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The phenotypes of plants can be influenced by the environmental conditions experienced by their parents. However, there is still much uncertainty about how common and how predictable such parental environmental effects really are. We carried out a comprehensive experimental test for parental effects, subjecting plants of multiple Arabidopsis thaliana genotypes to 24 different biotic or abiotic stresses, or combinations thereof, and comparing their offspring phenotypes in a common environment. The majority of environmental stresses caused significant parental effects, with -35% to +38% changes in offspring fitness. The expression of parental effects was strongly genotype-dependent, and multiple environmental stresses often acted non-additively when combined. The direction and magnitude of parental effects were unrelated to the direct effects on the parents: some environmental stresses did not affect the parents but caused substantial effects on offspring, while for others the situation was reversed. Our study demonstrates that parental environmental effects are common and often strong in A. thaliana, but they are genotype-dependent, act non-additively, and are difficult to predict. We should thus be cautious with generalizing from simple studies with single plant genotypes and/or only few individual environmental stresses. A thorough and general understanding of parental effects requires large multi-factorial experiments.

SummaryChanges in environments trigger plant DNA methylation responses, potentially modulating stress responses. Studies on stress-induced DNA methylation typically focus on single species, limiting our understanding of what are general and specific responses between species.Using reduced-representation bisulfite sequencing epiGBS, we compared DNA methylation stress-responses across seven plant species. Because methylation can be targeted at transposable elements (TEs) and because environmental plasticity may be particularly relevant in asexual species, we hypothesize that genome size and reproduction mode explain differences in methylation responses between species.We show that enrichment of environmentally-induced methylation responses in genes and TEs is a general feature across plant species. While previous studies have emphasized methylation responses in CHH-cytosines, we observed that cytosines in all sequence contexts (CG, CHG, CHH) are equally likely to respond to stress. Larger-genome species showed a higher proportion of stress-responding cytosines, and asexual species showed more cytosines with a strong methylation response to stress than sexually responding species.Our study highlights the role of TEs in methylome plasticity and identifies causes of variation in methylome plasticity. This provides guidance to extrapolating results from models to other plant species, and may contribute to better understanding of functionality of the response.

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.

Co-occurring tree functional types (TFTs) within forest–savanna transitions may differ in seedling responses to grass competition and fire in savannas. We performed a common garden experiment in the Guinea savanna of Ghana to test hypotheses related to competition effects on growth, allocation to root storage reserves, and subsequent survival responses to dry season fire for savanna-transitional TFT (i.e., species occurring both in forest and savanna) and forest TFT. The experiment included factorial combinations of TFT, comprising four species each of forest versus savanna-transitional trees, wet season grass competition (grass competition vs. grass no-grass competition), and dry season fire (burn vs. no-burn). Partly consistent with prediction, we found that grass competition suppressed tree seedling growth and caused a 17% reduction in root non-structural carbohydrates concentration [NSC] but had no effect on direct survival regardless of TFT at the end of the wet season. Post-fire survival averaged 6% for forest versus 91% for savanna-transitional TFTs across competition treatments. In contrast to our prediction on how grass competition influences post-fire seedling survival, a history of grass competition did not result in lower post-fire survival regardless of TFT, although plant mass, root mass fraction, and root [NSC] at the end of the dry season were lower for tree seedlings with a history of competition. Our results demonstrate that grass competition suppresses tree seedling growth and root storage reserves irrespective of TFT, and that competition alone (without fire) may not preclude the establishment of forest seedlings in savannas close to forests.

International audience; Species turnover is ubiquitous. However, it remains unknown whether certain types of species are consistently gained or lost across different habitats. Here, we analysed the trajectories of 1827 plant species over time intervals of up to 78 years at 141 sites across mountain summits, forests, and lowland grasslands in Europe. We found, albeit with relatively small effect sizes, displacements of smaller- by larger-ranged species across habitats. Communities shifted in parallel towards more nutrient-demanding species, with species from nutrient-rich habitats having larger ranges. Because these species are typically strong competitors, declines of smaller-ranged species could reflect not only abiotic drivers of global change, but also biotic pressure from increased competition. The ubiquitous component of turnover based on species range size we found here may partially reconcile findings of no net loss in local diversity with global species loss, and link community-scale turnover to macroecological processes such as biotic homogenisation.

The phenotypes of plants can be influenced by the environmental conditions experienced by their parents. In some cases, such parental effects have been found to be adaptive, which has led to much speculation about their ecological and evolutionary significance. However, there is still much uncertainty about how common and how predictable parental environmental effects really are. We carried out a comprehensive test for parental effects of different environmental stresses in the model plant Arabidopsis thaliana. We subjected plants of three Arabidopsis genotypes to a broad range of biotic or abiotic stresses, or combinations thereof, and compared their offspring phenotypes in a common environment. The majority of environmental stresses (16 out of 24 stress treatments) caused significant parental effects, in particular on plant biomass and reproduction, with positive or negative effects ranging from −35% to +38% changes in offspring fitness. The expression of parental effects was strongly genotype-dependent, with some effects only present in some genotypes but absent, or even in the opposite direction, in others. Parental effects of multiple environmental stresses were often non-additive, and their effects can thus not be predicted from what we know about the effects of individual stresses. Intriguingly, the direction and magnitude of parental effects were unrelated to the direct effects on the parents: some stresses did not affect the parents but caused substantial effects on offspring, while for others the situation was reversed. In summary, parental environmental effects are common and often strong in A. thaliana, but they are genotype-dependent and difficult to predict.SignificanceStress experienced by plants can alter the phenotypes of their offspring. To understand the ecological and evolutionary significance of such parental effects, we must know how common and how predictable they are. In a large experiment with Arabidopsis thaliana, we show that the majority of 24 environmental stresses cause significant, and often strong, positive or negative parental effects. However, we also find that parental effects are genotype-specific and unrelated to the direct effect of individual stresses, and that multiple stresses often act in non-additive ways across generations. Thus, parental effects appear to be common and strong, but difficult to predict. Our findings have important implications for the study of plant responses to environmental change, and the design of stress experiments.

We evaluated the genetic diversity of teak provenances at a newly established provenance trial with 52 provenances collected from Africa, South America and Asia in Tain II Forest Reserve in Central Ghana. This provenance collection / trial was established to widen the genetic basis for Teak establishment in West Africa. Using Genotyping by Sequencing (GBS) we analysed the genetic diversity of these provenances. Results of the study revealed that, although acquired from a wide geographical range, most Teak provenances in the trial belong to only two distinct groups that are closely related. The implication of this finding is that, for breeding, a wider range of provenances are needed from the original Teak distribution areas and more specifically from Southern India. We conclude that urgent protection of older existing sources of genetic variation in Teak as well as an improvement of international collaboration under the Nagoya protocol with countries with native teak populations is necessary.

epiGBS is an existing reduced representation bisulfite sequencing method to determine cytosine methylation and genetic polymorphisms de novo. Here, we present epiGBS2, an improved epiGBS laboratory protocol and user-friendly bioinformatics pipeline for a wide range of species with or without reference genome. epiGBS2 decreases costs and time investment and increases user-friendliness and reproducibility. The library protocol was adjusted to allow for a flexible choice of restriction enzymes and a double digest. Instead of fully methylated adapters, semi-methylated adapters are now used. The bioinformatics pipeline was improved in speed and integrated in the snakemake workflow management system, which now makes the pipeline easy to execute, modular, and parameter settings flexible. We also provide a detailed description of the laboratory protocol, 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.3819996), and example output.

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.

Populations often differ in phenotype and these differences can be caused by adaptation by natural selection, random neutral processes, and environmental responses. The most straightforward way to divide mechanisms that influence phenotypic variation is heritable variation and environmental-induced variation (e.g., plasticity). While genetic variation is responsible for most heritable phenotypic variation, part of this is also caused by nongenetic inheritance. Epigenetic processes may be one of the underlying mechanisms of plasticity and nongenetic inheritance and can therefore possibly contribute to heritable differences through drift and selection. Epigenetic variation may be influenced directly by the environment, and part of this variation can be transmitted to next generations. Field screenings combined with common garden experiments will add valuable insights into epigenetic differentiation, epigenetic memory and can help to reveal part of the relative importance of epigenetics in explaining trait variation. We explored both genetic and epigenetic diversity, structure and differentiation in the field and a common garden for five British and five French Scabiosa columbaria populations. Genetic and epigenetic variation was subsequently correlated with trait variation. Populations showed significant epigenetic differentiation between populations and countries in the field, but also when grown in a common garden. By comparing the epigenetic variation between field and common garden-grown plants, we showed that a considerable part of the epigenetic memory differed from the field-grown plants and was presumably environmentally induced. The memory component can consist of heritable variation in methylation that is not sensitive to environments and possibly genetically based, or environmentally induced variation that is heritable, or a combination of both. Additionally, random epimutations might be responsible for some differences as well. By comparing epigenetic variation in both the field and common environment, our study provides useful insight into the environmental and genetic components of epigenetic variation.

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.

Summary Transgenerational environmental effects can trigger strong phenotypic variation. However, it is unclear how cues from different preceding generations interact. Also, little is known about the genetic variation for these life history traits. Here, we present the effects of grandparental and parental mild heat, and their combination, on four traits of the third-generation phenotype of 14 Arabidopsis thaliana genotypes. We tested for correlations of these effects with climate and constructed a conceptual model to identify the environmental conditions that favour the parental effect on flowering time. We observed strong evidence for genotype-specific transgenerational effects. On average, A. thaliana accustomed to mild heat produced more seeds after two generations. Parental effects overruled grandparental effects in all traits except reproductive biomass. Flowering was generally accelerated by all transgenerational effects. Notably, the parental effect triggered earliest flowering in genotypes adapted to dry summers. Accordingly, this parental effect was favoured in the model when early summer heat terminated the growing season and environments were correlated across generations. Our results suggest that A. thaliana can partly accustom to mild heat over two generations and genotype-specific parental effects show non-random evolutionary divergence across populations that may support climate change adaptation in the Mediterranean.

Questions: Has plant species richness in semi‐natural grasslands changed over recent decades? Do the temporal trends of habitat specialists differ from those of habitat generalists? Has there been a homogenization of the grassland vegetation? Location: Different regions in Germany and the UK. Methods: We conducted a formal meta‐analysis of re‐survey vegetation studies of semi‐natural grasslands. In total, 23 data sets were compiled, spanning up to 75 years between the surveys, including 13 data sets from wet grasslands, six from dry grasslands and four from other grassland types. Edaphic conditions were assessed using mean Ellenberg indicator values for soil moisture, nitrogen and pH. Changes in species richness and environmental variables were evaluated using response ratios. Results: In most wet grasslands, total species richness declined over time, while habitat specialists almost completely vanished. The number of species losses increased with increasing time between the surveys and were associated with a strong decrease in soil moisture and higher soil nutrient contents. Wet grasslands in nature reserves showed no such changes or even opposite trends. In dry grasslands and other grassland types, total species richness did not consistently change, but the number or proportions of habitat specialists declined. There were also considerable changes in species composition, especially in wet grasslands that often have been converted into intensively managed, highly productive meadows or pastures. We did not find a general homogenization of the vegetation in any of the grassland types. Conclusions: The results document the widespread deterioration of semi‐natural grasslands, especially of those types that can easily be transformed to high production grasslands. The main causes for the loss of grassland specialists are changed management in combination with increased fertilization and nitrogen deposition. Dry grasslands are most resistant to change, but also show a long‐term trend towards an increase in more mesotrophic species.

Differential tree seedling recruitment across forest-savanna ecotones is poorly understood, but hypothesized to be influenced by vegetation cover and associated factors. In a 3-y-long field transplant experiment in the forest-savanna ecotone of Ghana, we assessed performance and root allocation of 864 seedlings for two forest (Khaya ivorensis and Terminalia superba) and two savanna (Khaya senegalensis and Terminalia macroptera) species in savanna woodland, closed-woodland and forest. Herbaceous vegetation biomass was significantly higher in savanna woodland (1.0 ± 0.4 kg m−2 vs 0.2 ± 0.1 kg m−2 in forest) and hence expected fire intensities, while some soil properties were improved in forest. Regardless, seedling survival declined significantly in the first-year dry-season for all species with huge declines for the forest species (50% vs 6% for Khaya and 16% vs 2% for Terminalia) by year 2. After 3 y, only savanna species survived in savanna woodland. However, best performance for savanna Khaya was in forest, but in savanna woodland for savanna Terminalia which also had the highest biomass fraction (0.8 ± 0.1 g g−1 vs 0.6 ± 0.1 g g−1 and 0.4 ± 0.1 g g−1) and starch concentration (27% ± 10% vs 15% ± 7% and 10% ± 4%) in roots relative to savanna and forest Khaya respectively. Our results demonstrate that tree cover variation has species-specific effects on tree seedling recruitment which is related to root storage functions.

Current approaches for assessing the effects of invasive alien species (IAS) are biased toward the negative effects of these species, resulting in an incomplete picture of their real effects. This can result in an inefficient IAS management. We address this issue by describing the INvasive Species Effects Assessment Tool (INSEAT) that enables expert elicitation for rapidly assessing the ecological consequences of IAS using the ecosystem services (ES) framework. INSEAT scores the ecosystem service “gains and losses” using a scale that accounted for the magnitude and the reversibility of its effects. We tested INSEAT on 18 IAS in Great Britain. Here, we highlighted four case studies: Harmonia axyridis (Harlequin ladybird), Astacus leptodactylus (Turkish crayfish), Pacifastacus leniusculus (Signal crayfish) and Impatiens glandulifera (Himalayan balsam). The results demonstrated that a collation of different experts’ opinions using INSEAT could yield valuable information on the invasive aliens’ ecological and social effects. The users can identify certain IAS as ES providers and the trade‐offs between the ES provision and loss associated with them. This practical tool can be useful for evidence‐based policy and management decisions that consider the potential role of invasive species in delivering human well‐being.

Biodiversity often increases plant productivity. In long-term grassland experiments, positive biodiversity effects on plant productivity commonly increase with time. Also, it has been shown that such positive biodiversity effects persist not only in the local environment but also when plants are transferred into a common environment. Thus, we hypothesized that community diversity had acted as a selective agent, resulting in the emergence of plant monoculture and mixture types with differing genetic composition. To test our hypothesis, we grew offspring from plants that were grown for eleven years in monoculture or mixture environments in a biodiversity experiment (Jena Experiment) under controlled glasshouse conditions in monocultures or two-species mixtures. We used epiGBS, a genotyping-by-sequencing approach combined with bisulfite conversion to provide integrative genetic and epigenetic data. We observed significant genetic and epigenetic divergence according to selection history in three out of five perennial grassland species, namelyGalium mollugo,Prunella vulgarisandVeronica chamaedrys, with epigenetic differences mostly reflecting the genetic differences. In addition, current diversity levels in the glasshouse had weak effects on epigenetic variation. However, given the limited genome coverage of the reference-free bisulfite method epiGBS, it remains unclear how much of this epigenetic divergence was independent of underlying genetic differences. Our results thus suggest that selection of genetic variants, and possibly epigenetic variants, caused the rapid emergence of monoculture and mixture types within plant species in the Jena Experiment.

Inbreeding depression plays a central role within the conservation genetics paradigm. Until now inbreeding depression is incorporated into models of population viability as a mean value (e.g. number of lethal equivalents) for all traits in a population. In this study of the locally threatened perennial plant species Scabiosa columbaria we investigated both the mean and the variance among families of inbreeding depression in eight life history traits for five natural populations varying in size from 300 to more than 120,000 individuals. Significant inbreeding depression was found in all populations and all traits. The mean inbreeding depression value per trait was never correlated to population size. Within each population, highly significant variation in inbreeding depression between families (VIFLID) was found. Per trait, families with inbreeding depression next to families with outbreeding depression were often found within the same population. Inbreeding depression at the family level was in many cases not correlated among traits and independent of correlations between traits themselves. VIFLID was negatively correlated with population size: in two traits these correlations were significant. The results underline that inbreeding depression is a complex, highly dynamic phenomenon. Models of viability should incorporate inbreeding depression distributions, with a trait specific mean and variance. Moreover, models of metapopulation dynamics should incorporate genotype quality as factor in colonization success.

Populations often differ in phenotype and these differences can be caused by adaptation by natural selection, random neutral processes, and environmental responses. The most straightforward way to divide mechanisms that influence phenotypic variation is heritable variation and environmental‐induced variation (e.g., plasticity). While genetic variation is responsible for most heritable phenotypic variation, part of this is also caused by nongenetic inheritance. Epigenetic processes may be one of the underlying mechanisms of plasticity and nongenetic inheritance and can therefore possibly contribute to heritable differences through drift and selection. Epigenetic variation may be influenced directly by the environment, and part of this variation can be transmitted to next generations. Field screenings combined with common garden experiments will add valuable insights into epigenetic differentiation, epigenetic memory and can help to reveal part of the relative importance of epigenetics in explaining trait variation. We explored both genetic and epigenetic diversity, structure and differentiation in the field and a common garden for five British and five French Scabiosa columbaria populations. Genetic and epigenetic variation was subsequently correlated with trait variation. Populations showed significant epigenetic differentiation between populations and countries in the field, but also when grown in a common garden. By comparing the epigenetic variation between field and common garden‐grown plants, we showed that a considerable part of the epigenetic memory differed from the field‐grown plants and was presumably environmentally induced. The memory component can consist of heritable variation in methylation that is not sensitive to environments and possibly genetically based, or environmentally induced variation that is heritable, or a combination of both. Additionally, random epimutations might be responsible for some differences as well. By comparing epigenetic variation in both the field and common environment, our study provides useful insight into the environmental and genetic components of epigenetic variation.

For plants with wide distributional areas, covering a wide range of ecologically distinct habitats, evolutionary divergence can lead to substantial phenotypic variation across a species' range. These intraspecific trait differences can be very informative about the nature of the selective environment as they potentially reflect different environmental selection pressures while controlling for other species characteristics. In this study, multiple regression and structural equation models were used to examine the relative importance of environmental, ecological, population size and population density effects for variation in growth, reproduction and leaf morphology among 36 populations of the perennial plant Arabidopsis lyrata ssp. petraea across its northwest European range. Substantial variation in temperature, soil nutrient levels and herbivory was observed across the species' range. In addition, large differences in flowering percentage and individual seed production were found. Leaf morphology varied considerably, with a substantial amount of variation in specific leaf area and trichome density among populations. Structural equation modeling suggested that this species is sensitive to small population sizes, eutrophication and herbivory. Reproductive output was negatively related to herbivory. In addition population size was negatively associated with soil nutrient concentrations. Leaf morphology was shown to be mainly associated with temperature and herbivory. Lower specific leaf areas and lower trichome densities were related to colder areas and high trichomes densities were related to high levels of herbivory. These model results are consistent with the interpretation that, in addition to changing environmental effects across its range, ecological effects such as herbivory contribute to the large variation in life history and morphology of this species. The results reveal a strong negative effect of herbivory on the reproductive output of this species, not only via direct effects of herbivory on flowers and seeds, but also indirectly via a shift in life history strategy.

Atmospheric nitrogen (N) deposition has been identified as a major threat to biodiversity, but field surveys of its effects have rarely focussed on sites which are actively managed to maintain characteristic species. We analysed permanent quadrat data from 106 plots in nature reserves on calcareous grassland sites in the United Kingdom collected during a survey between 1990 and 1993 and compared the data with the results from resurvey of 48 of these plots between 2006 and 2009. N deposition showed no significant spatial association with species richness, species diversity, or the frequency of species adapted to low nutrient conditions in the 1990–1993 dataset. However, temporal analysis showed that N deposition was significantly associated with changes in Shannon diversity and evenness. In plots with high rates of N deposition, there was a decrease in species diversity and evenness, a decline in the frequency of characteristic calcareous grassland species, and a lower number of rare and scarce species. As all sites had active management to maintain a high diversity and characteristic species, our results imply that even focussed management on nature conservation objectives cannot prevent adverse effects of high rates of N deposition. Structural equation modelling was used to compare different causal mechanisms to explain the observed effects. For change in Shannon diversity, direct effects of N deposition were the dominant mechanism and there was an independent effect of change in grazing intensity. In contrast, for change in herb species number, indirect effects on soil acidity, linked to both N and S deposition, were more important than direct effects of N deposition.

Habitat fragmentation and climate change are recognized as major threats to biodiversity. The major challenge for present day plant populations is how to adapt and cope with altered abiotic and biotic environments caused by climate change, when at the same time adaptive and evolutionary potential is decreased as habitat fragmentation reduces genetic variation and increases inbreeding. Although the ecological and evolutionary effects of fragmentation and climate change have been investigated separately, their combined effects remained largely unexplored. In this review, we will discuss the individual and joint effects of habitat fragmentation and climate change on plants and how the abilities and ways in which plants can respond and cope with climate change may be compromised due to habitat fragmentation.

Summary • The adaptive responses to atmospheric nitrogen deposition for different European accessions of Arabidopsis lyrata petraea were analysed using populations along a strong atmospheric N-deposition gradient. • Plants were exposed to three N-deposition rates, reflecting the rates at the different locations, in a full factorial design. • Differences between accessions in the response to N were found for important phenological and physiological response variables. For example, plants from low-deposition areas had higher nitrogen-use efficiencies (NUE) and C : N ratios than plants from areas high in N deposition when grown at low N-deposition rates. The NUE decreased in all accessions at higher experimental deposition rates. However, plants from high-deposition areas showed a limited capacity to increase their NUE at lower experimental deposition rates. Plants from low-deposition areas had faster growth rates, higher leaf turnover rates and shorter times to flowering, and showed a greater increase in growth rate in response to N deposition than those from high-deposition areas. • Indications for adaptation to N deposition were found, and results suggest that adaptation of plants from areas high in N deposition to increased N deposition has resulted in the loss of plasticity.

Background and Aims Differential capacity of species/genotypes to respond to increased atmospheric N deposition may be an important long-term driver of species composition change. In this study, differences in response to atmospheric N deposition were analysed for different accessions of Prunella vulgaris. Methods P. vulgaris plants deriving from different N deposition accessions (0–15, 15–25 and 25–35 kg N ha−1 yr−1) were exposed to different Ntreatments (10 or 30 kg N ha−1 yr−1), with and without competition with the grass Bromus erectus. After 8 months, plant performance and differencesin competitive ability were determined. Results Strong effects of accession were found, often in interaction with N treatment: P. vulgaris plants frompopulations with low N deposition backgrounds often outperformed plants from medium to high N backgrounds, illustrated by greater biomass at high N treatment and higher chlorophyll and leaf N concentrations at low N treatments. When grown in competition, however, a strong decrease in biomass was observed for plants from all accessions. Conclusion All accessions of P. vulgaris showed a low competitive ability. Plants from low N accessions proved to be most sensitive to competition in terms of root biomass. However, in the absence of competition, low N accessions showed a greater plasticity in nitrogenuse efficiency. These results illustrate the importance of including accession when considering effects of N deposition.

Plant phenotypes can be affected by environments experienced by their parents. Parental environmental effects are reported for the first offspring generation and some studies showed persisting environmental effects in second and further offspring generations. However, the expression of these transgenerational effects proved context-dependent and their reproducibility can be low. Here we study the context-dependency of transgenerational effects by evaluating parental and transgenerational effects under a range of parental induction and offspring evaluation conditions. We systematically evaluated two factors that can influence the expression of transgenerational effects: single- versus multiple-generation exposure and offspring environment. For this purpose, we exposed a single homozygous Arabidopsis thaliana Col-0 line to salt stress for up to three generations and evaluated offspring performance under control and salt conditions in a climate chamber and in a natural environment. Parental as well as transgenerational effects were observed in almost all traits and all environments and traced back as far as great-grandparental environments. The length of exposure exerted strong effects; multiple-generation exposure often reduced the expression of the parental effect compared to single-generation exposure. Furthermore, the expression of transgenerational effects strongly depended on offspring environment for rosette diameter and flowering time, with opposite effects observed in field and greenhouse evaluation environments. Our results provide important new insights into the occurrence of transgenerational effects and contribute to a better understanding of the context-dependency of these effects.

The letter by Garner et al. [1] continued an important discussion regarding the role genomics might play in conservation biology. In general, we do not see a dichotomy between our point of view [2] and that put forth by Garner et al. [1]. At the heart of the issue is how to define an actual impact of genomics on applied conservation and find suitable ways to remove existing barriers limiting the use of genomics for managing wild populations. The promised gains of identifying adaptive loci and the genes underlying phenotypes 3, 4 have in most systems not yet been realized and recent empirical work further highlights the challenges 5, 6. Thus, our take-home message boiled down to the application of genomics in wild populations being at an early developmental stage that is far from straightforward and far from regularly applied [2].

We describe epiGBSBS, a reduced representation bisulfite sequencing method for cost-effective exploration and comparative analysis of DNADNADNA methylation and genetic variation in hundreds of samples de novo. This method uses genotyping by sequencing of bisulfite-converted DNADNADNA followed by reliable de novo reference construction, mapping, variant calling, and distinction of single-nucleotide polymorphisms (SNSNPs) versus methylation variation (software is available at https://github.com/thomasvangurp/epiGBS). The output can be loaded directly into a genome browser for visualization and into RnBeads for analysis of differential methylation.

Summary 1 Small and isolated populations of species are susceptible to loss of genetic diversity, owing to random genetic drift and inbreeding. This loss of diversity may reduce the evolutionary potential to adapt to changing environments, and may cause immediate loss of fitness (cf. inbreeding depression). Together with other population size-dependent stochastic processes, this may lead to increased probabilities of population extinction. 2 This set of processes and theories forms the core of conservation genetics and has developed into the conservation genetics paradigm. Many empirical studies have concentrated on the relationship between population size and genetic diversity, and in many cases evidence was found that small populations of plants do indeed have lower levels of genetic diversity and increased homozygosity. Although less empirical attention has been given to the relationship between low genetic diversity, fitness and, in particular, evolutionary potential, the paradigm is now widely accepted. 3 Here we present five areas of the paradigm which could be refined, i.e. the ‘rough’ edges of the conservation genetics paradigm. 4 Treating population size and isolation not as interchangeable parameters but as separate parameters affecting population genetics in different ways could allow more accurate predictions of the effects of landscape fragmentation on the genetic diversity and viability of populations. 5 There is evidence that inbreeding depression may be a genotype-specific phenomenon, rather than a population parameter. This sheds new light on the link between population inbreeding depression and the expected increased probability of extinction. 6 Modern eco-genomics offers the opportunity to study the population genetics of functional genes, to the extent that the role of selection can be distinguished from the effects of drift, and allowing improved insights into the effects of loss of genetic diversity on evolutionary potential. 7 Incorporating multispecies considerations may result in the generally accepted notion that small populations are at peril being called into question. For instance, small populations may be less capable of sustaining parasites or herbivores. 8 Comparative studies of endangered, common and invasive species may be a valuable approach to developing conservation biology from a phenomenological case study discipline into one investigating the general principles of what sustains biodiversity. 9 The issues discussed set an agenda for further research within conservation genetics and may lead to a further refinement of our understanding and prediction of the genetic effects of habitat fragmentation. They also underline the need to integrate ecological and genetic approaches to the conservation of biodiversity, rather than regarding them as opposites.

In two natural heathland vegetations, we analysed the effect of turf cutting on spore numbers of arbuscular mycorrhizal fungi (AMF). Next to this, we performed a controlled factorial experiment to examine the role of AMF for germination and establishment of Arnica montana in both turf cut and non-turf cut situations. AMF spore numbers decreased with soil depth, and, along with the topsoil, almost all AMF spores were removed with the removal of the acidified and/or eutrophied organic layer. Recolonisation of AMF spore numbers after turf cutting was slow: spore numbers of approximately 60–95 spores g −1 dry soil were found two and a half years after turf cutting, corresponding with 55–70% of AMF spore numbers found in natural field populations of A. montana . Since AMF colonisation increased establishment and biomass, and decreased mortality of A. montana , it was suggested that lack of AMF after turf removal might complicate the establishment of this herbaceous species. Removal of organic material as a management measure should therefore carefully be applied, taking in consideration the low recolonisation rates of AMF as this can markedly effect the success of restoration.

The effects of increasing ammonium concentrations in combination with different pH levels were studied on five heathland plant species to determine whether their occurrence and decline could be attributed to ammonium toxicity and/or pH levels. Plants were grown in growth media amended with four different ammonium concentrations (10, 100, 500 and 1000 micromol l(-1)) and two pH levels resembling acidified (pH 3.5 or 4) and weakly buffered (pH 5 or 5.5) situations. Survival of Antennaria dioica and Succisa pratensis was reduced by low pH in combination with high ammonium concentrations. Biomass decreased with increased ammonium concentrations and decreasing pH levels. Internal pH of the plants decreased with increasing ammonium concentrations. Survival of Calluna vulgaris, Deschampsia flexuosa and Gentiana pneumonanthe was not affected by ammonium. Moreover, biomass increased with increasing ammonium concentrations. Biomass production of G. pneumonanthe reduced at low pH levels. A decline of acid-sensitive species in heathlands was attributed to ammonium toxicity effects in combination with a low pH.

he global loss of biodiversity continues at an alarming rate. Genomic approaches have been suggested as a promising tool for conservation practice as scaling up to genome-wide data can improve traditional conservation genetic inferences and provide qualitatively novel insights. However, the generation of genomic data and subsequent analyses and interpretations remain challenging and largely confined to academic research in ecology and evolution. This generates a gap between basic research and applicable solutions for conservation managers faced with multifaceted problems. Before the real-world conservation potential of genomic research can be realized, we suggest that current infrastructures need to be modified, methods must mature, analytical pipelines need to be developed, and successful case studies must be disseminated to practitioners.

Inbreeding depression (i.e. negative fitness effects of inbreeding) is central in evolutionary biology, affecting numerous aspects of population dynamics and demography, such as the evolution of mating systems, dispersal behaviour and the genetics of quantitative traits. Inbreeding depression is commonly observed in animals and plants. Here, we demonstrate that, in addition to genetic processes, epigenetic processes may play an important role in causing inbreeding effects. We compared epigenetic markers of outbred and inbred offspring of the perennial plant Scabiosa columbaria and found that inbreeding increases DNA methylation. Moreover, we found that inbreeding depression disappears when epigenetic variation is modified by treatment with a demethylation agent, linking inbreeding depression firmly to epigenetic variation. Our results suggest an as yet unknown mechanism for inbreeding effects and demonstrate the importance of evaluating the role of epigenetic processes in inbreeding depression.

While it is well established that ecosystems display strong responses to elevated nitrogen deposition, the importance of the ratio between the dominant forms of deposited nitrogen (NH(x) and NO(y)) in determining ecosystem response is poorly understood. As large changes in the ratio of oxidised and reduced nitrogen inputs are occurring, this oversight requires attention. One reason for this knowledge gap is that plants experience a different NH(x):NO(y) ratio in soil to that seen in atmospheric deposits because atmospheric inputs are modified by soil transformations, mediated by soil pH. Consequently species of neutral and alkaline habitats are less likely to encounter high NH(4)(+) concentrations than species from acid soils. We suggest that the response of vascular plant species to changing ratios of NH(x):NO(y) deposits will be driven primarily by a combination of soil pH and nitrification rates. Testing this hypothesis requires a combination of experimental and survey work in a range of systems.

Habitat fragmentation and climate change are recognized as major threats to biodiversity. The major challenge for present day plant populations is how to adapt and cope with altered abiotic and biotic environments caused by climate change, when at the same time adaptive and evolutionary potential is decreased as habitat fragmentation reduces genetic variation and increases inbreeding. Although the ecological and evolutionary effects of fragmentation and climate change have been investigated separately, their combined effects remained largely unexplored. In this review, we will discuss the individual and joint effects of habitat fragmentation and climate change on plants and how the abilities and ways in which plants can respond and cope with climate change may be compromised due to habitat fragmentation.

The basic premise of conservation genetics is that small populations may be genetically threatened. The two steps leading to this premise are: (1) due to prominent influence of random genetic drift and inbreeding allelic and genotypic diversity in small populations is expected to be low, and (2) low allelic diversity and high homozygosity are expected to lead to immediate fitness decreases (inbreeding depression) and a compromised potential for evolutionary adaptation. Conservation genetic research has been strongly stimulated by the application of neutral molecular markers like microsatellites and AFLPs. In general these marker studies have provided evidence for step 1. It is less evident how these markers may provide evidence for step 2. In this essay we argue that, in order to get detailed insight in step 2, adopting a conservation genomic approach, in which conservation genetics will use approaches from ecological and evolutionary functional genomics (ecogenomics), is both necessary and feasible. Conservation genomics is necessary for studying functional genomic variation as function of drift and inbreeding, for studying the mechanisms that relate low genetic variation to low fitness, for integrating environmental and genetic approaches to conservation biology, and for developing modern, fast monitoring tools. The rapid technical and financial developments in genomics currently make conservation genomics feasible, and will improve feasibility in the very near future even further. We therefore argue that conservation genomics personifies part of the near future of conservation genetics.

The early success of plant introduction as a function of source material and habitat quality was tested in Arnica montana to determine whether different introduction strategies could affect plant viability of the introduced population. Plants originating from related (single-family introductions) and unrelated (multi-family introductions) individuals were introduced into undisturbed sites and into sites which were limed, turf cut, or both turf cut and limed. For four consecutive years, we analysed plant performance by measuring survival time, growth, and reproductive capacity. Introduction success was found to be strongly affected by habitat quality. Turf cutting in combination with additional liming significantly increased reproductive capacity and stimulated early flowering. To restore eutrophic and acidified soil conditions, turf cutting with additional liming prior to introduction is recommended. Furthermore, a significant effect of multi-family introductions was observed. Multi-family introductions showed higher introduction success compared to single-family introductions. Although the long-term effects of multi-family introductions will emerge after several generations, the preliminary results suggest multi-family introductions as the most successful introduction strategy.

Item does not contain fulltext Plant biodiversity has declined seriously because of both habitat deterioration and habitat fragmentation. As a result, many species have been forced into small, fragmented, and isolated populations and are believed to suffer from higher extinction risks. Genetic reinforcement and the establishment of new populations are now widely used to prevent extinction. However, the genetic background of transplants may seriously affect the long-term success of these populations because increased genetic variation may reduce the risk of inbreeding or lead to better performance by restored heterozygosity levels (heterosis). Introduced transplants, however, may be poorly adapted to the new local conditions. We tested the initial success of alternative introduction strategies. We evaluated the potential for inbreeding, heterosis, and/or local adaptation after introduction of artificial populations of Succisa pratensis. We introduced individuals from local and distant artificial populations that were created from either small or large populations. We created the artificial populations with the same census population size but varying effective population sizes by adjusting the relatedness of individuals. We analyzed the demographic consequences of inbreeding, heterosis, and/or local adaptation of these artificial populations. Reduced performance after selfing was manifested by a reduction in seed production, seed weight, germination, and flowering percentage. Seed production, seed weight, flowering percentage, and number of flowerheads were negatively affected by small population size. Local adaptation increased biomass and flowering percentage for local individuals. Seed weight and seed production exhibited significant heterosis. Our results demonstrate that threatened populations can benefit from introduction and genetic reinforcement of individuals from related populations. Significant differences among the artificial populations for several measured performance components suggest that introduction or reinforcement is best achieved through material from a local population or, when unavailable, from several large populations.

Summary 1We studied the performance of 17 Dutch populations of the perennial Succisa pratensis, in relation to population size, genetic variation and habitat quality. We used a path-analytical model to analyse the possible relationships between these variables and performance. 2Plants in smaller populations produced fewer seeds per flower head. Their seeds had lower germination rates and higher seedling mortality, and more seeds were dormant or non-viable. 3Population size was also correlated with genetic measures. Small populations had higher inbreeding coefficients than large populations and observed heterozygosity was positively correlated with population size. The mean genetic diversity (expected heterozygosity) was relatively high (Hexp = 0.42), but not correlated with population size. 4Less eutrophic habitats appeared to support larger populations. High concentrations of NH4 and NO3 in the soil were significantly negatively correlated with population size. 5Path-analysis showed that Succisa pratensis is vulnerable to habitat deterioration (eutrophication). Population size was strongly influenced by habitat quality. Reduced performance, however, was better explained by direct genetic effects and by habitat deterioration rather than by effects of population size per se. Both habitat quality and genetic effects are thus important for population persistence, even in the short term. The results suggest that there will be a continuing decline of the small populations, due to deteriorating habitat conditions, decreased genetic variation and a reduced reproductive capacity.

Summary 1 Succisa pratensis, a characteristic plant species of biodiverse, slightly acidic grasslands (NARDETEA), has declined in the Netherlands over recent decades. This process has mainly been attributed to environmental stress caused by anthropogenic acidification and eutrophication, although genetic deterioration and decreasing population size may also be involved. 2 We analysed the combined effects of environmental stress, genetic variation and population size on the performance of Succisa pratensis. 3 Individual plant performance was negatively affected by eutrophication and, to a smaller extent, by acidification. Plants originating from smaller populations performed less well and showed less genetic variation and higher inbreeding coefficients than plants from large populations. Strong interactions between population size and stress treatments reflected the fact that plants from small populations suffered more from deteriorating soil conditions than those from large populations. 4 abitat fragmentation may reinforce the negative effects of environmental stress, leading to even the less effective treatment (acidification) having serious consequences.