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Abstract Over the last decade, a large effort has been made to understand how extreme climate events disrupt species interactions. Yet, it is unclear how these events affect plants and herbivores directly, via metabolic changes, and indirectly, via their subsequent altered interaction. We exposed common milkweed (Asclepias syriaca) and monarch caterpillars (Danaus plexippus) to control (26:14°C, day:night) or heat wave (HW) conditions (36:24°C, day:night) for 4 days and then moved each organism to a new control or HW partner to disentangle the direct and indirect effects of heat exposure on each organism. We found that the HW directly benefited plants in terms of growth and defence expression (increased latex exudation and total cardenolides) and insect her'bivores through faster larval development. Conversely, indirect HW effects caused both plant latex and total cardenolides to decrease after subsequent herbivory. Nonetheless, increasing trends of more toxic cardenolides and lower leaf nutritional quality after herbivory by HW caterpillars likely led to reduced plant damage compared to controls. Our findings reveal that indirect impacts of HWs may play a greater role in shaping plant‐herbivore interactions via changes in key physiological traits, providing valuable understanding of how ecological interactions may proceed in a changing world. 

Abstract Plants produce defensive toxins to deter herbivores. In response, some specialized herbivores evolved resistance and even the capacity to sequester toxins, affecting interactions at higher trophic levels. Here, we test the hypothesis that potential natural enemies of specialized herbivores are differentially affected by plant toxins depending on their level of adaptation to the plant-herbivore system. We focus on toxic cardiac glycosides (CGs) from milkweeds (Asclepiasspp.), which inhibit animal Na⁺/K⁺-ATPases, and two CG-resistant insects, the large milkweed bugOncopeltus fasciatusand a CRISPR-editedDrosophila melanogaster. Both have CG-resistant Na⁺/K⁺-ATPases through a set of key amino acid substitutions, which facilitate CG sequestration. We conducted infection experiments with entomopathogenic nematodes (Steinernema carpocapsae,S. feltiae, andS. hermaphroditum) as natural enemies on host insects containing mixtures of milkweed-derived CGs or purified CGs (ouabain, digoxin, and digitoxin) that vary in toxicity. The nematodeS. carpocapsaeis known to occur in soil near milkweed plants and naturally has several of the same Na⁺/K⁺-ATPase substitutions as the milkweed bugO. fasciatusand ourDrosophilamutant. This nematode not only exhibited higher fecundity in hosts that carried CGs relative to the other nematode species (which have sensitive Na⁺/K⁺-ATPases), but also showed attraction to mixtures of CGs in milkweed root extracts and to purified ouabain when tested on agar plates. A coiling phenotype, which is a symptom of neurotoxicity, was observed more frequently inS. feltiaeandS. hermaphroditumupon exposure to milkweed root extracts than inS. carpocapsae. Nematode behavior was further tested in sand, and while attraction to CGs was found forS. carpocapsae, nematodes of the other species tended to migrate away from milkweed root chemicals. Thus,S. carpocapsaecan tolerate CGs and may use these as chemical cues to locate insect hosts that live on or around milkweed plants. 

Abstract Plants have evolved multiple defensive traits in response to herbivory; in turn, herbivore specialists evolved adaptive behaviours to avoid or tolerate such defences. Here, we employ milkweeds (Asclepiasspp.) to test two defences, latex and trichomes, for their independent and interactive effects on behaviour and performance of monarch caterpillars (Danaus plexippus).Latex exuded upon damage and the density of leaf trichomes positively correlate across milkweed species, suggesting they may have evolved together as synergistic defences. Nonetheless, the complementary roles of these two traits have been little‐studied. We focus on two behaviours: shaving, or the removal of trichomes, and chewing, which encompasses both deactivation of latex and leaf consumption.In an experiment with seven milkweed species, with and without manipulated latex flow, we found latex to be the primary determinant of reducing chewing, while both defences positively predicted shaving behaviour in the first instar. Next, we conducted a factorial experiment throughout the first three instars, manipulating latex and trichomes on a high‐latex, high‐trichome species, the woolly milkweedAsclepias vestita. On plants with latex and trichomes intact, caterpillars spent the most time shaving and least time chewing of all treatment groups, suggesting a possible synergism. These defence‐driven behavioural effects decreased later in larval development.Latex and trichomes both impacted monarch performance, additively increasing mortality and reducing growth of survivors. Thus, latex and trichomes represent two important plant defences with effects on specialist herbivore behaviour and implications for insect fitness. 

Abstract Urbanization is altering landscapes globally at an unprecedented rate. While ecological differences between urban and rural environments often promote phenotypic divergence among populations, it is unclear to what degree these trait differences arise from genetic divergence as opposed to phenotypic plasticity. Furthermore, little is known about how specific landscape elements, such as green corridors, impact genetic divergence in urban environments. We tested the hypotheses that: (1) urbanization, and (2) proximity to an urban green corridor influence genetic divergence in common milkweed (Asclepias syriaca) populations for phenotypic traits. Using seeds from 52 populations along three urban-to-rural subtransects in the Greater Toronto Area, Canada, one of which followed a green corridor, we grew ~ 1000 plants in a common garden setup and measured > 20 ecologically-important traits associated with plant defense/damage, reproduction, and growth over four years. We found significant heritable variation for nine traits within common milkweed populations and weak phenotypic divergence among populations. However, neither urbanization nor an urban green corridor influenced genetic divergence in individual traits or multivariate phenotype. These findings contrast with the expanding literature demonstrating that urbanization promotes rapid evolutionary change and offer preliminary insights into the eco-evolutionary role of green corridors in urban environments. 

Abstract Herbivores that sequester toxins are thought to have cracked the code of plant defences. Nonetheless, coevolutionary theory predicts that plants should evolve toxic variants that also negatively impact specialists. We propose and test the selective sequestration hypothesis, that specialists preferentially sequester compounds that are less toxic to themselves while maintaining toxicity to enemies. Using chemically distinct plants, we show that monarch butterflies sequester only a subset of cardenolides from milkweed leaves that are less potent against their target enzyme (Na⁺/K⁺‐ATPase) compared to several dominant cardenolides from leaves. However, sequestered compounds remain highly potent against sensitive Na⁺/K⁺‐ATPases found in most predators. We confirmed this differential toxicity with mixtures of purified cardenolides from leaves and butterflies. The genetic basis of monarch adaptation to sequestered cardenolides was also confirmed with transgenicDrosophilathat were CRISPR‐edited with the monarch's Na⁺/K⁺‐ATPase. Thus, the monarch's selective sequestration appears to reduce self‐harm while maintaining protection from enemies. 

Summary The chemical arms race between plants and insects is foundational to the generation and maintenance of biological diversity. We asked how the evolution of a novel defensive compound in an already well‐defended plant lineage impacts interactions with diverse herbivores.Erysimum cheiranthoides(Brassicaceae), which produces both ancestral glucosinolates and novel cardiac glycosides, served as a model.We analyzed gene expression to identify cardiac glycoside biosynthetic enzymes inE. cheiranthoidesand characterized these enzymes via heterologous expression and CRISPR/Cas9 knockout. UsingE. cheiranthoidescardiac glycoside‐deficient lines, we conducted insect experiments in both the laboratory and field.EcCYP87A126 initiates cardiac glycoside biosynthesis via sterol side‐chain cleavage, andEcCYP716A418 has a role in cardiac glycoside hydroxylation. In EcCYP87A126knockout lines, cardiac glycoside production was eliminated. Laboratory experiments with these lines revealed that cardiac glycosides were highly effective defenses against two species of glucosinolate‐tolerant specialist herbivores, but did not protect against all crucifer‐feeding specialist herbivores in the field. Cardiac glycosides had lesser to no effect on two broad generalist herbivores.These results begin elucidation of theE. cheiranthoidescardiac glycoside biosynthetic pathway and demonstratein vivothat cardiac glycoside production allowsErysimumto escape from some, but not all, specialist herbivores. 

Abstract Extreme heat events are becoming more frequent and intense as climate variability increases, and these events inherently vary in their timing. We predicted that the timing of a heat wave would determine its consequences for insect communities owing to temporal variation in the susceptibility of host plants to heat stress. We subjected common milkweed (Asclepias syriaca) plants to in‐field experimental heat waves to investigate how the timing of heat waves, both seasonally and relative to a biotic stressor (experimental herbivory), affected their ecological consequences. We found that heat waves had multiyear, timing‐specific effects on plant–insect communities. Early‐season heat waves led to greater and more persistent effects on plants and herbivore communities than late‐season heat waves. Heat waves following experimental herbivory had reduced consequences. Our results show that extreme climate events can have complex, lasting ecological effects beyond the year of the event—and that timing is key to understanding those effects. 

Abstract Despite long‐standing theory for classifying plant ecological strategies, limited data directly link organismal traits to whole‐plant growth rates (GRs). We compared trait‐growth relationships based on three prominent theories: growth analysis, Grime's competitive–stress tolerant–ruderal (CSR) triangle, and the leaf economics spectrum (LES). Under these schemes, growth is hypothesized to be predicted by traits related to relative biomass investment, leaf structure, or gas exchange, respectively. We also considered traits not included in these theories but that might provide potential alternative best predictors of growth. In phylogenetic analyses of 30 diverse milkweeds (Asclepiasspp.) and 21 morphological and physiological traits, GR (total biomass produced per day) varied 50‐fold and was best predicted by biomass allocation to leaves (as predicted by growth analysis) and the CSR traits of leaf size and leaf dry matter content. Total leaf area (LA) and plant height were also excellent predictors of whole‐plant GRs. Despite two LES traits correlating with growth (mass‐based leaf nitrogen and area‐based leaf phosphorus contents), these were in the opposite direction of that predicted by LES, such that higher N and P contents corresponded to slower growth. The remaining LES traits (e.g., leaf gas exchange) were not predictive of plant GRs. Overall, differences in GR were driven more by whole‐plant characteristics such as biomass fractions and total LA than individual leaf‐level traits such as photosynthetic rate or specific leaf area. Our results are most consistent with classical growth analysis—combining leaf traits with whole‐plant allocation to best predict growth. However, given that destructive biomass measures are often not feasible, applying easy‐to‐measure leaf traits associated with the CSR classification appear more predictive of whole‐plant growth than LES traits. Testing the generality of this result across additional taxa would further improve our ability to predict whole‐plant growth from functional traits across scales. 

Abstract As a general rule, plants defend against herbivores with multiple traits. The defense synergy hypothesis posits that some traits are more effective when co‐expressed with others compared to their independent efficacy. However, this hypothesis has rarely been tested outside of phytochemical mixtures, and seldom under field conditions. We tested for synergies between multiple defense traits of common milkweed (Asclepias syriaca) by assaying the performance of two specialist chewing herbivores on plants in natural populations. We employed regression and a novel application of random forests to identify synergies and antagonisms between defense traits. We found the first direct empirical evidence for two previously hypothesized defense synergies in milkweed (latex by secondary metabolites, latex by trichomes) and identified numerous other potential synergies and antagonisms. Our strongest evidence for a defense synergy was between leaf mass per area and low nitrogen content; given that these “leaf economic” traits typically covary in milkweed, a defense synergy could reinforce their co‐expression. We report that each of the plant defense traits showed context‐dependent effects on herbivores, and increased trait expression could well be beneficial to herbivores for some ranges of observed expression. The novel methods and findings presented here complement more mechanistic approaches to the study of plant defense diversity and provide some of the best evidence to date that multiple classes of plant defense synergize in their impact on insects. Plant defense synergies against highly specialized herbivores, as shown here, are consistent with ongoing reciprocal evolution between these antagonists. 

Abstract Coevolution between plants and herbivores often involves escalation of defence‐offence strategies, but attack by multiple herbivores may obscure the match of plant defence to any one attacker. As herbivores often specialize on distinct plant parts, we hypothesized that defence‐offence interactions in coevolved systems may become physiologically and evolutionarily compartmentalized between plant tissues. We report that roots, leaves, flower buds and seeds of the tropical milkweed (Asclepias curassavica) show increasing concentrations of cardenolide toxins acropetally, with latex showing the highest concentration. In vitro assays of the physiological target of cardenolides, the Na⁺/K⁺–ATPase (hereafter “sodium pump”), of three specialized milkweed herbivores (root‐feedingTetraopes tetrophthalmus, leaf‐feedingDanaus plexippus, and seed‐feedingOncopeltus fasciatus) show that they are proportionally tolerant to the cardenolide concentrations of the tissues they eat. Indeed, molecular substitutions in the insects’ sodium pumps predicted their tolerance to toxins from their target tissues. Nonetheless, the relative inhibition of the sodium pumps of these specialists by the concentration versus composition (inhibition controlled for concentration, what we term “potency”) of cardenolides from their target versus nontarget plant tissues revealed different degrees of insect adaptation to tissue‐specific toxins. In addition, a trade‐off between toxin concentration and potency emerged across plant tissues, potentially reflecting coevolutionary history or plant physiological constraints. Our findings suggest that tissue‐specific coevolutionary dynamics may be proceeding between the plant and its specialized community of herbivores. This novel finding may be common in nature, contributing to ways in which coevolution proceeds in multispecies communities.