Test of Specificity in Signalling between Potato Plants in Response to Infection by Fusarium Solani and Phytophthora Infestans.

Plant-plant signalling via volatile organic compounds (VOCs) in response to insect herbivory has been widely studied, but its occurrence and specificity in response to pathogen attack has received much less attention. To fill this gap, we carried out a greenhouse experiment using two fungal pathogens (Fusarium solani and Phytophthora infestans) to test for specificity in VOC induction and signalling between potato plants (Solanum tuberosum). We paired potato plants in plastic cages, one acting as VOC emitter and the other as receiver, and subjected emitters to one of the following treatments: no infection (control), infected by F. solani, or infected by P. infestans. We measured total emission and composition of VOCs released by emitter plants to test for pathogen-specificity in VOC induction, and then conducted a pathogen infection bioassay to assess resistance levels on receiver plants by subjecting half of the receivers of each emitter treatment to F. solani infection and the other half to P. infestans infection. This allowed us to test for specificity in plant VOC signalling by comparing its effects on conspecific and heterospecific sequential infections. Results showed that infection by neither F. solani or P. infestans produced quantitative (total emissions) or qualitative (compositional) changes in VOC emissions. Mirroring these patterns, emitter infection treatment (control vs. pathogen infection) did not produce a significant change in pathogen infection levels on receiver plants in any case (i.e., either for conspecific or heterospecific sequential infections), indicating a lack of signalling effects which precluded pathogen-based specificity in signalling. We discuss possible mechanisms for lack of pathogen effects on VOC emissions and call for future work testing for pathogen specificity in plant-plant signalling and its implications for plant-pathogen interactions under ecologically relevant scenarios involving infections by multiple pathogens.

Effect of herbivore load on VOC-mediated plant communication in potato.

MAIN CONCLUSION: VOC emissions increased with herbivore load, but this did not result in concomitant increases in resistance in neighbouring plants, suggesting that communication occurred independently of herbivore load in emitter plants. Herbivore-damaged plants emit volatile organic compounds (VOCs) that can alert neighbours and boost their resistance. While VOC-mediated plant communication has been shown to be herbivore-specific, we know little about its contingency on variation in herbivore load. To address this knowledge gap, we tested herbivore load effects on VOC-mediated communication between potato plants (Solanum tuberosum) using the generalist herbivore Spodoptera exigua. First, we tested whether herbivore load (three levels: undamaged control, low, and high load) affected total VOC emissions and composition. Second, we matched emitter and receiver plants and subjected emitters to the same herbivore load treatments. Finally, we performed a bioassay with S. exigua on receivers to test for induced resistance due to VOC-mediated communication. We found that herbivory significantly increased total VOC emissions relative to control plants, and that such increase was greater under high herbivore load. In contrast, we found no detectable effect of herbivory, regardless of the load, on VOC composition. The communication experiment showed that VOCs released by herbivore-induced emitters boosted resistance in receivers (i.e., lower leaf damage than receivers exposed to VOCs released by control emitters), but the magnitude of such effect was similar for both levels of emitter herbivore load. These findings suggest that changes in VOCs due to variation in herbivore load do not modify the outcomes of plant communication.

Aphid and caterpillar feeding drive similar patterns of induced defences and resistance to subsequent herbivory in wild cotton.

MAIN CONCLUSION: Our results indicate caterpillars and aphids cause similar levels of induced defences and resistance against caterpillars in wild cotton plants. These symmetrical effects are not consistent with patterns predicted by plant defensive signaling crosstalk and call for further work addressing the biochemical mechanisms underpinning these results. Plant-induced responses to attack often mediate interactions between different species of insect herbivores. These effects are predicted to be contingent on the herbivore's feeding guild, whereby prior feeding by insects should negatively impact subsequent feeding by insects of the same guild (induced resistance) but may positively influence insects of a different guild (induced susceptibility) due to interfering crosstalk between plant biochemical pathways specific to each feeding guild. We compared the effects of prior feeding by leaf-chewing caterpillars (Spodoptera frugiperda) vs. sap-sucking aphids (Aphis gossypii) on induced defences in wild cotton (Gossypium hirsutum) and the consequences of these attacks on subsequently feeding caterpillars (S. frugiperda). To this end, we conducted a greenhouse experiment where cotton plants were either left undamaged or first exposed to caterpillar or aphid feeding, and we subsequently placed caterpillars on the plants to assess their performance. We also collected leaves to assess the induction of chemical defences in response to herbivory. We found that prior feeding by both aphids and caterpillars resulted in reductions in consumed leaf area, caterpillar mass gain, and caterpillar survival compared with control plants. Concomitantly, prior aphid and caterpillar herbivory caused similar increases in phenolic compounds (flavonoids and hydroxycinnamic acids) and defensive terpenoids (hemigossypolone) compared with control plants. Overall, these findings indicate that these insects confer a similar mode and level of induced resistance in wild cotton plants, calling for further work addressing the biochemical mechanisms underpinning these effects.

Volatile-Mediated Signalling Between Potato Plants in Response to Insect Herbivory is not Contingent on Soil Nutrients.

Plant-plant signalling via volatile organic compounds (VOCs) has been studied intensively, but its contingency on abiotic conditions (e.g., soil nutrients, drought, warming) is poorly understood. To address this gap, we carried out a greenhouse experiment testing whether soil nutrients influenced signalling between potato (Solanum tuberosum) plants in response to insect leaf herbivory by the generalist caterpillar Spodoptera exigua. We placed pairs of plants in plastic cages, where one plant acted as a VOC emitter and the other as a receiver. We factorially manipulated soil nutrients for both emitter and receiver plants, namely: unfertilized (baseline soil nutrients) vs. fertilized (augmented nutrients). Then, to test for signalling effects, half of the emitters within each fertilization level were damaged by S. exigua larvae and the other half remained undamaged. Three days after placing larvae, we collected VOCs from emitter plants to test for herbivory and fertilization effects on VOC emissions and placed S. exigua larvae on receivers to test for signalling effects on leaf consumption and larval mass gain as proxies of induced resistance. We found that herbivory increased total VOC emissions and altered VOC composition by emitter plants, but these effects were not contingent on fertilization. In addition, bioassay results showed that receivers exposed to VOCs from herbivore-damaged emitters had lower levels of herbivory compared to receivers exposed to undamaged emitters. However, and consistent with VOC results, fertilization did not influence herbivore-induced signalling effects on receiver resistance to herbivory. In sum, we found evidence of S. exigua-induced signalling effects on resistance to herbivory in potato plants but such effects were not affected by increased soil nutrients. These results call for further work testing signalling effects under broader range of nutrient concentration levels (including nutrient limitation), teasing apart the effects of specific nutrients, and incorporating other abiotic factors likely to interact or covary with soil nutrients.

Variation in the Outcome of Plant-Mediated Pathogen Interactions in Potato: Effects of Initial Infections on Conspecific vs. Heterospecific Subsequent Infections.

Plants are often attacked sequentially by multiple enemies. Pathogen sequential co-infections can lead to indirect interactions mediated by plant induced responses whose outcome is contingent on differences in the magnitude and type of plant induced defences elicited by different species or guilds. To date, however, most studies have tested unidirectional effects of one pathogen on another, not discerning between conspecific vs. heterospecific infections, and often not measuring plant induced responses underlying such outcomes. To address this, we conducted a greenhouse experiment testing for the impact of initial infection by two leaf pathogens (Alternaria solani and Phytophthora infestans) on subsequent infection by each of these pathogens on potato (Solanum tuberosum) plants, and also measured induced plant defences (phenolic compounds) to inform on interaction outcomes. We found contrasting results depending on the identity of the initially infecting pathogen. Specifically, initial infection by A. solani drove induced resistance (lower necrosis) by subsequently infecting A. solani (conspecific induced resistance) but had no effect on subsequent infection by P. infestans. In contrast, initial infection by P. infestans drove induced resistance to subsequent infection by both conspecifics and A. solani. Patterns of plant induced defences correlated with (and potentially explained) induced resistance to conspecific but not heterospecific (e.g., in the case of P. infestans) subsequent infection. Overall, these results further our understanding of plant-mediated pathogen interactions by showing that plant-mediated interactions between pathogen species can be asymmetrical and in some cases not reciprocal, that pathogen species can vary in the importance of conspecific vs. heterospecific effects, and shed mechanistic insight into the role of plant induced responses driving such interactions.

The co-existence of multiple oak leaf flushes contributes to the large within-tree variation in chemistry, insect attack and pathogen infection.

Many plant species produce multiple leaf flushes during the growing season, which might have major consequences for within-plant variation in chemistry and species interactions. Yet, we lack a theoretical or empirical framework for how differences among leaf flushes might shape variation in damage by insects and diseases. We assessed the impact of leaf flush identity on leaf chemistry, insect attack and pathogen infection on the pedunculate oak Quercus robur by sampling leaves from each leaf flush in 20 populations across seven European countries during an entire growing season. The first leaf flush had higher levels of primary compounds, and lower levels of secondary compounds, than the second flush, whereas plant chemistry was highly variable in the third flush. Insect attack decreased from the first to the third flush, whereas infection by oak powdery mildew was lowest on leaves from the first flush. The relationship between plant chemistry, insect attack and pathogen infection varied strongly among leaf flushes and seasons. Our findings demonstrate the importance of considering differences among leaf flushes for our understanding of within-tree variation in chemistry, insect attack and disease levels, something particularly relevant given the expected increase in the number of leaf flushes with climate change.

Tree Diversity and Forest Resistance to Insect Pests: Patterns, Mechanisms, and Prospects.

Ecological research conducted over the past five decades has shown that increasing tree species richness at forest stands can improve tree resistance to insect pest damage. However, the commonality of this finding is still under debate. In this review, we provide a quantitative assessment (i.e., a meta-analysis) of tree diversity effects on insect herbivory and discuss plausible mechanisms underlying the observed patterns. We provide recommendations and working hypotheses that can serve to lay the groundwork for research to come. Based on more than 600 study cases, our quantitative review indicates that insect herbivory was, on average, lower in mixed forest stands than in pure stands, but these diversity effects were contingent on herbivore diet breadth and tree species composition. In particular, tree species diversity mainly reduced damage of specialist insect herbivores in mixed stands with phylogenetically distant tree species. Overall, our findings provide essential guidance for forest pest management.

Apparent inhibition of induced plant volatiles by a fungal pathogen prevents airborne communication between potato plants.

Plant communication in response to insect herbivory has been increasingly studied, whereas that involving pathogen attack has received much less attention. We tested for communication between potato (Solanum tuberosum) plants in response to leaf infection by the fungal pathogen Sclerotinia sclerotiorum. To this end, we measured the total amount and composition of volatile organic compounds (VOCs) produced by control and infected emitter plants, as well as tested for induced resistance of receiver plants exposed to VOCs from emitters. We further tested for changes in the expression of defensive genes due to pathogen infection. Fungal infection did not significantly affect the total amount or composition of VOCs produced by emitter plants. Correspondingly, we found no evidence of higher resistance to the pathogen in receiver plants exposed to VOCs from infected emitters relative to control emitters. Molecular analyses indicated that pathogen infection drove a down-regulation of genes coding for VOC precursors, potentially explaining the absence of pathogen effects on VOC emissions and thus of communication. Overall, these results indicate no evidence of airborne communication between potato plants in response to fungal infection and point at pathogen inhibition of VOC emissions as a likely explanation for this result.

Effects of latitude and conspecific plant density on insect leaf herbivory in oak saplings and seedlings.

PREMISE: Abiotic factors and plant species traits have been shown to drive latitudinal gradients in herbivory, and yet, population-level factors have been largely overlooked within this context. One such factor is plant density, which may influence the strength of herbivory and may vary with latitude. METHODS: We measured insect herbivory and conspecific plant density (CPD) of oak (Quercus robur) seedlings and saplings along a 17° latitudinal gradient (2700 km) to test whether herbivory exhibited a latitudinal gradient, whether herbivory was associated with CPD, and whether such an association changed with latitude. RESULTS: We found a positive but saturating association between latitude and leaf herbivory. Furthermore, we found no significant relationship between CPD and herbivory, and such lack of density effects remained consistent throughout the sampled latitudinal gradient. CONCLUSIONS: Despite the apparently negligible influence of plant density on herbivory for Q. robur, further research with other plant taxa and in different types of plant communities are needed to investigate density-dependent processes shaping geographical variation in plant-herbivore interactions.

Leaf chemical defences and insect herbivory in oak: accounting for canopy position unravels marked genetic relatedness effects.

BACKGROUND AND AIMS: Highly controlled experiments document that plant genetic diversity and relatedness can shape herbivore communities and patterns of herbivory. Evidence from the field is, however, scarce and inconsistent. We assessed whether a genetic signal underlying herbivory can be detected in oak woodlands when accounting for variation at smaller (within-tree) and larger (among-stand) scales. METHODS: We tested relationships between tree genetic relatedness, leaf chemical defences and insect herbivory for different canopy layers in 240 trees from 15 pedunculate oak (Quercus robur) forest stands. We partitioned sources of variability in herbivory and defences among stands, individuals and branches. KEY RESULTS: Leaf defences, insect herbivory and their relationship differed systematically between the upper and the lower tree canopy. When accounting for this canopy effect, the variation explained by tree genetic relatedness rose from 2.8 to 34.1 % for herbivory and from 7.1 to 13.8 % for leaf defences. The effect was driven by markedly stronger relationships in the upper canopy. CONCLUSIONS: Our findings illustrate that considerable effects of the host plant genotype on levels of leaf chemical defences and associated insect herbivory can be detected in natural tree populations when within-individual variation is properly accounted for.

Latitudinal variation in seed predation correlates with latitudinal variation in seed defensive and nutritional traits in a widespread oak species.

BACKGROUND AND AIMS: Classic theory on geographical gradients in plant-herbivore interactions assumes that herbivore pressure and plant defences increase towards warmer and more stable climates found at lower latitudes. However, the generality of these expectations has been recently called into question by conflicting empirical evidence. One possible explanation for this ambiguity is that most studies have reported on patterns of either herbivory or plant defences whereas few have measured both, thus preventing a full understanding of the implications of observed patterns for plant-herbivore interactions. In addition, studies have typically not measured climatic factors affecting plant-herbivore interactions, despite their expected influence on plant and herbivore traits. METHODS: Here we tested for latitudinal variation in insect seed predation and seed traits putatively associated with insect attack across 36 Quercus robur populations distributed along a 20° latitudinal gradient. We then further investigated the associations between climatic factors, seed traits and seed predation to test for climate-based mechanisms of latitudinal variation in seed predation. KEY RESULTS: We found strong but contrasting latitudinal clines in seed predation and seed traits, whereby seed predation increased whereas seed phenolics and phosphorus decreased towards lower latitudes. We also found a strong direct association between temperature and seed predation, with the latter increasing towards warmer climates. In addition, temperature was negatively associated with seed traits, with populations at warmer sites having lower levels of total phenolics and phosphorus. In turn, these negative associations between temperature and seed traits led to a positive indirect association between temperature and seed predation. CONCLUSIONS: These results help unravel how plant-herbivore interactions play out along latitudinal gradients and expose the role of climate in driving these outcomes through its dual effects on plant defences and herbivores. Accordingly, this emphasizes the need to account for abiotic variation while testing concurrently for latitudinal variation in plant traits and herbivore pressure.

Parallel increases in insect herbivory and defenses with increasing elevation for both saplings and adult trees of oak (Quercus) species.

PREMISE: Herbivory is predicted to increase toward warmer and more stable climates found at lower elevations, and this increase should select for higher plant defenses. Still, a number of recent studies have reported either no evidence of such gradients or reverse patterns. One source of inconsistency may be that plant ontogenetic variation is usually not accounted for and may influence levels of plant defenses and herbivory. METHODS: We tested for elevational gradients in insect leaf herbivory and leaf traits putatively associated with herbivore resistance across eight oak (Quercus, Fagaceae) species and compared these patterns for saplings and adult trees. To this end, we surveyed insect leaf herbivory and leaf traits (phenolic compounds, toughness and nutrients) in naturally occurring populations of each oak species at low-, mid- or high-elevation sites throughout the Iberian Peninsula. RESULTS: Leaf herbivory and chemical defenses (lignins) were unexpectedly higher at mid- and high-elevation sites than at low-elevation sites. In addition, leaf chemical defenses (lignins and condensed tannins) were higher for saplings than adult trees, whereas herbivory did not significantly differ between ontogenetic stages. Overall, elevational variation in herbivory and plant chemical defenses were consistent across ontogenetic stages (i.e., elevational gradients were not contingent upon tree ontogeny), and herbivory and leaf traits were not associated across elevations. CONCLUSIONS: These findings suggest disassociated patterns of elevational variation in herbivory and leaf traits, which, in turn, are independent of plant ontogenetic stage.

Greater phylogenetic distance from native oaks predicts escape from insect leaf herbivores by non-native oak saplings.

PREMISE: Non-native plant species have been hypothesized to experience lower herbivory in novel environments as a function of their phylogenetic distance from native plant species. Although recent work has found support for this prediction, the plant traits responsible for such patterns have been largely overlooked. METHODS: In a common garden experiment in northwestern Spain, we tested whether oak species (Quercus spp.) not native to this region that are phylogenetically more distantly related to native species exhibit less insect leaf herbivory. In addition, we also investigated plant traits potentially correlated with any such effect of phylogenetic distance. RESULTS: As expected, phylogenetic distance from native species negatively predicted insect leaf herbivory on non-native oaks. In addition, we found that the leaf traits, namely phosphorus and condensed tannins, were significantly associated with herbivory, suggesting that they are associated with the effect of phylogenetic distance on leaf herbivory on non-native oak species. CONCLUSIONS: This study contributes to a better understanding of how evolutionary relationships (relatedness) between native and non-native plant species determine the latter's success in novel environments via locally shared enemies, and encourages more work investigating the plant traits that mediate the effects of phylogenetic distance on enemy escape.

Weather cues associated with masting behavior dampen the negative autocorrelation between past and current reproduction in oaks.

PREMISE OF THE STUDY: The influence of weather conditions on masting and the ecological advantages of this reproductive behavior have been the subject of much interest. Weather conditions act as cues influencing reproduction of individual plants, and similar responses expressed across many individuals lead to population-level synchrony in reproductive output. In turn, synchrony leads to benefits from economies of scale such as enhanced pollination success and seed predator satiation. However, there may also be individual-level benefits from reproductive responses to weather cues, which may explain the origin of masting in the absence of economies of scale. In a previous study, we found support for a mechanism whereby individual responses to weather cues attenuate the negative autocorrelation between past and current annual seed production-a pattern typically attributed to resource limitation and reproductive tradeoffs among years. METHODS: Here we provide a follow-up and more robust evaluation of this hypothesis in 12 species of oaks (Quercus spp.), testing for a negative autocorrelation (tradeoff) between past and current reproduction and whether responses to weather cues associated with masting reduce the strength of this negative autocorrelation. KEY RESULTS: Our results showed a strong negative autocorrelation for 11 of the species, and that species-specific reproductive responses to weather cues dampened this negative autocorrelation in 10 of them. CONCLUSIONS: This dampening effect presumably reflects a reduction in resource limitation or increased resource use associated with weather conditions, and suggests that responses to weather cues conferring these advantages should be selected for based on individual benefits.

Bottom-up control of geographic variation in insect herbivory on wild cotton (Gossypium hirsutum) by plant defenses and climate.

PREMISE: The occurrence and amount of herbivory are shaped by bottom-up forces, primarily plant traits (e.g., defenses), and by abiotic factors. Addressing these concurrent effects in a spatial context has been useful in efforts to understand the mechanisms governing variation in plant-herbivore interactions. Still, few studies have evaluated the simultaneous influence of multiple sources of bottom-up variation on spatial variation in herbivory. METHODS: We tested to what extent chemical (phenolics, production of terpenoid glands) and physical (pubescence) defensive plant traits and climatic factors are associated with variation in herbivory by leaf-chewing insects across populations of wild cotton (Gossypium hirsutum). RESULTS: We found substantial population variation in cotton leaf defenses and insect leaf herbivory. Leaf pubescence, but not gossypol gland density or phenolic content, was significantly negatively associated with herbivory by leaf-chewing insects. In addition, there were direct effects of climate on defenses and herbivory, with leaf pubescence increasing toward drier conditions and leaf damage increasing toward wetter and cooler conditions. There was no evidence, however, of indirect effects (via plant defenses) of climate on herbivory. CONCLUSIONS: These results suggest that spatial variation in insect herbivory on wild G. hirsutum is predominantly driven by concurrent and independent influences of population variation in leaf pubescence and climatic factors.

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.

Herbivore specificity and the chemical basis of plant-plant communication in Baccharis salicifolia (Asteraceae).

It is well known that plant damage by leaf-chewing herbivores can induce resistance in neighbouring plants. It is unknown whether such communication occurs in response to sap-feeding herbivores, whether communication is specific to herbivore identity, and the chemical basis of communication, including specificity. We carried out glasshouse experiments using the California-native shrub Baccharis salicifolia and two ecologically distinct aphid species (one a dietary generalist and the other a specialist) to test for specificity of plant-plant communication and to document the underlying volatile organic compounds (VOCs). We show specificity of plant-plant communication to herbivore identity, as each aphid-damaged plant only induced resistance in neighbours against the same aphid species. The amount and composition of induced VOCs were markedly different between plants attacked by the two aphid species, providing a putative chemical mechanism for this specificity. Furthermore, a synthetic blend of the five major aphid-induced VOCs (ethanone, limonene, methyl salicylate, myrcene, ocimene) triggered resistance in receiving plants of comparable magnitude to aphid damage of neighbours, and the effects of the blend exceeded those of individual compounds. This study significantly advances our understanding of plant-plant communication by demonstrating the importance of sap-feeding herbivores and herbivore identity, as well as the chemical basis for such effects.

Specificity of plant-plant communication for Baccharis salicifolia sexes but not genotypes.

Plants are able to adjust their anti-herbivore defenses in response to the volatile organic compounds (VOCs) emitted by herbivore-damaged neighbors, and some of these changes increase resistance against subsequent herbivory. This phenomenon of plant-plant communication is thought to be widespread, but recent investigations have cautioned that it can be context dependent, including variation in the strength of communication based on the identity of plants and their associated herbivores. Here, we performed three greenhouse experiments using multiple male and female genotypes of the dioecious woody shrub Baccharis salicifolia and its specialist aphid Uroleucon macolai to test for specificity of plant-plant communication with respect to plant sex and genotype. Moreover, we evaluated plant sexual dimorphism and genotypic variation in VOC emissions (i.e., the "speaking" side of the interaction) and response of plants to VOC exposure (i.e., the "listening" side of the interaction) in order to identify the chemical mechanisms underlying such specificity. We did not find genotypic specificity of communication; emitter plants damaged by U. macolai significantly reduced subsequent U. macolai performance on receivers, but these effects were indistinguishable for communication within vs. among genotypes. In contrast, we found sex specificity of communication; male emitter plants reduced subsequent U. macolai performance on male and female receiver plants equally, while female emitter plants only did so for female receivers. We found sexual (but not genotypic) dimorphism in speaking but not listening; of the seven compounds induced by U. macolai feeding (speaking), pinocarvone was approximately fivefold greater in female than in male plants, while exposure of plants to pinocarvone emissions (listening) reduced U. macolai performance equally in both male and female plants. Together, our study demonstrates novel evidence for sexually dimorphic specificity of plant-plant communication and the chemical mechanism underlying this effect.

Host plant frequency and secondary metabolites are concurrently associated with insect herbivory in a dominant riparian tree.

Herbivory is strongly influenced by different sources of plant variation, from traits such as secondary metabolites to features associated with population- and community-level variation. However, most studies have assessed the influence of these drivers in isolation. We conducted a large-scale study to evaluate the associations between multiple types of plant-based variation and insect leaf herbivory in alder ( Alnus glutinosa) trees sampled in riparian forests throughout northwestern Spain. We assessed the associations between insect leaf herbivory and alder mean production of leaf secondary metabolites (phenolic compounds), variation among neighbouring alder trees in leaf phenolics and community-related features including alder relative size and frequency and tree species phylogenetic diversity. Structural equation modelling indicated that increasing concentrations of alder leaf flavonoids (but not other types of phenolic compounds) and increasing variation in phenolics among neighbouring alders were both significantly negatively associated with herbivory. In addition, increasing relative frequency of alder was positively associated with leaf damage, whereas the size of alders relative to other trees and phylogenetic diversity were not significantly associated with herbivory. These results demonstrate the concurrent and independent influences of different sources of plant-based variation on insect herbivory and argue for further future work simultaneously addressing multiple plant-based bottom-up controls.