Screening of leaf extraction and storage conditions for eco-metabolomics studies.

Mass spectrometry-based plant metabolomics is frequently used to identify novel natural products or study the effect of specific treatments on a plant's metabolism. Reliable sample handling is required to avoid artifacts, which is why most protocols mandate shock freezing of plant tissue in liquid nitrogen and an uninterrupted cooling chain. However, the logistical challenges of this approach make it infeasible for many ecological studies. Especially for research in the tropics, permanent cooling poses a challenge, which is why many of those studies use dried leaf tissue instead. We screened a total of 10 extraction and storage approaches for plant metabolites extracted from maize leaf tissue across two cropping seasons to develop a methodology for agroecological studies in logistically challenging tropical locations. All methods were evaluated based on changes in the metabolite profile across a 2-month storage period at different temperatures with the goal of reproducing the metabolite profile of the living plant as closely as possible. We show that our newly developed on-site liquid-liquid extraction protocol provides a good compromise between sample replicability, extraction efficiency, material logistics, and metabolite profile stability. We further discuss alternative methods which showed promising results and feasibility of on-site sample handling for field studies.

Damage and recovery from drift of synthetic-auxin herbicide dicamba depends on concentration and varies among floral, vegetative, and lifetime traits in rapid cycling Brassica rapa.

Herbicides can drift from intended plants onto non-target species. It remains unclear how drift impacts plant functional traits that are important for fitness. To address this gap, we conducted an experiment where fast cycling Brassica rapa plants were exposed to one of three drift concentrations (0.5%, 1%, 10%) of synthetic-auxin dicamba. We evaluated damage to and capacity of floral and vegetative traits to recover as well as lifetime fitness by comparing treated plants to controls. Response to dicamba exposure was concentration-dependent across all traits but varied with trait type. At 0.5% dicamba, three out of five floral traits were affected, while at 1% dicamba, four floral traits and one out of two vegetative traits were negatively impacted. At 10% dicamba all floral and vegetative traits were stunted. Overall, floral traits were more responsive to all dicamba drift concentrations than vegetative traits and displayed a wide range of variation ranging from no response (e.g., pistil length) to up to 84% reduction (ovule number). However, despite floral traits were more affected across the dicamba drift concentrations they were also more likely to recover than the vegetative traits. There was also variation among lifetime traits; the onset of flowering was delayed, and reproductive fitness was negatively affected in a concentration-dependent manner, but the final biomass and total flower production were not affected. Altogether, we show substantial variation across plant traits in their response to dicamba and conclude that accounting for this variation is essential to understand the full impact of herbicide drift on plants and the ecological interactions these traits mediate.

Herbivory and pollination impact on the evolution of herbivore-induced plasticity in defense and floral traits.

Theory predicts that herbivory should primarily determine the evolution of herbivore-induced plasticity in plant defenses, but little is known about the influence of other interactions such as pollination. Pollinators may exert negative selection on the herbivore-induced plasticity of chemical defenses when floral signals and rewards are indirectly affected, provoking deterrent effects on these mutualists. We investigated the influence of constant herbivory and pollination on the evolved patterns and degree of herbivore-induced plasticity in chemical plant defenses and floral morphometry and volatiles in fast-cycling Brassica rapa plants. To do this, we used plants from an evolution experiment that had evolved under bee/hand pollination and herbivory manipulated in a 2 × 2 factorial design during six generations, producing four selection treatments. We grew sibling plant pairs from each of the four selection treatments of the last generation and infested one group with herbivores and left the other uninfested. Herbivore-induced plasticity was analyzed within- and between-selection treatments. We found support for the hypothesis that constant herbivory favors the evolution of higher constitutive yet lower herbivore-induced plasticity in defenses. However, this only occurred in plants that evolved under hand pollination and constant herbivory. Bee pollination had a strong influence on the evolution of herbivore-induced plasticity of all traits studied. Plants that evolved under bee pollination, with and without constant herbivory, showed remarkably similar patterns of herbivore-induced plasticity in their defense- and floral traits and had a higher number of plastic responses compared to plants with hand pollination. Such patterns support the hypothesis that bee pollination influenced the evolution of herbivore-induced plasticity, most likely via indirect effects, such as links between defense- and floral traits. We conclude that interactions other than herbivory, such as pollination, may impact herbivore-induced plasticity, through indirect effects and metabolic trade-offs, when it contributes to trait evolution in plants.

Digest: Floral evolution through the lens of the community context.

How do emergent properties of natural plant communities affect floral evolution? In this issue, Eisen et al. explored this question by studying selection on floral traits in natural communities of Clarkia species. They found that two community properties, namely congeneric species richness and floral density (of conspecifics and heterospecifics), influenced the patterns of selection, although not through the expected means of pollinator-mediated selection. Instead, the authors suggest that additional factors like competition and facilitation between plants are responsible. The results support the hypothesis that, beside pollinators, other factors of the community context can also determine floral evolution.

Rapid plant evolution driven by the interaction of pollination and herbivory.

Pollination and herbivory are both key drivers of plant diversity but are traditionally studied in isolation from each other. We investigated real-time evolutionary changes in plant traits over six generations by using fast-cycling Brassica rapa plants and manipulating the presence and absence of bumble bee pollinators and leaf herbivores. We found that plants under selection by bee pollinators evolved increased floral attractiveness, but this process was compromised by the presence of herbivores. Plants under selection from both bee pollinators and herbivores evolved higher degrees of self-compatibility and autonomous selfing, as well as reduced spatial separation of sexual organs (herkogamy). Overall, the evolution of most traits was affected by the interaction of bee pollination and herbivory, emphasizing the importance of the cross-talk between both types of interactions for plant evolution.