fingerprints. The use of ambient conditions and the fact that no sample preparation was required, established LAESI-MSI as an ideal technique for untargeted metabolomics and for direct correlation of the acquired data to the underlying metabolomic complexity present in intact plant samples.