Variation in induced responses in volatile and non-volatile metabolites among six willow species: Do willow species share responses to herbivory?

Chemical variation is a critical aspect affecting performance among co-occurring plants. High chemical variation in metabolites with direct effects on insect herbivores supports chemical niche partitioning, and it can reduce the number of herbivores shared by co-occurring plant species. In contrast, low intraspecific variation in metabolites with indirect effects, such as induced volatile organic compounds (VOCs), may improve the attraction of specialist predators or parasitoids as they show high specificity to insect herbivores. We explored whether induced chemical variation following herbivory by various insect herbivores differs between VOCs vs. secondary non-volatile metabolites (non-VOCs) and salicinoids with direct effects on herbivores in six closely related willow species. Willow species identity explained most variation in VOCs (18.4%), secondary non-VOCs (41.1%) and salicinoids (60.7%). The variation explained by the independent effect of the herbivore treatment was higher in VOCs (2.8%) compared to secondary non-VOCs (0.5%) and salicinoids (0.5%). At the level of individual VOCs, willow species formed groups, as some responded similarly to the same herbivores. Most non-VOCs and salicinoids were upregulated by sap-suckers compared to other herbivore treatments and control across the willow species. In contrast, induced responses in non-VOCs and salicinoids to other herbivores largely differed between the willows. Our results suggest that induced responses broadly differ between various types of chemical defences, with VOCs and non-VOCs showing different levels of specificity and similarity across plant species. This may further contribute to flexible plant responses to herbivory and affect how closely related plants share or partition their chemical niches.

Effects of individual traits vs. trait syndromes on assemblages of various herbivore guilds associated with central European Salix.

Plants employ diverse anti-herbivore defences that can covary to form syndromes consisting of multiple traits. Such syndromes are hypothesized to impact herbivores more than individual defences. We studied 16 species of lowland willows occurring in central Europe and explored if their chemical and physical traits form detectable syndromes. We tested for phylogenetic trends in the syndromes and explored whether three herbivore guilds (i.e., generalist leaf-chewers, specialist leaf-chewers, and gallers) are affected more by the detected syndromes or individual traits. The recovered syndromes showed low phylogenetic signal and were mainly defined by investment in concentration, richness, or uniqueness of structurally related phenolic metabolites. Resource acquisition traits or inducible volatile organic compounds exhibited a limited correlation with the syndromes. Individual traits composing the syndromes showed various correlations to the assemblages of herbivores from the three studied guilds. In turn, we found some support for the hypothesis that defence syndromes are composed of traits that provide defence against various herbivores. However, individual traits rather than trait syndromes explained more variation for all studied herbivore assemblages. The detected negative correlations between various phenolics suggest that investment trade-offs may occur primarily among plant metabolites with shared metabolic pathways that may compete for their precursors. Moreover, several traits characterizing the recovered syndromes play additional roles in willows other than defence from herbivory. Taken together, our findings suggest that the detected syndromes did not solely evolve as an anti-herbivore defence.

Challenge accepted: Evolutionary lineages versus taxonomic classification of North American shrub willows (Salix).

PREMISE: The huge diversity of Salix subgenus Chamaetia/Vetrix clade in North America and the lack of phylogenetic resolution within this clade has presented a difficult but fascinating challenge for taxonomists to resolve. Here we tested the existing taxonomic classification with molecular tools. METHODS: In this study, 132 samples representing 46 species from 22 described sections of shrub willows from the United States and Canada were analyzed and combined with 67 samples from Eurasia. The ploidy levels of the samples were determined using flow cytometry and nQuire. Sequences were produced using a RAD sequencing approach and subsequently analyzed with ipyrad, then used for phylogenetic reconstructions (RAxML, SplitsTree), dating analyses (BEAST, SNAPPER), and character evolution analyses of 14 selected morphological traits (Mesquite). RESULTS: The RAD sequencing approach allowed the production of a well-resolved phylogeny of shrub willows. The resulting tree showed an exclusively North American (NA) clade in sister position to a Eurasian clade, which included some North American endemics. The NA clade began to diversify in the Miocene. Polyploid species appeared in each observed clade. Character evolution analyses revealed that adaptive traits such as habit and adaxial nectaries evolved multiple times independently. CONCLUSIONS: The diversity in shrub willows was shaped by an evolutionary radiation in North America. Most species were monophyletic, but the existing sectional classification could not be supported by molecular data. Nevertheless, monophyletic lineages share several morphological characters, which might be useful in the revision of the taxonomic classification of shrub willows.

High intraspecific variability and previous experience affect polyphenol metabolism in polyphagous Lymantria mathura caterpillars.

Polyphagous insect herbivores feed on multiple host-plant species and face a highly variable chemical landscape. Comparative studies of polyphagous herbivore metabolism across a range of plants is an ideal approach for exploring how intra- and interspecific chemical variation shapes species interactions. We used polyphagous caterpillars of Lymantria mathura (Erebidae, Lepidoptera) to explore mechanisms that may contribute to its ability to feed on various hosts. We focused on intraspecific variation in polyphenol metabolism, the fates of individual polyphenols, and the role of previous feeding experience on polyphenol metabolism and leaf consumption. We collected the caterpillars from Acer amoenum (Sapindaceae), Carpinus cordata (Betulaceae), and Quercus crispula (Fagaceae). We first fed the larvae with the leaves of their original host and characterized the polyphenol profiles in leaves and frass. We then transferred a subset of larvae to a different host species and quantified how host shifting affected their leaf consumption and polyphenol metabolism. There was high intraspecific variation in frass composition, even among caterpillars fed with one host. While polyphenols had various fates when ingested by the caterpillars, most of them were passively excreted. When we transferred the caterpillars to a new host, their previous experience influenced how they metabolized polyphenols. The one-host larvae metabolized a larger quantity of ingested polyphenols than two-host caterpillars. Some of these metabolites could have been sequestered, others were probably activated in the gut. One-host caterpillars retained more of the ingested leaf biomass than transferred caterpillars. The pronounced intraspecific variation in polyphenol metabolism, an ability to excrete ingested metabolites and potential dietary habituation are factors that may contribute to the ability of L. mathura to feed across multiple hosts. Further comparative studies can help identify if these mechanisms are related to differential host-choice and response to host-plant traits in specialist and generalist insect herbivores.

Simulated Herbivory Affects the Volatile Emissions of Oak Saplings, while Neighbourhood Affects Flavan-3-ols Content of Their Leaves.

To what extent particular plant defences against herbivorous insects are constitutive or inducible will depend on the costs and benefits in their neighbourhood. Some defensive chemicals in leaves are thought to be costly and hard to produce rapidly, while others, including volatile organic compounds that attract natural enemies, might be cheaper and can be released rapidly. When surrounding tree species are more closely related, trees can face an increased abundance of both specialist herbivores and their parasitoids, potentially increasing the benefits of constitutive and inducible defences. To test if oaks (Quercus robur) respond more to herbivore attacks with volatile emission than with changes in leaf phenolic chemistry and carbon to nitrogen ratio (C: N), and whether oaks respond to the neighbouring tree species, we performed an experiment in a forest in Poland. Oak saplings were placed in neighbourhoods dominated by oak, beech, or pine trees, and half of them were treated with the phytohormone methyl jasmonate (elicitor of anti-herbivore responses). Oaks responded to the treatment by emitting a different volatile blend within 24 h, while leaf phenolic chemistry and C: N remained largely unaffected after 16 days and multiple treatments. Leaf phenolics were subtly affected by the neighbouring trees with elevated flavan-3-ols concentrations in pine-dominated plots. Our results suggest that these oaks rely on phenols as a constitutive defence and when attacked emit volatiles to attract natural enemies. Further studies might determine if the small effect of the neighbourhood on leaf phenolics is a response to different levels of shading, or if oaks use volatile cues to assess the composition of their neighbourhood.

Contrasting levels of ß-diversity and underlying phylogenetic trends indicate different paths to chemical diversity in highland and lowland willow species.

Diverse specialised metabolites contributed to the success of vascular plants in colonising most terrestrial habitats. Understanding how distinct aspects of chemical diversity arise through heterogeneous environmental pressures can help us understand the effects of abiotic and biotic stress on plant evolution and community assembly. We examined highland and lowland willow species within a phylogenetic framework to test for trends in their chemical α-diversity (richness) and ß-diversity (variation among species sympatric in elevation). We show that differences in chemistry among willows growing at different elevations occur mainly through shifts in chemical ß-diversity and due to convergence or divergence among species sharing their elevation level. We also detect contrasting phylogenetic trends in concentration and α-diversity of metabolites in highland and lowland willow species. The resulting elevational patterns contribute to the chemical diversity of willows and suggest that variable selective pressure across ecological gradients may, more generally, underpin complex changes in plant chemistry.

Leaf volatile and nonvolatile metabolites show different levels of specificity in response to herbivory.

Plants produce diverse chemical defenses with contrasting effects on different insect herbivores. Deploying herbivore-specific responses can help plants increase their defensive efficiency. Here, we explore how variation in induced plant responses correlates with herbivore species, order, feeding guild, and level of specialization. In a greenhouse experiment, we exposed 149 plants of Salix fragilis (Linnaeus, 1753) to 22 herbivore species naturally associated with this host. The insects belonged to four orders (Coleoptera, Lepidoptera, Hemiptera, and Hymenoptera), three feeding guilds (external leaf-chewers, leaf-tying chewers, and sap-sucking), and included both dietary specialists and generalists. Following herbivory, we quantified induced changes in volatiles and nonvolatile leaf metabolites. We performed multivariate analyses to assess the correlation between herbivore order, feeding guild, dietary specialization, chewing damage by herbivores, and induced responses. The volatile composition was best explained by chewing damage and insect order, with Coleoptera and Lepidoptera eliciting significantly different responses. Furthermore, we recorded significant differences in elicited volatiles among some species within the two orders. Variation in nonvolatile leaf metabolites was mainly explained by the presence of insects, as plants exposed to herbivores showed significantly different metabolites from controls. Herbivore order also played a role to some extent, with beetles eliciting different responses than other herbivores. The induction of volatile and nonvolatile leaf metabolites shows different levels of specificity. The specificity in volatiles could potentially serve as an important cue to specialized predators or parasitoids, increasing the efficacy of volatiles as indirect defenses. By contrast, the induction of nonvolatile leaf metabolites was largely unaffected by herbivore identity. Most nonvolatile metabolites were downregulated, possibly indicating that plants redirected their resources from leaves in response to herbivory. Our results demonstrate how diverse responses to herbivores can contribute to the diversity of plant defensive strategies.

What Goes in Must Come Out? The Metabolic Profile of Plants and Caterpillars, Frass, And Adults of Asota (Erebidae: Aganainae) Feeding on Ficus (Moraceae) in New Guinea.

Insect herbivores have evolved a broad spectrum of adaptations in response to the diversity of chemical defences employed by plants. Here we focus on two species of New Guinean Asota and determine how these specialist moths deal with the leaf alkaloids of their fig (Ficus) hosts. As each focal Asota species is restricted to one of three chemically distinct species of Ficus, we also test whether these specialized interactions lead to similar alkaloid profiles in both Asota species. We reared Asota caterpillars on their respective Ficus hosts in natural conditions and analyzed the alkaloid profiles of leaf, frass, caterpillar, and adult moth samples using UHPLC-MS/MS analyses. We identified 43 alkaloids in our samples. Leaf alkaloids showed various fates. Some were excreted in frass or found in caterpillars and adult moths. We also found two apparently novel indole alkaloids-likely synthesized de novo by the moths or their microbiota-in both caterpillar and adult tissue but not in leaves or frass. Overall, alkaloids unique or largely restricted to insect tissue were shared across moth species despite feeding on different hosts. This indicates that a limited number of plant compounds have a direct ecological function that is conserved among the studied species. Our results provide evidence for the importance of phytochemistry and metabolic strategies in the formation of plant-insect interactions and food webs in general. Furthermore, we provide a new potential example of insects acquiring chemicals for their benefit in an ecologically relevant insect genus.

A mosaic of induced and non-induced branches promotes variation in leaf traits, predation and insect herbivore assemblages in canopy trees.

Forest canopies are complex and highly diverse environments. Their diversity is affected by pronounced gradients in abiotic and biotic conditions, including variation in leaf chemistry. We hypothesised that branch-localised defence induction and vertical stratification in mature oaks constitute sources of chemical variation that extend across trophic levels. To test this, we combined manipulation of plant defences, predation monitoring, food-choice trials with herbivores and sampling of herbivore assemblages. Both induction and vertical stratification affected branch chemistry, but the effect of induction was stronger. Induction increased predation in the canopy and reduced herbivory in bioassays. The effects of increased predation affected herbivore assemblages by decreasing their abundance, and indirectly, their richness. In turn, we show that there are multiple factors contributing to variation across canopies. Branch-localised induction, variation between tree individuals and predation may be the ones with particularly strong effects on diverse assemblages of insects in temperate forests.

Highly Diverse Shrub Willows (Salix L.) Share Highly Similar Plastomes.

Plastome phylogenomics is used in a broad range of studies where single markers do not bear enough information. Phylogenetic reconstruction in the genus Salix is difficult due to the lack of informative characters and reticulate evolution. Here, we use a genome skimming approach to reconstruct 41 complete plastomes of 32 Eurasian and North American Salix species representing different lineages, different ploidy levels, and separate geographic regions. We combined our plastomes with published data from Genbank to build a comprehensive phylogeny of 61 samples (50 species) using RAxML (Randomized Axelerated Maximum Likelihood). Additionally, haplotype networks for two observed subclades were calculated, and 72 genes were tested to be under selection. The results revealed a highly conserved structure of the observed plastomes. Within the genus, we observed a variation of 1.68%, most of which separated subg. Salix from the subgeneric Chamaetia/Vetrix clade. Our data generally confirm previous plastid phylogenies, however, within Chamaetia/Vetrix phylogenetic results represented neither taxonomical classifications nor geographical regions. Non-coding DNA regions were responsible for most of the observed variation within subclades and 5.6% of the analyzed genes showed signals of diversifying selection. A comparison of nuclear restriction site associated DNA (RAD) sequencing and plastome data on a subset of 10 species showed discrepancies in topology and resolution. We assume that a combination of (i) a very low mutation rate due to efficient mechanisms preventing mutagenesis, (ii) reticulate evolution, including ancient and ongoing hybridization, and (iii) homoplasy has shaped plastome evolution in willows.

Branch-Localized Induction Promotes Efficacy of Volatile Defences and Herbivore Predation in Trees.

Induction of plant defences can show various levels of localization, which can optimize their efficiency. Locally induced responses may be particularly important in large plants, such as trees, that show high variability in traits and herbivory rates across their canopies. We studied the branch-localized induction of polyphenols, volatiles (VOCs), and changes in leaf protein content in Carpinus betulus L., Quercus robur L., and Tilia cordata L. in a common garden experiment. To induce the trees, we treated ten individuals per species on one branch with methyl jasmonate. Five other individuals per species served as controls. We measured the traits in the treated branches, in control branches on treated trees, and in control trees. Additionally, we ran predation assays and caterpillar food-choice trials to assess the effects of our treatment on other trophic levels. Induced VOCs included mainly mono- and sesquiterpenes. Their production was strongly localized to the treated branches in all three tree species studied. Treated trees showed more predation events than control trees. The polyphenol levels and total protein content showed a limited response to the treatment. Yet, winter moth caterpillars preferred leaves from control branches over leaves from treated branches within C. betulus individuals and leaves from control Q. robur individuals over leaves from treated Q. robur individuals. Our results suggest that there is a significant level of localization in induction of VOCs and probably also in unknown traits with direct effects on herbivores. Such localization allows trees to upregulate defences wherever and whenever they are needed.

Spatial covariance of herbivorous and predatory guilds of forest canopy arthropods along a latitudinal gradient.

In arthropod community ecology, species richness studies tend to be prioritised over those investigating patterns of abundance. Consequently, the biotic and abiotic drivers of arboreal arthropod abundance are still relatively poorly known. In this cross-continental study, we employ a theoretical framework in order to examine patterns of covariance among herbivorous and predatory arthropod guilds. Leaf-chewing and leaf-mining herbivores, and predatory ants and spiders, were censused on > 1000 trees in nine 0.1 ha forest plots. After controlling for tree size and season, we found no negative pairwise correlations between guild abundances per plot, suggestive of weak signals of both inter-guild competition and top-down regulation of herbivores by predators. Inter-guild interaction strengths did not vary with mean annual temperature, thus opposing the hypothesis that biotic interactions intensify towards the equator. We find evidence for the bottom-up limitation of arthropod abundances via resources and abiotic factors, rather than for competition and predation.

Compound Specific Trends of Chemical Defences in Ficus Along an Elevational Gradient Reflect a Complex Selective Landscape.

Elevational gradients affect the production of plant secondary metabolites through changes in both biotic and abiotic conditions. Previous studies have suggested both elevational increases and decreases in host-plant chemical defences. We analysed the correlation of alkaloids and polyphenols with elevation in a community of nine Ficus species along a continuously forested elevational gradient in Papua New Guinea. We sampled 204 insect species feeding on the leaves of these hosts and correlated their community structure to the focal compounds. Additionally, we explored species richness of folivorous mammals along the gradient. When we accounted for Ficus species identity, we found a general elevational increase in flavonoids and alkaloids. Elevational trends in non-flavonol polyphenols were less pronounced or showed non-linear correlations with elevation. Polyphenols responded more strongly to changes in temperature and humidity than alkaloids. The abundance of insect herbivores decreased with elevation, while the species richness of folivorous mammals showed an elevational increase. Insect community structure was affected mainly by alkaloid concentration and diversity. Although our results show an elevational increase in several groups of metabolites, the drivers behind these trends likely differ. Flavonoids may provide figs with protection against abiotic stressors. In contrast, alkaloids affect insect herbivores and may provide protection against mammalian herbivores and pathogens. Concurrent analysis of multiple compound groups alongside ecological data is an important approach for understanding the selective landscape that shapes plant defences.

Plant phylogeny drives arboreal caterpillar assemblages across the Holarctic.

Assemblages of insect herbivores are structured by plant traits such as nutrient content, secondary metabolites, physical traits, and phenology. Many of these traits are phylogenetically conserved, implying a decrease in trait similarity with increasing phylogenetic distance of the host plant taxa. Thus, a metric of phylogenetic distances and relationships can be considered a proxy for phylogenetically conserved plant traits and used to predict variation in herbivorous insect assemblages among co-occurring plant species.Using a Holarctic dataset of exposed-feeding and shelter-building caterpillars, we aimed at showing how phylogenetic relationships among host plants explain compositional changes and characteristics of herbivore assemblages.Our plant-caterpillar network data derived from plot-based samplings at three different continents included >28,000 individual caterpillar-plant interactions. We tested whether increasing phylogenetic distance of the host plants leads to a decrease in caterpillar assemblage overlap. We further investigated to what degree phylogenetic isolation of a host tree species within the local community explains abundance, density, richness, and mean specialization of its associated caterpillar assemblage.The overlap of caterpillar assemblages decreased with increasing phylogenetic distance among the host tree species. Phylogenetic isolation of a host plant within the local plant community was correlated with lower richness and mean specialization of the associated caterpillar assemblages. Phylogenetic isolation had no effect on caterpillar abundance or density. The effects of plant phylogeny were consistent across exposed-feeding and shelter-building caterpillars.Our study reveals that distance metrics obtained from host plant phylogeny are useful predictors to explain compositional turnover among hosts and host-specific variations in richness and mean specialization of associated insect herbivore assemblages in temperate broadleaf forests. As phylogenetic information of plant communities is becoming increasingly available, further large-scale studies are needed to investigate to what degree plant phylogeny structures herbivore assemblages in other biomes and ecosystems.

Vertical stratification of a temperate forest caterpillar community in eastern North America.

Vertical niche partitioning might be one of the main driving forces explaining the high diversity of forest ecosystems. However, the forest's vertical dimension has received limited investigation, especially in temperate forests. Thus, our knowledge about how communities are vertically structured remains limited for temperate forest ecosystems. In this study, we investigated the vertical structuring of an arboreal caterpillar community in a temperate deciduous forest of eastern North America. Within a 0.2-ha forest stand, all deciduous trees ≥ 5 cm diameter at breast height (DBH) were felled and systematically searched for caterpillars. Sampled caterpillars were assigned to a specific stratum (i.e. understory, midstory, or canopy) depending on their vertical position and classified into feeding guild as either exposed feeders or shelter builders (i.e. leaf rollers, leaf tiers, webbers). In total, 3892 caterpillars representing 215 species of butterflies and moths were collected and identified. While stratum had no effect on caterpillar density, feeding guild composition changed significantly with shelter-building caterpillars becoming the dominant guild in the canopy. Species richness and diversity were found to be highest in the understory and midstory and declined strongly in the canopy. Family and species composition changed significantly among the strata; understory and canopy showed the lowest similarity. Food web analyses further revealed an increasing network specialization towards the canopy, caused by an increase in specialization of the caterpillar community. In summary, our study revealed a pronounced stratification of a temperate forest caterpillar community, unveiling a distinctly different assemblage of caterpillars dwelling in the canopy stratum.

Quantitative assessment of plant-arthropod interactions in forest canopies: A plot-based approach.

Research on canopy arthropods has progressed from species inventories to the study of their interactions and networks, enhancing our understanding of how hyper-diverse communities are maintained. Previous studies often focused on sampling individual tree species, individual trees or their parts. We argue that such selective sampling is not ideal when analyzing interaction network structure, and may lead to erroneous conclusions. We developed practical and reproducible sampling guidelines for the plot-based analysis of arthropod interaction networks in forest canopies. Our sampling protocol focused on insect herbivores (leaf-chewing insect larvae, miners and gallers) and non-flying invertebrate predators (spiders and ants). We quantitatively sampled the focal arthropods from felled trees, or from trees accessed by canopy cranes or cherry pickers in 53 0.1 ha forest plots in five biogeographic regions, comprising 6,280 trees in total. All three methods required a similar sampling effort and provided good foliage accessibility. Furthermore, we compared interaction networks derived from plot-based data to interaction networks derived from simulated non-plot-based data focusing either on common tree species or a representative selection of tree families. All types of non-plot-based data showed highly biased network structure towards higher connectance, higher web asymmetry, and higher nestedness temperature when compared with plot-based data. Furthermore, some types of non-plot-based data showed biased diversity of the associated herbivore species and specificity of their interactions. Plot-based sampling thus appears to be the most rigorous approach for reconstructing realistic, quantitative plant-arthropod interaction networks that are comparable across sites and regions. Studies of plant interactions have greatly benefited from a plot-based approach and we argue that studies of arthropod interactions would benefit in the same way. We conclude that plot-based studies on canopy arthropods would yield important insights into the processes of interaction network assembly and dynamics, which could be maximised via a coordinated network of plot-based study sites.

Chemical cues and genetic divergence in insects on plants: conceptual cross pollination between mutualistic and antagonistic systems.

Cascading or reciprocal genetic diversification of herbivores, parasitoids, and pollinators can track chemotypic variation in host resources, and can lead to non-overlapping communities. Because plants simultaneously interact with both pollinators and herbivores, models investigating the genetic divergence of antagonistic herbivores and mutualistic pollinators should be merged in order to study how both processes interact using a common conceptual and methodological approach. We expect insects to mediate divergence in many systems, with outcomes depending on the level of pollinator or herbivore specialisation, and the relative selective pressures they impose. Applying approaches widely used to study insect pollinators, for example genomic tools and integration of behavioural, genetic and chemical data, to both pollinators and herbivores in the same system will facilitate our understanding of patterns of genetic divergence across multiple interacting species.