High outcrossing rates in a self-compatible and highly aggregated host-generalist mistletoe.

Plants have evolved various strategies to avoid inbreeding, but the mass flowering displayed by many plants predisposes them to within-plant pollen movements and self-pollination. Mistletoes often aggregate at multiple spatial scales. Their bird pollinators often visit several flowers of the same individual and of others on the same host tree. We hypothesized that hermaphroditic mistletoes have self-incompatibility mechanisms that reduce or prevent selfing. Whether their spatial distribution, affected by host specificity, host distribution, and the behaviour of seed dispersers, influences their mating system and population genetic structure remains unclear. We studied how mating system and spatial distribution affect genetic structure in four populations of the host-generalist mistletoe Dendrophthoe pentandra in southwestern China using microsatellite markers and progeny arrays. We also characterized the fine-scale spatial genetic structure among 166 mistletoes from four host trees in one population. Prevalence and intensity of infection both appeared to vary among host species, strongly affecting the degree of aggregation. Host tree size had a strong effect on infection intensity. Surprisingly, manual pollination experiments indicated that D. pentandra is self-compatible, but genetic analyses revealed that outcrossing rates were higher than expected in all four populations (MLTR tm 0.83-1.20, Bayesian tm 0.772-0.952). Spatial genetic structure was associated with distance between host trees but not at shorter scales (within hosts). Our results demonstrate that the combination of bird pollination, bird-mediated seed dispersal, and post-dispersal processes result in outcrossing and maintain relatively high diversity in the presence of biparental inbreeding, despite very high local densities and possible self-compatibility.

Quantifying heritability and estimating evolutionary potential in the wild when individuals that share genes also share environments.

Accurate heritability estimates for fitness-related traits are required to predict an organism's ability to respond to global change. Heritability estimates are theoretically expected to be inflated if, due to limited dispersal, individuals that share genes are also likely to share similar environments. However, if relatives occupy similar environments due, at least partly, to genetic variation for habitat selection, then accounting for environmental similarity in quantitative genetic models may result in diminished heritability estimates in wild populations. This potential issue has been pointed out in the literature, but has not been evaluated by empirical studies. Here, we investigate whether environmental similarity among individuals can be partly explained by genetic variation for habitat selection, and how this link potentially blurs estimates for heritability in fitness-related traits. Using intensive GPS monitoring, we quantified home-range habitat composition for 293 roe deer inhabiting a heterogeneous landscape to assess environmental similarity. To investigate if environmental similarity might harbour genetic variation, we combined genome-wide data in a quantitative genetic framework to evaluate genetic variation for home-range habitat composition, which is partly the result of habitat selection at settlement. Finally, we explored how environmental similarity affects heritability estimates for behaviours related to the risk avoidance-resource acquisition trade-off (i.e. being in open habitat and distance to roads) and proxies of individual performance (i.e. body mass and hind foot length). We found substantial heritability for home-range habitat composition, with estimates ranging from 0.40 (proportion of meadows) to 0.85 (proportion of refuge habitat). Accounting for similarity in habitat composition between relatives decreased the heritability estimates for both behavioural and morphological traits (reduction ranging from 55% to 100% and from 22% to 41% respectively). As a consequence, only half of these heritability estimates remained significantly different from zero. Our results show that similar genotypes occupy similar environments, which could lead to heritable variation being incorrectly attributed to environmental effects. To accurately distinguish the sources of phenotypic variation and predict the ability of organisms to respond to global change, it is necessary to develop quantitative genetic studies investigating the mechanisms underpinning environmental similarity among relatives.

Natural selection fluctuates at an extremely fine spatial scale inside a wild population of snapdragon plants.

Spatial variation in natural selection is expected to shape phenotypic variation of wild populations and drive their evolution. Although evidence of phenotypic divergence across populations experiencing different selection regimes is abundant, investigations of intrapopulation variation in selection pressures remain rare. Fine-grained spatial environmental heterogeneity can be expected to influence selective forces within a wild population and thereby alter its fitness function by producing multiple fitness optima at a fine spatial scale. Here, we tested this hypothesis in a wild population of snapdragon plants living on an extremely small island in southern France (about 7500 m2 ). We estimated the spline-based fitness function linking individuals' fitness and five morphological traits in interaction with three spatially variable ecological drivers. We found that selection acting on several traits varied both in magnitude and direction in response to environmental variables at the scale of a meter. Our findings illustrate how different phenotypes can be selected at different locations within a population in response to environmental variation. Investigating spatial variation in selection within a population, in association with ecological conditions, represents an opportunity to identify putative ecological drivers of selection in the wild.

Non-reproducible signals of adaptation to elevation between open and understorey microhabitats in snapdragon plants.

Experimental studies on local adaptation rarely investigate how different environmental variables might modify signals of adaptation or maladaptation. In plant common garden experiments, signals of adaptation or maladaptation to elevation are usually investigated in open habitats under full light. However, most plants inhabit heterogeneous habitats where environmental conditions differ. Understorey microhabitats are common and differ in terms of tree shade, temperature, water availability, microbiota, allelochemicals etc. Germination is a fitness-related trait of major importance for the adaptation of plants to contrasted climate conditions. It is affected by shade in snapdragon plants (Antirrhinum majus) and many other plant species. Here, we tested for the reproducibility of signals extrapolated from germination results between open and understorey microhabitats in two parapatric snapdragon plant subspecies (A. m. striatum and A. m. pseudomajus) characterized by a similar elevation range by using common garden experiments at different elevations. Signals observed under one microhabitat systematically differed in the other. Most scenarios could be inferred, with signals either shifting, appearing or disappearing between different environments. Our findings imply that caution should be taken when extrapolating the evolutionary significance of these types of experimental signals because they are not stable from one local environmental condition to the next. Forecasting the ability of plants to adapt to environmental changes based on common garden and reciprocal transplant experiments must account for the multivariate nature of the environment.

Thyroid hormones regulate the formation and environmental plasticity of white bars in clownfishes.

Determining how plasticity of developmental traits responds to environmental conditions is a challenge that must combine evolutionary sciences, ecology, and developmental biology. During metamorphosis, fish alter their morphology and color pattern according to environmental cues. We observed that juvenile clownfish (Amphiprion percula) modulate the developmental timing of their adult white bar formation during metamorphosis depending on the sea anemone species in which they are recruited. We observed an earlier formation of white bars when clownfish developed with Stichodactyla gigantea (Sg) than with Heteractis magnifica (Hm). As these bars, composed of iridophores, form during metamorphosis, we hypothesized that timing of their development may be thyroid hormone (TH) dependent. We treated clownfish larvae with TH and found that white bars developed earlier than in control fish. We further observed higher TH levels, associated with rapid white bar formation, in juveniles recruited in Sg than in Hm, explaining the faster white bar formation. Transcriptomic analysis of Sg recruits revealed higher expression of duox, a dual oxidase implicated in TH production as compared to Hm recruits. Finally, we showed that duox is an essential regulator of iridophore pattern timing in zebrafish. Taken together, our results suggest that TH controls the timing of adult color pattern formation and that shifts in duox expression and TH levels are associated with ecological differences resulting in divergent ontogenetic trajectories in color pattern development.

Phenotypic Response to Light Versus Shade Associated with DNA Methylation Changes in Snapdragon Plants (Antirrhinum majus).

The phenotypic plasticity of plants in response to change in their light environment, and in particularly, to shade is a schoolbook example of ecologically relevant phenotypic plasticity with evolutionary adaptive implications. Epigenetic variation is known to potentially underlie plant phenotypic plasticity. Yet, little is known about its role in ecologically and evolutionary relevant mechanisms shaping the diversity of plant populations in nature. Here we used a reference-free reduced representation bisulfite sequencing method for non-model organisms (epiGBS) to investigate changes in DNA methylation patterns across the genome in snapdragon plants (Antirrhinum majus L.). We exposed plants to sunlight versus artificially induced shade in four highly inbred lines to exclude genetic confounding effects. Our results showed that phenotypic plasticity in response to light versus shade shaped vegetative traits. They also showed that DNA methylation patterns were modified under light versus shade, with a trend towards global effects over the genome but with large effects found on a restricted portion. We also detected the existence of a correlation between phenotypic and epigenetic variation that neither supported nor rejected its potential role in plasticity. While our findings imply epigenetic changes in response to light versus shade environments in snapdragon plants, whether these changes are directly involved in the phenotypic plastic response of plants remains to be investigated. Our approach contributed to this new finding but illustrates the limits in terms of sample size and statistical power of population epigenetic approaches in non-model organisms. Pushing this boundary will be necessary before the relationship between environmentally induced epigenetic changes and phenotypic plasticity is clarified for ecologically relevant mechanisms with evolutionary implications.

Wild snapdragon plant pedigree sheds light on limited connectivity enhanced by higher migrant reproductive success in a fragmented landscape.

Background: In contrast with historical knowledge, a recent view posits that a non-negligible proportion of populations thrive in a fragmented landscape. One underlying mechanism is the maintenance of functional connectivity, i.e., the net flow of individuals or their genes moving among suitable habitat patches. Alternatively, functional connectivity might be typically limited but enhanced by a higher reproductive success of migrants. Methods: We tested for this hypothesis in wild snapdragon plants inhabiting six patches separated by seawater in a fragmented Mediterranean scrubland landscape. We reconstructed their pedigree by using a parentage assignment method based on microsatellite genetic markers. We then estimated functional connectivity and the reproductive success of plants resulting from between-patch dispersal events. Results: We found that wild snapdragon plants thrived in this fragmented landscape, although functional connectivity between habitat patches was low (i.e. 2.9%). The progeny resulting from between-patch dispersal events had a higher reproductive success than residents. Conclusion: Our findings imply that low functional connectivity in a fragmented landscapes may have been enhanced by higher reproductive success after migration. This original mechanisms might be partly compensating the negative impact of fragmentation.

Potential adaptive divergence between subspecies and populations of snapdragon plants inferred from QST -FST comparisons.

Phenotypic divergence among natural populations can be explained by natural selection or by neutral processes such as drift. Many examples in the literature compare putatively neutral (FST ) and quantitative genetic (QST ) differentiation in multiple populations to assess their evolutionary signature and identify candidate traits involved with local adaptation. Investigating these signatures in closely related or recently diversified species has the potential to shed light on the divergence processes acting at the interspecific level. Here, we conducted this comparison in two subspecies of snapdragon plants (eight populations of Antirrhinum majus pseudomajus and five populations of A. m. striatum) in a common garden experiment. We also tested whether altitude was involved with population phenotypic divergence. Our results identified candidate phenological and morphological traits involved with local adaptation. Most of these traits were identified in one subspecies but not the other. Phenotypic divergence increased with altitude for a few biomass-related traits, but only in A. m. striatum. These traits therefore potentially reflect A. m. striatum adaptation to altitude. Our findings imply that adaptive processes potentially differ at the scale of A. majus subspecies.

Intraspecific difference among herbivore lineages and their host-plant specialization drive the strength of trophic cascades.

Trophic cascades - the indirect effect of predators on non-adjacent lower trophic levels - are important drivers of the structure and dynamics of ecological communities. However, the influence of intraspecific trait variation on the strength of trophic cascade remains largely unexplored, which limits our understanding of the mechanisms underlying ecological networks. Here we experimentally investigated how intraspecific difference among herbivore lineages specialized on different host plants influences trophic cascade strength in a terrestrial tri-trophic system. We found that the occurrence and strength of the trophic cascade are strongly influenced by herbivores' lineage and host-plant specialization but are not associated with density-dependent effects mediated by the growth rate of herbivore populations. Our findings stress the importance of intraspecific heterogeneities and evolutionary specialization as drivers of trophic cascade strength and underline that intraspecific variation should not be overlooked to decipher the joint influence of evolutionary and ecological factors on the functioning of multi-trophic interactions.

Pedigree-free quantitative genetic approach provides evidence for heritability of movement tactics in wild roe deer.

Assessing the evolutionary potential of animal populations in the wild is crucial to understanding how they may respond to selection mediated by rapid environmental change (e.g. habitat loss and fragmentation). A growing number of studies have investigated the adaptive role of behaviour, but assessments of its genetic basis in a natural setting remain scarce. We combined intensive biologging technology with genome-wide data and a pedigree-free quantitative genetic approach to quantify repeatability, heritability and evolvability for a suite of behaviours related to the risk avoidance-resource acquisition trade-off in a wild roe deer (Capreolus capreolus) population inhabiting a heterogeneous, human-dominated landscape. These traits, linked to the stress response, movement and space-use behaviour, were all moderately to highly repeatable. Furthermore, the repeatable among-individual component of variation in these traits was partly due to additive genetic variance, with heritability estimates ranging from 0.21 ± 0.08 to 0.70 ± 0.11 and evolvability ranging from 1.1% to 4.3%. Changes in the trait mean can therefore occur under hypothetical directional selection over just a few generations. To the best of our knowledge, this is the first empirical demonstration of additive genetic variation in space-use behaviour in a free-ranging population based on genomic relatedness data. We conclude that wild animal populations may have the potential to adjust their spatial behaviour to human-driven environmental modifications through microevolutionary change.

Strong habitat and weak genetic effects shape the lifetime reproductive success in a wild clownfish population.

The relative contributions of environmental, maternal and additive genetic factors to the Lifetime reproductive success (LRS) determine whether species can adapt to rapid environmental change. Yet to date, studies quantifying LRS across multiple generations in marine species in the wild are non-existent. Here we used 10-year pedigrees resolved for a wild orange clownfish population from Kimbe Island (PNG) and a quantitative genetic linear mixed model approach to quantify the additive genetic, maternal and environmental contributions to variation in LRS for the self-recruiting portion of the population. We found that the habitat of the breeder, including the anemone species and geographic location, made the greatest contribution to LRS. There were low to negligible contributions of genetic and maternal factors equating with low heritability and evolvability. Our findings imply that our population will be susceptible to short-term, small-scale changes in habitat structure and may have limited capacity to adapt to these changes.

Different phenotypic plastic responses to predators observed among aphid lineages specialized on different host plants.

The role of intraspecific variation in the magnitude and direction of plastic responses in ecology and evolution is increasingly recognized. However, the factors underlying intraspecific variation in plastic responses remain largely unexplored, particularly for the hypothesis that the herbivores' phenotypic response to predators might vary amongst lineages associated with different host plants. Here, we tested whether plant-specialized lineages of the pea aphid, Acyrthosiphon pisum, differed in their transgenerational phenotypic response to ladybird predators (i.e., the asexual production of winged offspring by wingless mothers). In a full factorial laboratory experiment, we found that six aphid clonal lineages each specialized either on alfalfa or clover significantly differed in their transgenerational phenotypic response to predators. Some lineages produced an increased number of winged aphids in predator presence while others did not respond. Aphid lineages specialized on alfalfa had stronger phenotypic responses to predators than those specialized on clover. Although we tested only six aphid lineages from two biotypes, our results imply that intraspecific variation in prey phenotypic response of herbivores to predators differs amongst lineages specialized on different host plants. Our findings therefore raise the question of the influence of plant specialization in shaping herbivore phenotypic responses, and highlight the need to consider multi-trophic interactions to understand the causes and consequences of intraspecific variation in complex phenotypic traits.

RAD-sequencing for estimating genomic relatedness matrix-based heritability in the wild: A case study in roe deer.

Estimating the evolutionary potential of quantitative traits and reliably predicting responses to selection in wild populations are important challenges in evolutionary biology. The genomic revolution has opened up opportunities for measuring relatedness among individuals with precision, enabling pedigree-free estimation of trait heritabilities in wild populations. However, until now, most quantitative genetic studies based on a genomic relatedness matrix (GRM) have focused on long-term monitored populations for which traditional pedigrees were also available, and have often had access to knowledge of genome sequence and variability. Here, we investigated the potential of RAD-sequencing for estimating heritability in a free-ranging roe deer (Capreolous capreolus) population for which no prior genomic resources were available. We propose a step-by-step analytical framework to optimize the quality and quantity of the genomic data and explore the impact of the single nucleotide polymorphism (SNP) calling and filtering processes on the GRM structure and GRM-based heritability estimates. As expected, our results show that sequence coverage strongly affects the number of recovered loci, the genotyping error rate and the amount of missing data. Ultimately, this had little effect on heritability estimates and their standard errors, provided that the GRM was built from a minimum number of loci (above 7,000). Genomic relatedness matrix-based heritability estimates thus appear robust to a moderate level of genotyping errors in the SNP data set. We also showed that quality filters, such as the removal of low-frequency variants, affect the relatedness structure of the GRM, generating lower h2 estimates. Our work illustrates the huge potential of RAD-sequencing for estimating GRM-based heritability in virtually any natural population.

Assessing Global DNA Methylation Changes Associated with Plasticity in Seven Highly Inbred Lines of Snapdragon Plants (Antirrhinum majus).

Genetic and epigenetic variations are commonly known to underlie phenotypic plastic responses to environmental cues. However, the role of epigenetic variation in plastic responses harboring ecological significance in nature remains to be assessed. The shade avoidance response (SAR) of plants is one of the most prevalent examples of phenotypic plasticity. It is a phenotypic syndrome including stem elongation and multiple other traits. Its ecological significance is widely acknowledged, and it can be adaptive in the presence of competition for light. Underlying genes and pathways were identified, but evidence for its epigenetic basis remains scarce. We used a proven and accessible approach at the population level and compared global DNA methylation between plants exposed to regular light and three different magnitudes of shade in seven highly inbred lines of snapdragon plants (Antirrhinum majus) grown in a greenhouse. Our results brought evidence of a strong SAR syndrome for which magnitude did not vary between lines. They also brought evidence that its magnitude was not associated with the global DNA methylation percentage for five of the six traits under study. The magnitude of stem elongation was significantly associated with global DNA demethylation. We discuss the limits of this approach and why caution must be taken with such results. In-depth approaches at the DNA sequence level will be necessary to better understand the molecular basis of the SAR syndrome.

Environmental variations mediate duckweed (Lemna minor L.) sensitivity to copper exposure through phenotypic plasticity.

Environmentally mediated sensitivity of Lemna minor to copper (Cu) was evaluated for the first time in three experiments: the effects of two levels of nutrient concentration, light irradiance or Cu pre-exposure were tested. Various Cu concentrations (ranging from 0.05 to 0.25 mg/L) were used to assess the sensitivity of L. minor to this metal, using one common strain previously acclimatized to two different levels of light intensity, nutrient enrichment and Cu pre-exposure. Our results showed a phenotypic plastic response of the relative growth rates based on frond number and fresh mass production, and maximum quantum yield of photosystem II (Fv/Fm). Growth was affected by the three environmental conditions both prior and during Cu exposure, whereas Fv/Fm was mostly affected during Cu exposure. Copper significantly influenced all the parameters measured in the three experiments. Environmental conditions significantly modified L. minor sensitivity to Cu in all experiments, with up to twofold difference depending on the treatment. Growth rate was the parameter that was most impacted. Our study revealed for the first time the existence of phenotypic plasticity in L. minor sensitivity to chemical contamination, and implies that environmental context needs to be taken into account for a relevant risk assessment.

A guide to using a multiple-matrix animal model to disentangle genetic and nongenetic causes of phenotypic variance.

Non-genetic influences on phenotypic traits can affect our interpretation of genetic variance and the evolutionary potential of populations to respond to selection, with consequences for our ability to predict the outcomes of selection. Long-term population surveys and experiments have shown that quantitative genetic estimates are influenced by nongenetic effects, including shared environmental effects, epigenetic effects, and social interactions. Recent developments to the "animal model" of quantitative genetics can now allow us to calculate precise individual-based measures of non-genetic phenotypic variance. These models can be applied to a much broader range of contexts and data types than used previously, with the potential to greatly expand our understanding of nongenetic effects on evolutionary potential. Here, we provide the first practical guide for researchers interested in distinguishing between genetic and nongenetic causes of phenotypic variation in the animal model. The methods use matrices describing individual similarity in nongenetic effects, analogous to the additive genetic relatedness matrix. In a simulation of various phenotypic traits, accounting for environmental, epigenetic, or cultural resemblance between individuals reduced estimates of additive genetic variance, changing the interpretation of evolutionary potential. These variances were estimable for both direct and parental nongenetic variances. Our tutorial outlines an easy way to account for these effects in both wild and experimental populations. These models have the potential to add to our understanding of the effects of genetic and nongenetic effects on evolutionary potential. This should be of interest both to those studying heritability, and those who wish to understand nongenetic variance.

Evolution without standing genetic variation: change in transgenerational plastic response under persistent predation pressure.

Transgenerational phenotypic plasticity is a fast non-genetic response to environmental modifications that can buffer the effects of environmental stresses on populations. However, little is known about the evolution of plasticity in the absence of standing genetic variation although several non-genetic inheritance mechanisms have now been identified. Here we monitored the pea aphid transgenerational phenotypic response to ladybird predators (production of winged offspring) during 27 generations of experimental evolution in the absence of initial genetic variation (clonal multiplication starting from a single individual). We found that the frequency of winged aphids first increased rapidly in response to predators and then remained stable over 25 generations, implying a stable phenotypic reconstruction at each generation. We also found that the high frequency of winged aphids persisted for one generation after removing predators. Winged aphid frequency then entered a refractory phase during which it dropped below the level of control lines for at least two generations before returning to it. Interestingly, the persistence of the winged phenotype decreased and the refractory phase lasted longer with the increasing number of generations of exposure to predators. Finally, we found that aphids continuously exposed to predators for 22 generations evolved a significantly weaker plastic response than aphids never exposed to predators, which, in turn, increased their fitness in presence of predators. Our findings therefore showcased an example of experimental evolution of plasticity in the absence of initial genetic variation and highlight the importance of integrating several components of non-genetic inheritance to detect evolutionary responses to environmental changes.

The Missing Response to Selection in the Wild.

Although there are many examples of contemporary directional selection, evidence for responses to selection that match predictions are often missing in quantitative genetic studies of wild populations. This is despite the presence of genetic variation and selection pressures - theoretical prerequisites for the response to selection. This conundrum can be explained by statistical issues with accurate parameter estimation, and by biological mechanisms that interfere with the response to selection. These biological mechanisms can accelerate or constrain this response. These mechanisms are generally studied independently but might act simultaneously. We therefore integrated these mechanisms to explore their potential combined effect. This has implications for explaining the apparent evolutionary stasis of wild populations and the conservation of wildlife.