Unraveling the secrets of plant roots: Simplified method for large scale root exudate sampling and analysis in Arabidopsis thaliana.

Background: Plants exude a plethora of compounds to communicate with their environment. Although much is known about above-ground plant communication, we are only beginning to fathom the complexities of below-ground chemical communication channels. Studying root-exuded compounds and their role in plant communication has been difficult due to the lack of standardized methodologies. Here, we develop an interdisciplinary workflow to explore the natural variation in root exudate chemical composition of the model plant Arabidopsis thaliana. We highlight key challenges associated with sampling strategies and develop a framework for analyzing both narrow- and broad-scale patterns of root exudate composition in a large set of natural A. thaliana accessions. Methods: Our method involves cultivating individual seedlings in vitro inside a plastic mesh, followed by a short hydroponic sampling period in small quantities of ultrapure water. The mesh makes it easy to handle plants of different sizes and allows for large-scale characterization of individual plant root exudates under axenic conditions. This setup can also be easily extended for prolonged temporal exudate collection experiments. Furthermore, the short sampling time minimizes the duration of the experiment while still providing sufficient signal even with small volume of the sampling solution. We used ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) for untargeted metabolic profiling, followed by tentative compound identification using MZmine3 and SIRIUS 5 software, to capture a broad overview of root exudate composition in A. thaliana accessions. Results: Based on 28 replicates of the Columbia genotype (Col-0) compared with 10 random controls, MZmine3 annotated 354 metabolites to be present only in Col-0 by negative ionization. Of these, 254 compounds could be annotated by SIRIUS 5 software. Conclusions: The methodology developed in this study can be used to broadly investigate the role of root exudates as chemical signals in plant belowground interactions.

Plants exude many compounds to communicate with their surroundings. For decades, our understanding of this chemical communication was limited to studying the aboveground parts of plants, as roots are hidden within the soil, which makes them difficult to study. We are only beginning to comprehend the complexities and importance of below ground communication channels (including plant-microbes, plant-insects, and plant-plants). Identifying the chemical compounds plant exude belowground (called root exudates) is important for us to fully comprehend their potential roles in a plant´s life. Here, we developed a simplified and easy-to-manage setup for collecting and analyzing root exudates from individual Arabidopsis thaliana plants.

From genotype to phenotype: Genetic redundancy and the maintenance of an adaptive polymorphism in the context of high gene flow.

A central question in evolution is how several adaptive phenotypes are maintained within a species. Theory predicts that the genetic determination of a trait, and in particular the amounts of redundancy in the mapping of genotypes to phenotypes, mediates evolutionary outcomes of phenotypic selection. In Mediterranean wild thyme, numerous discrete chemical phenotypes (chemotypes) occur in close geographic proximity. Chemotypes are defined by the predominant monoterpene produced by individual plants in their essential oil. In this study, we analyze the ecological genetics of six chemotypes nested within two well-established chemical families (hereafter ecotypes). Ecotypes, and chemotypes within ecotypes, are spatially segregated, and their distributions track local differences in the abiotic environment. By combining population genomic, phenotypic, and environmental data from 700 individuals, we show how the genetics of ecotype determination mediates this evolutionary response. Variation in three terpene-synthase loci explains variation in ecotype identity, with one single locus accounting for as much as 78% of this variation. Phenotypic selection combined with low segregating genotypic redundancy of ecotypes leaves a clear footprint at the genomic level: alleles associated with ecotype identity track environmental variation despite extensive gene flow. Different chemotypes within each ecotype differentially track environmental variation. Their identity is determined by multiple loci and displays a wider range of genotypic redundancy that dilutes phenotypic selection on their characteristic alleles. Our study thus provides a novel illustration of how genetic redundancy of a phenotype modulates the ability of selection to maintain adaptive differentiation. Identifying the precise genetics of the chemical polymorphism in thyme is the next crucial step for our understanding of the origin and maintenance of a polymorphism that is present in many aromatic plants.

Ongoing decline in insect-pollinated plants across Danish grasslands.

Loss of habitat, eutrophication and reduced grazing intensity are known drivers of landscape-level changes in plant species composition; however, consequences of the massive decline in insect abundance are still to be understood. Pollinator decline can reduce seed set in plants relying on insects for successful reproduction. This may result in a reduced recruitment of insect-pollinated plant species with associated changes in species composition. So far, large-scale studies addressing this issue have relied on few data points-typically consisting of 'historic' records of numbers of insect-pollinated plants compared to present-day records. Such comparisons can provide information as to whether the diversity of insect-pollinated plants has changed, but not whether the process is still ongoing. Here, we use nationwide monitoring data of plant species richness in Danish grasslands from the period 2004-2014, covering 244 grassland sites and encompassing more than 790 flowering plant species. We show an ongoing decrease in insect-pollinated, but not wind-pollinated, plant species across different habitat types. In both dry calcareous and Nardus grasslands, loss of insect-pollinated plants was greatest at sites with low grazing intensity. However, insect-pollinated plants also declined from sites with higher grazing intensity, and plants requiring more specialized insect pollination tended to decline most. In addition to changes in plant diversity driven by land-use intensification, loss of pollinators may also play a role in reducing the richness of insect-pollinated plants. Ongoing reduction in floral richness could further increase the threat to insects relying on these plants as a food source.

Plant Secondary Compounds in Soil and Their Role in Belowground Species Interactions.

Knowledge of the effect of plant secondary compounds (PSCs) on belowground interactions in the more diffuse community of species living outside the rhizosphere is sparse compared with what we know about how PSCs affect aboveground interactions. We illustrate here that PSCs from foliar tissue, root exudates, and leaf litter effectively influence such belowground plant-plant, plant-microorganism, and plant-soil invertebrate interactions. Climatic factors can induce PSC production and select for different plant chemical types. Therefore, climate change can alter both quantitative and qualitative PSC production, and how these compounds move in the soil. This can change the soil chemical environment, with cascading effects on both the ecology and evolution of belowground species interactions and, ultimately, soil functioning.

Species-specific interference exerted by the shrub Cistus clusii Dunal in a semi-arid Mediterranean gypsum plant community.

BACKGROUND: The gypsovag shrub Cistus clusii is locally dominant in semi-arid gypsum plant communities of North-Eastern Spain. This species commonly grows in species-poor patches even though it has nurse potential, suggesting interference on neighbouring species. Other Cistus species exert a chemically mediated interference on plant communities, suggesting that it might be a common phenomenon in this genus. This study aimed investigating whether C. clusii exerts chemically mediated interference on neighbouring species in gypsum plant communities. We tested in a greenhouse whether aqueous extracts from C. clusii leaves (L), roots (R) and a mixture of both (RL) affected germination, seedling survival, and growth of nine native species of gypsum communities, including C. clusii itself. We further assessed in the field richness and abundance of plants under the canopy of C. clusii compared to Gypsophila struthium (shrub with a similar architecture having a nurse role) and in open patches. Finally, we specifically assessed in the field the influence of C. clusii on the presence of the species tested in the greenhouse experiment. RESULTS: Aqueous extracts from C. clusii (R and RL) negatively affected either germination or survival in four of nine species. In the field, richness and abundance of plants were lower under the canopy of C. clusii than under G. struthium, but higher than in open patches. Specifically, five of nine species were less frequent than expected under the canopy of C. clusii. CONCLUSIONS: Cistus clusii shows species-specific interference with neighbouring species in the community, which may be at least partially attributable to its phytotoxic activity. To our knowledge, this is the first report of species-specific interference by C. clusii.

Patterns of Genome-Wide Nucleotide Diversity in the Gynodioecious Plant Thymus vulgaris Are Compatible with Recent Sweeps of Cytoplasmic Genes.

Gynodioecy is a sexual dimorphism where females coexist with hermaphrodite individuals. In most cases, this dimorphism involves the interaction of cytoplasmic male sterility (CMS) genes and nuclear restorer genes. Two scenarios can account for how these interactions maintain gynodioecy. Either CMS genes recurrently enter populations at low frequency via mutation or migration and go to fixation unimpeded (successive sweeps), or CMS genes maintain polymorphism over evolutionary time through interactions with a nuclear restorer allele (balanced polymorphism). To distinguish between these scenarios, we used transcriptome sequencing in gynodioecious Thymus vulgaris and surveyed genome-wide diversity in 18 naturally occurring individuals sampled from populations at a local geographic scale. We contrast the amount and patterns of nucleotide diversity in the nuclear and cytoplasmic genome, and find ample diversity at the nuclear level (π = 0.019 at synonymous sites) but reduced genetic diversity and an excess of rare polymorphisms in the cytoplasmic genome relative to the nuclear genome. Our finding is incompatible with the maintenance of gynodioecy via scenarios invoking long-term balancing selection, and instead suggests the recent fixation of CMS lineages in the populations studied.

Covariation and phenotypic integration in chemical communication displays: biosynthetic constraints and eco-evolutionary implications.

Chemical communication is ubiquitous. The identification of conserved structural elements in visual and acoustic communication is well established, but comparable information on chemical communication displays (CCDs) is lacking. We assessed the phenotypic integration of CCDs in a meta-analysis to characterize patterns of covariation in CCDs and identified functional or biosynthetically constrained modules. Poorly integrated plant CCDs (i.e. low covariation between scent compounds) support the notion that plants often utilize one or few key compounds to repel antagonists or to attract pollinators and enemies of herbivores. Animal CCDs (mostly insect pheromones) were usually more integrated than those of plants (i.e. stronger covariation), suggesting that animals communicate via fixed proportions among compounds. Both plant and animal CCDs were composed of modules, which are groups of strongly covarying compounds. Biosynthetic similarity of compounds revealed biosynthetic constraints in the covariation patterns of plant CCDs. We provide a novel perspective on chemical communication and a basis for future investigations on structural properties of CCDs. This will facilitate identifying modules and biosynthetic constraints that may affect the outcome of selection and thus provide a predictive framework for evolutionary trajectories of CCDs in plants and animals.

A replicated climate change field experiment reveals rapid evolutionary response in an ecologically important soil invertebrate.

Whether species can respond evolutionarily to current climate change is crucial for the persistence of many species. Yet, very few studies have examined genetic responses to climate change in manipulated experiments carried out in natural field conditions. We examined the evolutionary response to climate change in a common annelid worm using a controlled replicated experiment where climatic conditions were manipulated in a natural setting. Analyzing the transcribed genome of 15 local populations, we found that about 12% of the genetic polymorphisms exhibit differences in allele frequencies associated to changes in soil temperature and soil moisture. This shows an evolutionary response to realistic climate change happening over short-time scale, and calls for incorporating evolution into models predicting future response of species to climate change. It also shows that designed climate change experiments coupled with genome sequencing offer great potential to test for the occurrence (or lack) of an evolutionary response.

Intraspecific genetic variation and species coexistence in plant communities.

Many studies report that intraspecific genetic variation in plants can affect community composition and coexistence. However, less is known about which traits are responsible and the mechanisms by which variation in these traits affect the associated community. Focusing on plant-plant interactions, we review empirical studies exemplifying how intraspecific genetic variation in functional traits impacts plant coexistence. Intraspecific variation in chemical and architectural traits promotes species coexistence, by both increasing habitat heterogeneity and altering competitive hierarchies. Decomposing species interactions into interactions between genotypes shows that genotype × genotype interactions are often intransitive. The outcome of plant-plant interactions varies with local adaptation to the environment and with dominant neighbour genotypes, and some plants can recognize the genetic identity of neighbour plants if they have a common history of coexistence. Taken together, this reveals a very dynamic nature of coexistence. We outline how more traits mediating plant-plant interactions may be identified, and how future studies could use population genetic surveys of genotype distribution in nature and methods from trait-based ecology to better quantify the impact of intraspecific genetic variation on plant coexistence.

"Ménage à trois": the presence/absence of thyme shapes the mutualistic interaction between the host plant Medicago truncatula (Fabaceae) and its symbiotic bacterium Sinorhizobium meliloti.

The long-term maintenance of specialized mutualisms remains an evolutionary puzzle. Recent focus has been on factors governing the stability of these mutualisms, including sanctions by the host, partner choice, and coevolutionary constraint, that is, the genetic correlation (r(G)) between fitness of both partners. So far these studies have been typically carried out in a single environment. Here, we ask if the genetic correlation between fitness of the host plant Medicago truncatula (Fabaceae) and its bacterial symbiont Sinorhizobium meliloti is affected by the presence/absence of a monoterpene (carvacrol) leached into the soil by Thymus vulgaris-a common plant of the Mediterranean vegetation, often co-occuring with Medicago. We show that the presence of carvacrol in the soil dramatically affects fitness of the rhizobial partner and increases the magnitude of r(G) between plant and rhizobia fitness (r(G) = 0.02 ± 0.05 vs. r(G) = 0.57 ± 0.02). This finding emphasizes the importance of heterogeneity in the biotic environment for understanding the evolution of species interactions.

Soil microorganisms alleviate the allelochemical effects of a thyme monoterpene on the performance of an associated grass species.

BACKGROUND: Plant allelochemicals released into the soil can significantly impact the performance of associated plant species thereby affecting their competitive ability. Soil microbes can potentially affect the interaction between plant and plant chemicals by degrading the allelochemicals. However, most often plant-plant chemical interactions are studied using filter paper bioassays examining the pair-wise interaction between a plant and a plant chemical, not taking into account the potential role of soil microorganisms. METHODOLOGY/PRINCIPAL FINDINGS: To explore if the allelopathic effects on a grass by the common thyme monoterpene "carvacrol" are affected by soil microorganisms. Seedlings of the grass Agrostis capillaris originating from 3 different thyme sites were raised in the greenhouse. Seedlings were grown under four different soil treatments in a 2*2 fully factorial experiment. The monoterpene carvacrol was either added to standard greenhouse soil or left out, and soil was either sterilized (no soil microorganisms) or not (soil microorganisms present in soil). The presence of carvacrol in the soil strongly increased mortality of Agrostis plants, and this increase was highest on sterile soil. Plant biomass was reduced on soil amended with carvacrol, but only when the soil was also sterilized. Plants originating from sites where thyme produces essential oils containing mostly carvacrol had higher survival on soil treated with that monoterpene than plants originating from a site where thyme produced different types of terpenes, suggesting an adaptive response to the locally occurring terpene. CONCLUSIONS/SIGNIFICANCE: The study shows that presence of soil microorganisms can alleviate the negative effect of a common thyme monoterpene on the performance of an associated plant species, emphasizing the role of soil microbes in modulating plant-plant chemical interactions.

When gametophytic self-incompatibility meets gynodioecy.

The occurrence of gynodioecy among angiosperms appears to be associated with self-compatibility. We use individual-based simulations to investigate the conditions for breakdown of a gametophytic self-incompatibility system in gynodioecious populations and make a comparison with hermaphroditic populations where the conditions are well known. We study three types of mutations causing self-compatibility. We track the fate of these mutations in both gynodioecious and hermaphroditic populations, where we vary the number of S-alleles, inbreeding depression and selfing rate. We find that the conditions for breakdown are less stringent if the population is gynodioecious and that the breakdown of self-incompatibility tends to promote stability of gynodioecious populations since it results in a higher frequency of females. We also find that fecundity selection has a large effect on the probability of breakdown of self-incompatibility, in particular if caused by a mutation destroying the female function of the S-locus.

'Inconstant males' and the maintenance of labile sex expression in subdioecious plants.

* Here, we evaluate the role of pollen limitation and selfing in the maintenance of labile sex expression in subdioecious plant species. * We used a literature survey to explore which factors correlated with a significant occurrence of hermaphrodites in dioecious species. We developed models to explore the selective maintenance of labile sex expression. The models had similar ecological assumptions but differed in the genetic basis of sex lability. * We found that a significant frequency of hermaphrodites was associated with animal pollination, and that hermaphrodites were 'inconstant' males with perfect flowers, suggesting evolution through the gynodioecious pathway. Models showed that a modifier converting pure males into inconstant males could be maintained under a wide range of reduction in both male and female fitness. Pollen limitation and self-fertilization facilitated invasion of the modifier. Depending on the genetics of sex determination, we found pure dioecy, stable subdioecy (trioecy), and situations where inconstant males coexisted with either pure females or pure males. Under selfing and pollen limitation, certain conditions selected for inconstant males which will drive populations to extinction. * We discuss our results in relation to the evolution towards, and the breakdown of, dioecy, and the ecological and evolutionary implications of labile sex expression.

Sex inheritance in gynodioecious species: a polygenic view.

Gynodioecy is defined as the coexistence of two different sexual morphs in a population: females and hermaphrodites. This breeding system is found among many different families of angiosperms and is usually under nucleo-cytoplasmic inheritance, with maternally inherited genes causing male sterility and nuclear factors restoring male fertility. Numerous theoretical models have investigated the conditions for the stable coexistence of females and hermaphrodites. To date, all models rest on the assumption that restoration of a given male sterile genotype is controlled by a single Mendelian factor. Here, we review data bearing on the genetic determinism of sex inheritance in three gynodiecious plant species. We suggest that restoration of male fertility is probably best viewed as a quantitative trait controlled by many loci. We develop a threshold model that accommodates an underlying polygenic trait, which is resolved at the phenotypic level in discrete sexual morphs. We use this model to reanalyse data in Thymus vulgaris, Silene vulgaris and Plantago coronopus. A simple Mendelian inheritance of sex determinism is unlikely in all three species. We discuss how our model can shed additional light on the genetics of restoration and point towards future efforts in the modelling of gynodioecy.

Do co-occurring plant species adapt to one another? The response of Bromus erectus to the presence of different Thymus vulgaris chemotypes.

Local modification of the soil environment by individual plants may affect the performance and composition of associated plant species. The aromatic plant Thymus vulgaris has the potential to modify the soil through leaching of water-soluble compounds from leaves and litter decomposition. In southern France, six different thyme chemotypes can be distinguished based on the dominant monoterpene in the essential oil, which is either phenolic or non-phenolic in structure. We examine how soils from within and away from thyme patches in sites dominated by either phenolic or non-phenolic chemotypes affect germination, growth and reproduction of the associated grass species Bromus erectus. To do so, we collected seeds of B. erectus from three phenolic and three non-phenolic sites. Seeds and seedlings were grown on soils from these sites in a reciprocal transplant type experiment in the glasshouse. Brome of non-phenolic origin performed significantly better on its home soil than on soil from a different non-phenolic or a phenolic site. This response to local chemotypes was only observed on soil collected directly underneath thyme plants and not on soil in the same site (<5 m away) but where no thyme plants were present. This is preliminary evidence that brome plants show an adaptive response to soil modifications mediated by the local thyme chemotypes. Reproductive effort was consistently higher in brome of phenolic origin than in brome of non-phenolic origin (on both thyme- and grass-soil), indicating that life-history variation may be related to environmental factors which also contribute to the spatial differentiation of thyme chemotypes. Moreover, we found that brome growing on thyme-soil in general was heavier than when growing on grass-soil, regardless of the origin of the brome plants. This is concordant with thyme-soil containing higher amounts of organic matter and nitrogen than grass-soil. Our results indicate that patterns of genetic differentiation and local adaptation may modify competitive interactions and possible facilitation effects in natural communities.