Variation in insect herbivory across an urbanization gradient: The role of abiotic factors and leaf secondary metabolites.

Urbanization impacts plant-herbivore interactions, which are crucial for ecosystem functions such as carbon sequestration and nutrient cycling. While some studies have reported reductions in insect herbivory in urban areas (relative to rural or natural forests), this trend is not consistent and the underlying causes for such variation remain unclear. We conducted a continental-scale study on insect herbivory along urbanization gradients for three European tree species: Quercus robur, Tilia cordata, and Fraxinus excelsior, and further investigated their biotic and abiotic correlates to get at mechanisms. To this end, we quantified insect leaf herbivory and foliar secondary metabolites (phenolics, terpenoids, alkaloids) for 176 trees across eight European cities. Additionally, we collected data on microclimate (air temperature) and soil characteristics (pH, carbon, nutrients) to test for abiotic correlates of urbanization effects directly or indirectly (through changes in plant secondary chemistry) linked to herbivory. Our results showed that urbanization was negatively associated with herbivory for Q. robur and F. excelsior, but not for T. cordata. In addition, urbanization was positively associated with secondary metabolite concentrations, but only for Q. robur. Urbanization was positively associated with air temperature for Q. robur and F. excelsior, and negatively with soil nutrients (magnesium) in the case of F. excelsior, but these abiotic variables were not associated with herbivory. Contrary to expectations, we found no evidence for indirect effects of abiotic factors via plant defences on herbivory for either Q. robur or F. excelsior. Additional biotic or abiotic drivers must therefore be accounted for to explain observed urbanization gradients in herbivory and their interspecific variation.

plantMASST - Community-driven chemotaxonomic digitization of plants.

Understanding the distribution of hundreds of thousands of plant metabolites across the plant kingdom presents a challenge. To address this, we curated publicly available LC-MS/MS data from 19,075 plant extracts and developed the plantMASST reference database encompassing 246 botanical families, 1,469 genera, and 2,793 species. This taxonomically focused database facilitates the exploration of plant-derived molecules using tandem mass spectrometry (MS/MS) spectra. This tool will aid in drug discovery, biosynthesis, (chemo)taxonomy, and the evolutionary ecology of herbivore interactions.

Leaf metabolic traits reveal hidden dimensions of plant form and function.

The metabolome is the biochemical basis of plant form and function, but we know little about its macroecological variation across the plant kingdom. Here, we used the plant functional trait concept to interpret leaf metabolome variation among 457 tropical and 339 temperate plant species. Distilling metabolite chemistry into five metabolic functional traits reveals that plants vary on two major axes of leaf metabolic specialization-a leaf chemical defense spectrum and an expression of leaf longevity. Axes are similar for tropical and temperate species, with many trait combinations being viable. However, metabolic traits vary orthogonally to life-history strategies described by widely used functional traits. The metabolome thus expands the functional trait concept by providing additional axes of metabolic specialization for examining plant form and function.

Reductions in home-range size and social interactions among dehorned black rhinoceroses (Diceros bicornis).

Poaching for horns and tusks is driving declines of megaherbivores worldwide, including the critically endangered African black rhinoceros (Diceros bicornis). By proactively dehorning entire rhinoceros populations, conservationists aim to deter poaching and prevent species loss. However, such conservation interventions may have hidden and underestimated effects on animals' behavior and ecology. Here, we combine >15 y of black rhino-monitoring data across 10 South African game reserves, comprising >24,000 sightings of 368 individuals, to determine the consequences of dehorning for black rhino space use and social interactions. While preventative dehorning at these reserves coincided with a nationwide decrease in black rhino mortality from poaching and did not infer increased natural mortality, dehorned black rhinos decreased their home range area by, on average, 11.7 km2 (45.5%) and were 37% less likely to engage in social encounters. We conclude that dehorning black rhinos as an antipoaching measure alters their behavioral ecology, although the potential population-level effects of these changes remain to be determined.

Eight key rules for successful data-dependent acquisition in mass spectrometry-based metabolomics.

In recent years, metabolomics has emerged as a pivotal approach for the holistic analysis of metabolites in biological systems. The rapid progress in analytical equipment, coupled to the rise of powerful data processing tools, now provides unprecedented opportunities to deepen our understanding of the relationships between biochemical processes and physiological or phenotypic conditions in living organisms. However, to obtain unbiased data coverage of hundreds or thousands of metabolites remains a challenging task. Among the panel of available analytical methods, targeted and untargeted mass spectrometry approaches are among the most commonly used. While targeted metabolomics usually relies on multiple-reaction monitoring acquisition, untargeted metabolomics use either data-independent acquisition (DIA) or data-dependent acquisition (DDA) methods. Unlike DIA, DDA offers the possibility to get real, selective MS/MS spectra and thus to improve metabolite assignment when performing untargeted metabolomics. Yet, DDA settings are more complex to establish than DIA settings, and as a result, DDA is more prone to errors in method development and application. Here, we present a tutorial which provides guidelines on how to optimize the technical parameters essential for proper DDA experiments in metabolomics applications. This tutorial is organized as a series of rules describing the impact of the different parameters on data acquisition and data quality. It is primarily intended to metabolomics users and mass spectrometrists that wish to acquire both theoretical background and practical tips for developing effective DDA methods.

DNA-based networks reveal the ecological determinants of plant-herbivore interactions along environmental gradients.

Understanding the ecological rules structuring the organization of species interactions is a prerequisite to predicting how ecosystems respond to environmental changes. While the ecological determinants of single networks have been documented, it remains unclear whether network ecological rules are conserved along spatial and environmental gradients. To address this gap, we reconstructed 48 plant-herbivore interaction networks along six elevation gradients in the Central European Alps in Switzerland, using DNA metabarcoding on orthoptera faeces. We developed hypotheses on the ecological mechanisms expected to structure interaction networks, based on plant phylogeny, plant abundance, leaf toughness, leaf nitrogen content and plant metabolomics. We show that plant phylogenetic relationships and species abundance have the greatest explanatory power regarding the structure of the ecological networks. Moreover, we found that leaf nitrogen content is a key determinant of interactions in warmer environments, while phenolic compounds and tannins are more important in colder environments, suggesting that determinants of species interactions can shift along environmental gradients. With this work, we propose an approach to study the mechanisms that structure the way species interact with each other between bioregions and ecosystems.

Metabolomics in Ecology and Bioactive Natural Products Discovery: Challenges and Prospects for a Comprehensive Study of the Specialised Metabolome.

Metabolomics is playing an increasingly prominent role in chemical ecology and in the discovery of bioactive natural products (NPs). The identification of metabolites is a common/central objective in both research fields. NPs have significant biological properties and play roles in multiple chemical-ecological interactions. Classically, in pharmacognosy, their chemical structure is determined after a complex process of isolating and interpreting spectroscopic data. With the advent of powerful analytical techniques such as liquid chromatography-mass spectrometry (LC-MS) the annotation process of the specialised metabolome of plants and microorganisms has improved considerably. In this article, we summarise the possibilities opened by these advances and illustrate how we harnessed them in our own research to automate annotations of NPs and target the isolation of key compounds. In addition, we are also discussing the analytical and computational challenges associated with these emerging approaches and their perspective.

Open and reusable annotated mass spectrometry dataset of a chemodiverse collection of 1,600 plant extracts.

As privileged structures, natural products often display potent biological activities. However, the discovery of novel bioactive scaffolds is often hampered by the chemical complexity of the biological matrices they are found in. Large natural extract collections are thus extremely valuable for their chemical novelty potential but also complicated to exploit in the frame of drug-discovery projects. In the end, it is the pure chemical substances that are desired for structural determination purposes and bioactivity evaluation. Researchers interested in the exploration of large and chemodiverse extract collections should thus establish strategies aiming to efficiently tackle such chemical complexity and access these structures. Establishing carefully crafted digital layers documenting the spectral and chemical complexity as well as bioactivity results of natural extracts collections can help prioritize time-consuming but mandatory isolation efforts. In this note, we report the results of our initial exploration of a collection of 1,600 plant extracts in the frame of a drug-discovery effort. After describing the taxonomic coverage of this collection, we present the results of its liquid chromatography high-resolution mass spectrometric profiling and the exploitation of these profiles using computational solutions. The resulting annotated mass spectral dataset and associated chemical and taxonomic metadata are made available to the community, and data reuse cases are proposed. We are currently continuing our exploration of this plant extract collection for drug-discovery purposes (notably looking for novel antitrypanosomatids, anti-infective and prometabolic compounds) and ecometabolomics insights. We believe that such a dataset can be exploited and reused by researchers interested in computational natural products exploration.

Spatial and evolutionary predictability of phytochemical diversity.

To cope with environmental challenges, plants produce a wide diversity of phytochemicals, which are also the source of numerous medicines. Despite decades of research in chemical ecology, we still lack an understanding of the organization of plant chemical diversity across species and ecosystems. To address this challenge, we hypothesized that molecular diversity is not only related to species diversity, but also constrained by trophic, climatic, and topographical factors. We screened the metabolome of 416 vascular plant species encompassing the entire alpine elevation range and four alpine bioclimatic regions in order to characterize their phytochemical diversity. We show that by coupling phylogenetic information, topographic, edaphic, and climatic variables, we predict phytochemical diversity, and its inherent composition, of plant communities throughout landscape. Spatial mapping of phytochemical diversity further revealed that plant assemblages found in low to midelevation habitats, with more alkaline soils, possessed greater phytochemical diversity, whereas alpine habitats possessed higher phytochemical endemism. Altogether, we present a general tool that can be used for predicting hotspots of phytochemical diversity in the landscape, independently of plant species taxonomic identity. Such an approach offers promising perspectives in both drug discovery programs and conservation efforts worldwide.

Ecological convergence of secondary phytochemicals along elevational gradients.

Biologists still strive to identify the ecological and evolutionary drivers of phytochemical variation that mediate biotic interactions. We hypothesized that plant species growing at sites characterized by high herbivore pressure would converge to produce highly toxic blends of secondary metabolites, independent of phylogenetic constraints. To address the role of shared evolutionary history and ecological niches in driving variation in plant phytochemistry, we combined targeted metabolomics with insect herbivore bioassays and with a set of growth-related traits of several Cardamine species growing along the entire elevational gradient of the Alps. We observed that Cardamine phytochemical profiles grouped according to previously established growth form categorizations within specific abiotic conditions, independently of phylogenetic relationship. We also showed that novel indices summarizing functional phytochemical diversity better explain plant resistance against chewing and sap-feeding herbivores than classic diversity indices. We conclude that multiple functional axes of phytochemical diversity should be integrated with the functional axis of plant growth forms to study phenotypic convergence along large-scale ecological gradients.

Novel trophic interactions under climate change promote alpine plant coexistence.

Herbivory and plant defenses exhibit a coupled decline along elevation gradients. However, the current ecological equilibrium could be disrupted under climate change, with a faster upward range shift of animals than plants. Here, we experimentally simulated this upward herbivore range shift by translocating low-elevation herbivore insects to alpine grasslands. We report that the introduction of novel herbivores and increased herbivory disrupted the vertical functional organization of the plant canopy. By feeding preferentially on alpine plants with functional traits matching their low-elevation host plants, herbivores reduced the biomass of dominant alpine plant species and favored encroachment of herbivore-resistant small-stature plant species, inflating species richness. Supplementing a direct effect of temperature, novel biotic interactions represent a neglected but major driver of ecosystem modifications under climate change.

Correlated Induction of Phytohormones and Glucosinolates Shapes Insect Herbivore Resistance of Cardamine Species Along Elevational Gradients.

Although the production of phytohormones has been commonly associated with production of plant defence and stress-related traits, few studies have simultaneously investigated this phenomenon across several plant species that grow along large-scale ecological gradients. To address these knowledge gaps, we performed a common garden experiment with six Cardamine species, which collectively encompass an elevational gradient of 2000 m. We quantified constitutive and Pieris brassicae caterpillars-induced phytohormones and chemical defences in leaves. We found a correlated expression of phytohormone production and the subsequent induction of chemical defences, and this correlated expression reduced herbivore performance. Furthermore, we found that abiotic conditions associated with the optimal elevation range of each species influenced the production of phytohormones and chemical defences, as well as plant growth and productivity. In particular, we found that plant species adapted to milder abiotic conditions at low elevations grew faster, were more productive and produced greater levels of chemical defences. In contrast, plant species adapted to harsher abiotic conditions at high elevations tended to produce greater levels of defence-related oxylipins. Overall, these findings highlight the importance of disentangling the role of phytohormones in mediating plant adaptations to shifting biotic and abiotic conditions.

The unfolding of plant growth form-defence syndromes along elevation gradients.

Understanding the functional economics that drives plant investment of resources requires investigating the interface between plant phenotypes and the variation in ecological conditions. While allocation to defence represents a large portion of the carbon budget, this axis is usually neglected in the study of plant economic spectrum. Using a novel geometrical approach, we analysed the co-variation in a comprehensive set of functional traits related to plant growth strategies, as well as chemical defences against herbivores on all 15 Cardamine species present in the Swiss Alps. By extracting geometrical information of the functional space, we observed clustering of plants into three main syndromes. Those different strategies of growth form and defence were also distributed within distinct elevational bands demonstrating an association between the functional space and the ecological conditions. We conclude that plant strategies converge into clear syndromes that trade off abiotic tolerance, growth and defence within each elevation zone.

Biological Control beneath the Feet: A Review of Crop Protection against Insect Root Herbivores.

Sustainable agriculture is certainly one of the most important challenges at present, considering both human population demography and evidence showing that crop productivity based on chemical control is plateauing. While the environmental and health threats of conventional agriculture are increasing, ecological research is offering promising solutions for crop protection against herbivore pests. While most research has focused on aboveground systems, several major crop pests are uniquely feeding on roots. We here aim at documenting the current and potential use of several biological control agents, including micro-organisms (viruses, bacteria, fungi, and nematodes) and invertebrates included among the macrofauna of soils (arthropods and annelids) that are used against root herbivores. In addition, we discuss the synergistic action of different bio-control agents when co-inoculated in soil and how the induction and priming of plant chemical defense could be synergized with the use of the bio-control agents described above to optimize root pest control. Finally, we highlight the gaps in the research for optimizing a more sustainable management of root pests.

Leaf and root-associated fungal assemblages do not follow similar elevational diversity patterns.

The diversity of fungi along environmental gradients has been little explored in contrast to plants and animals. Consequently, environmental factors influencing the composition of fungal assemblages are poorly understood. The aim of this study was to determine whether the diversity and composition of leaf and root-associated fungal assemblages vary with elevation and to investigate potential explanatory variables. High-throughput sequencing of the Internal Transcribed Spacer 1 region was used to explore fungal assemblages along three elevation gradients, located in French mountainous regions. Beech forest was selected as a study system to minimise the host effect. The variation in species richness and specific composition was investigated for ascomycetes and basidiomycetes assemblages with a particular focus on root-associated ectomycorrhizal fungi. The richness of fungal communities associated with leaves or roots did not significantly relate to any of the tested environmental drivers, i.e. elevation, mean temperature, precipitation or edaphic variables such as soil pH or the ratio carbon∶nitrogen. Nevertheless, the ascomycete species richness peaked at mid-temperature, illustrating a mid-domain effect model. We found that leaf and root-associated fungal assemblages did not follow similar patterns of composition with elevation. While the composition of the leaf-associated fungal assemblage correlated primarily with the mean annual temperature, the composition of root-associated fungal assemblage was explained equally by soil pH and by temperature. The ectomycorrhizal composition was also related to these variables. Our results therefore suggest that above and below-ground fungal assemblages are not controlled by the same main environmental variables. This may be due to the larger amplitude of climatic variables in the tree foliage compared to the soil environment.

Do interactions between plant and soil biota change with elevation? A study on Fagus sylvatica.

Theoretical models predict weakening of negative biotic interactions and strengthening of positive interactions with increasing abiotic stress. However, most empirical tests have been restricted to plant-plant interactions. No empirical study has examined theoretical predictions of interactions between plants and below-ground micro-organisms, although soil biota strongly regulates plant community composition and dynamics. We examined variability in soil biota effects on tree regeneration across an abiotic gradient. Our candidate tree species was European beech (Fagus sylvatica L.), whose regeneration is extremely responsive to soil biota activity. In a greenhouse experiment, we measured tree survival in sterilized and non-sterilized soils collected across an elevation gradient in the French Alps. Negative effects of soil biota on tree survival decreased with elevation, similar to shifts observed in plant-plant interactions. Hence, soil biota effects must be included in theoretical models of plant biotic interactions to accurately represent and predict the effects of abiotic gradient on plant communities.

Plant-ants feed their host plant, but above all a fungal symbiont to recycle nitrogen.

In ant-plant symbioses, plants provide symbiotic ants with food and specialized nesting cavities (called domatia). In many ant-plant symbioses, a fungal patch grows within each domatium. The symbiotic nature of the fungal association has been shown in the ant-plant Leonardoxa africana and its protective mutualist ant Petalomyrmex phylax. To decipher trophic fluxes among the three partners, food enriched in (13)C and (15)N was given to the ants and tracked in the different parts of the symbiosis up to 660 days later. The plant received a small, but significant, amount of nitrogen from the ants. However, the ants fed more intensively the fungus. The pattern of isotope enrichment in the system indicated an ant behaviour that functions specifically to feed the fungus. After 660 days, the introduced nitrogen was still present in the system and homogeneously distributed among ant, plant and fungal compartments, indicating efficient recycling within the symbiosis. Another experiment showed that the plant surface absorbed nutrients (in the form of simple molecules) whether or not it is coated by fungus. Our study provides arguments for a mutualistic status of the fungal associate and a framework for investigating the previously unsuspected complexity of food webs in ant-plant mutualisms.

Ant-plants and fungi: a new threeway symbiosis.

Symbioses between plants and fungi, fungi and ants, and ants and plants all play important roles in ecosystems. Symbioses involving all three partners appear to be rare. Here, we describe a novel tripartite symbiosis in which ants and a fungus inhabit domatia of an ant-plant, and present evidence that such interactions are widespread. We investigated 139 individuals of the African ant-plant Leonardoxa africana for occurrence of fungus. Behaviour of mutualist ants toward the fungus within domatia was observed using a video camera fitted with an endoscope. Fungi were identified by sequencing a fragment of their ribosomal DNA. Fungi were always present in domatia occupied by mutualist ants but never in domatia occupied by opportunistic or parasitic ants. Ants appear to favour the propagation, removal and maintenance of the fungus. Similar fungi were associated with other ant-plants in Cameroon. All belong to the ascomycete order Chaetothyriales; those from L. africana formed a monophyletic clade. These new plant-ant-fungus associations seem to be specific, as demonstrated within Leonardoxa and as suggested by fungal phyletic identities. Such tripartite associations are widespread in African ant-plants but have long been overlooked. Taking fungal partners into account will greatly enhance our understanding of symbiotic ant-plant mutualisms.