Tissue and toxin-specific divergent evolution in plant defense Evolución divergente específica de tejido y toxina en defensa de plantas.

A major predicted constraint on the evolution of anti-herbivore defense in plants is the nonindependent expression of traits mediating resistance. Since herbivore attack can be highly variable across plant tissues, we hypothesized that correlations in toxin expression within and between plant tissues may limit population differentiation and, thus, plant adaptation. Using full-sib families from two nearby (<1 km) common milkweed (Asclepias syriaca) populations, we investigated genetic correlations among 28 distinct cardenolide toxins within and between roots, leaves, and seeds and examined signatures of tissue-specific divergent selection between populations by QST-FST comparisons. The prevalence, direction, and strength of genetic correlations among cardenolides were tissue specific, and concentrations of individual cardenolides were moderately correlated between tissues; nonetheless, the direction and strength of correlations were population specific. Population divergence in the cardenolide chemistry was stronger in roots than in leaves and seeds. Divergent selection on individual cardenolides was tissue and toxin specific, except for a single highly toxic cardenolide (labriformin), that showed divergent selection across all plant tissues. Heterogeneous evolution of cardenolides within and between tissues across populations appears possible due to their highly independent expression. This independence may be common in nature, especially in specialized interactions in which distinct herbivores feed on different plant tissues.

Tissue-specific plant toxins and adaptation in a specialist root herbivore.

In coevolution between plants and insects, reciprocal selection often leads to phenotype matching between chemical defense and herbivore offense. Nonetheless, it is not well understood whether distinct plant parts are differentially defended and how herbivores adapted to those parts cope with tissue-specific defense. Milkweed plants produce a diversity of cardenolide toxins and specialist herbivores have substitutions in their target enzyme (Na+/K+-ATPase), each playing a central role in milkweed-insect coevolution. The four-eyed milkweed beetle (Tetraopes tetrophthalmus) is an abundant toxin-sequestering herbivore that feeds exclusively on milkweed roots as larvae and less so on milkweed leaves as adults. Accordingly, we tested the tolerance of this beetle's Na+/K+-ATPase to cardenolide extracts from roots versus leaves of its main host (Asclepias syriaca), along with sequestered cardenolides from beetle tissues. We additionally purified and tested the inhibitory activity of dominant cardenolides from roots (syrioside) and leaves (glycosylated aspecioside). Tetraopes' enzyme was threefold more tolerant of root extracts and syrioside than leaf cardenolides. Nonetheless, beetle-sequestered cardenolides were more potent than those in roots, suggesting selective uptake or dependence on compartmentalization of toxins away from the beetle's enzymatic target. Because Tetraopes has two functionally validated amino acid substitutions in its Na+/K+-ATPase compared to the ancestral form in other insects, we compared its cardenolide tolerance to that of wild-type Drosophila and CRISPR-edited Drosophila with Tetraopes' Na+/K+-ATPase genotype. Those two amino acid substitutions accounted for >50% of Tetraopes' enhanced enzymatic tolerance of cardenolides. Thus, milkweed's tissue-specific expression of root toxins is matched by physiological adaptations in its specialist root herbivore.

Spatial metabolomics reveal divergent cardenolide processing in the monarch (Danaus plexippus) and the common crow butterfly (Euploea core).

Although being famous for sequestering milkweed cardenolides, the mechanism of sequestration and where cardenolides are localized in caterpillars of the monarch butterfly (Danaus plexippus, Lepidoptera: Danaini) is still unknown. While monarchs tolerate cardenolides by a resistant Na+ /K+ -ATPase, it is unclear how closely related species such as the nonsequestering common crow butterfly (Euploea core, Lepidoptera: Danaini) cope with these toxins. Using novel atmospheric-pressure scanning microprobe matrix-assisted laser/desorption ionization mass spectrometry imaging, we compared the distribution of cardenolides in caterpillars of D. plexippus and E. core. Specifically, we tested at which physiological scale quantitative differences between both species are mediated and how cardenolides distribute across body tissues. Whereas D. plexippus sequestered most cardenolides from milkweed (Asclepias curassavica), no cardenolides were found in the tissues of E. core. Remarkably, quantitative differences already manifest in the gut lumen: while monarchs retain and accumulate cardenolides above plant concentrations, the toxins are degraded in the gut lumen of crows. We visualized cardenolide transport over the monarch midgut epithelium and identified integument cells as the final site of storage where defences might be perceived by predators. Our study provides molecular insight into cardenolide sequestration and highlights the great potential of mass spectrometry imaging for understanding the kinetics of multiple compounds including endogenous metabolites, plant toxins, or insecticides in insects.

Compound-Specific Behavioral and Enzymatic Resistance to Toxic Milkweed Cardenolides in a Generalist Bumblebee Pollinator.

Plant secondary metabolites that defend leaves from herbivores also occur in floral nectar. While specialist herbivores often have adaptations providing resistance to these compounds in leaves, many social insect pollinators are generalists, and therefore are not expected to be as resistant to such compounds. The milkweeds, Asclepias spp., contain toxic cardenolides in all tissues including floral nectar. We compared the concentrations and identities of cardenolides between tissues of the North American common milkweed Asclepias syriaca, and then studied the effect of the predominant cardenolide in nectar, glycosylated aspecioside, on an abundant pollinator. We show that a generalist bumblebee, Bombus impatiens, a common pollinator in eastern North America, consumes less nectar with experimental addition of ouabain (a standard cardenolide derived from Apocynacid plants native to east Africa) but not with addition of glycosylated aspecioside from milkweeds. At a concentration matching that of the maximum in the natural range, both cardenolides reduced activity levels of bees after four days of consumption, demonstrating toxicity despite variation in behavioral deterrence (i.e., consumption). In vitro enzymatic assays of Na+/K+-ATPase, the target site of cardenolides, showed lower toxicity of the milkweed cardenolide than ouabain for B. impatiens, indicating that the lower deterrence may be due to greater tolerance to glycosylated aspecioside. In contrast, there was no difference between the two cardenolides in toxicity to the Na+/K+-ATPase from a control insect, the fruit fly Drosophila melanogaster. Accordingly, this work reveals that even generalist pollinators such as B. impatiens may have adaptations to reduce the toxicity of specific plant secondary metabolites that occur in nectar, despite visiting flowers from a wide variety of plants over the colony's lifespan.

Plant defense synergies and antagonisms affect performance of specialist herbivores of common milkweed.

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.

Pyrethroid insecticide and milkweed cardenolide interactions on detoxification enzyme activity and expression in monarch caterpillars.

Declines of the monarch butterfly population have prompted large-scale plantings of milkweed to restore the population. In North America, there are >73 species of milkweed to choose from for these nationwide plantings. However, it is unclear how different milkweed species affect monarch caterpillar physiology, particularly detoxification enzyme activity and gene expression, given the highly variable cardenolide composition across milkweed species. Here, we investigate the effects of a high cardenolide, tropical milkweed species and a low cardenolide, swamp milkweed species on pyrethroid sensitivity as well as detoxification enzyme activity and expression in monarch caterpillars. Caterpillars fed on each species through the fifth-instar stage and were topically treated with bifenthrin after reaching this final-instar stage. Esterase, glutathione S-transferase, and cytochrome P450 monooxygenase activities were quantified as well as the expression of selected esterase, glutathione S-transferase, ABC transporter, and cytochrome P450 monooxygenase transcripts. There were no significant differences in survival 24 h after treatment with bifenthrin. However, bifenthrin significantly increased glutathione S-transferase activity in caterpillars feeding on tropical milkweed and significantly decreased esterase activity in caterpillars feeding on tropical and swamp milkweed. Significant differential expression of ABC transporter, glutathione S-transferase, and esterase genes was observed for caterpillars feeding on tropical and swamp milkweed and not receiving bifenthrin treatment. Furthermore, significant differential expression of glutathione S-transferase and esterase genes was observed for bifenthrin-treated and -untreated caterpillars feeding on tropical milkweed relative to swamp milkweed. These results suggest that feeding on different milkweed species can affect detoxification and development mechanisms with which monarch caterpillars rely on to cope with their environment.

Functional evidence supports adaptive plant chemical defense along a geographical cline.

Environmental clines in organismal defensive traits are usually attributed to stronger selection by enemies at lower latitudes or near the host's range center. Nonetheless, little functional evidence has supported this hypothesis, especially for coevolving plants and herbivores. We quantified cardenolide toxins in seeds of 24 populations of common milkweed (Asclepias syriaca) across 13 degrees of latitude, revealing a pattern of increasing cardenolide concentrations toward the host's range center. The unusual nitrogen-containing cardenolide labriformin was an exception and peaked at higher latitudes. Milkweed seeds are eaten by specialist lygaeid bugs that are even more tolerant of cardenolides than the monarch butterfly, concentrating most cardenolides (but not labriformin) from seeds into their bodies. Accordingly, whether cardenolides defend seeds against these specialist bugs is unclear. We demonstrate that Oncopeltus fasciatus (Lygaeidae) metabolized two major compounds (glycosylated aspecioside and labriformin) into distinct products that were sequestered without impairing growth. We next tested several isolated cardenolides in vitro on the physiological target of cardenolides (Na+/K+-ATPase); there was little variation among compounds in inhibition of an unadapted Na+/K+-ATPase, but tremendous variation in impacts on that of monarchs and Oncopeltus. Labriformin was the most inhibitive compound tested for both insects, but Oncopeltus had the greater advantage over monarchs in tolerating labriformin compared to other compounds. Three metabolized (and stored) cardenolides were less toxic than their parent compounds found in seeds. Our results suggest that a potent plant defense is evolving by natural selection along a geographical cline and targets specialist herbivores, but is met by insect tolerance, detoxification, and sequestration.

Cardenolides, toxicity, and the costs of sequestration in the coevolutionary interaction between monarchs and milkweeds.

For highly specialized insect herbivores, plant chemical defenses are often co-opted as cues for oviposition and sequestration. In such interactions, can plants evolve novel defenses, pushing herbivores to trade off benefits of specialization with costs of coping with toxins? We tested how variation in milkweed toxins (cardenolides) impacted monarch butterfly (Danaus plexippus) growth, sequestration, and oviposition when consuming tropical milkweed (Asclepias curassavica), one of two critical host plants worldwide. The most abundant leaf toxin, highly apolar and thiazolidine ring-containing voruscharin, accounted for 40% of leaf cardenolides, negatively predicted caterpillar growth, and was not sequestered. Using whole plants and purified voruscharin, we show that monarch caterpillars convert voruscharin to calotropin and calactin in vivo, imposing a burden on growth. As shown by in vitro experiments, this conversion is facilitated by temperature and alkaline pH. We next employed toxin-target site experiments with isolated cardenolides and the monarch's neural Na+/K+-ATPase, revealing that voruscharin is highly inhibitory compared with several standards and sequestered cardenolides. The monarch's typical >50-fold enhanced resistance to cardenolides compared with sensitive animals was absent for voruscharin, suggesting highly specific plant defense. Finally, oviposition was greatest on intermediate cardenolide plants, supporting the notion of a trade-off between benefits and costs of sequestration for this highly specialized herbivore. There is apparently ample opportunity for continued coevolution between monarchs and milkweeds, although the diffuse nature of the interaction, due to migration and interaction with multiple milkweeds, may limit the ability of monarchs to counteradapt.

Mycorrhizae Alter Constitutive and Herbivore-Induced Volatile Emissions by Milkweeds.

Plants use volatile organic compounds (VOCs) to cue natural enemies to their herbivore prey on plants. Simultaneously, herbivores utilize volatile cues to identify appropriate hosts. Despite extensive efforts to understand sources of variation in plant communication by VOCs, we lack an understanding of how ubiquitous belowground mutualists, such as arbuscular mycorrhizal fungi (AMF), influence plant VOC emissions. In a full factorial experiment, we subjected plants of two milkweed (Asclepias) species under three levels of AMF availability to damage by aphids (Aphis nerii). We then measured plant headspace volatiles and chemical defenses (cardenolides) and compared these to VOCs emitted and cardenolides produced by plants without herbivores. We found that AMF have plant species-specific effects on constitutive and aphid-induced VOC emissions. High AMF availability increased emissions of total VOCs, two green leaf volatiles (3-hexenyl acetate and hexyl acetate), and methyl salicylate in A. curassavica, but did not affect emissions in A. incarnata. In contrast, aphids consistently increased emissions of 6-methyl-5-hepten-2-one and benzeneacetaldehyde in both species, independent of AMF availability. Both high AMF availability and aphids alone suppressed emissions of individual terpenes. However, aphid damage on plants under high AMF availability increased, or did not affect, emissions of those terpenes. Lastly, aphid feeding suppressed cardenolide concentrations only in A. curassavica, and AMF did not affect cardenolides in either plant species. Our findings suggest that by altering milkweed VOC profiles, AMF may affect both herbivore performance and natural enemy attraction.

Population Variation, Environmental Gradients, and the Evolutionary Ecology of Plant Defense against Herbivory.

A central tenet of plant defense theory is that adaptation to the abiotic environment sets the template for defense strategies, imposing a trade-off between plant growth and defense. Yet this trade-off, commonly found among species occupying divergent resource environments, may not occur across populations of single species. We hypothesized that more favorable climates and higher levels of herbivory would lead to increases in growth and defense across plant populations. We evaluated whether plant growth and defense traits covaried across 18 populations of showy milkweed (Asclepias speciosa) inhabiting an east-west climate gradient spanning 25° of longitude. A suite of traits impacting defense (e.g., latex, cardenolides), growth (e.g., size), or both (e.g., specific leaf area [SLA], trichomes) were measured in natural populations and in a common garden, allowing us to evaluate plastic and genetically based variation in these traits. In natural populations, herbivore pressure increased toward warmer sites with longer growing seasons. Growth and defense traits showed strong clinal patterns and were positively correlated. In a common garden, clines with climatic origin were recapitulated only for defense traits. Correlations between growth and defense traits were also weaker and more negative in the common garden than in the natural populations. Thus, our data suggest that climatically favorable sites likely facilitate the evolution of greater defense at minimal costs to growth, likely because of increased resource acquisition.

Belowground annual ring growth coordinates with aboveground phenology and timing of carbon storage in two tallgrass prairie forb species.

PREMISE OF THE STUDY: Herb chronology, the study of belowground annual growth rings in perennial forbs, has much potential as a tool for monitoring plant growth as a function of environment. To harness this potential, understanding of the coordination between ring ontogeny, aboveground phenology, and the temporal allocation of carbon products belowground in herbaceous forbs must be improved. METHODS: We investigated these relationships in two southern United States tallgrass prairie perennial forb species, Asclepias viridis and Lespedeza stuevei, making monthly excavations for a year. KEY RESULTS: Belowground xylogenesis began when starch reserves were at their seasonal low in the spring as shoots reached maximum height. The highest relative radial growth of the ring occurred concurrently with replenishment of root starch reserves in early summer. Xylogenesis concluded with leaf senescence in late summer and belowground starch reserves near saturation. CONCLUSIONS: By demonstrating that ring ontogeny is tied to early summer starch replenishment, our results illustrate the mechanisms behind previous findings where ring width was highly correlated with summer climatic conditions for these two species. This study provides a new physiological link between how ring chronologies in herbs often accord with growing-season environment; further dissecting this phenomenon is vital in unlocking the potential of herb chronology.

The Effects of Milkweed Induced Defense on Parasite Resistance in Monarch Butterflies, Danaus plexippus.

Many plants express induced defenses against herbivores through increasing the production of toxic secondary chemicals following damage. Phytochemical induction can directly or indirectly affect other organisms within the community. In tri-trophic systems, increased concentrations of plant toxins could be detrimental to plants if herbivores can sequester these toxins as protective chemicals for themselves. Thus, through trophic interactions, induction can lead to either positive or negative effects on plant fitness. We examined the effects of milkweed (Asclepias spp.) induced defenses on the resistance of monarch caterpillars (Danaus plexippus) to a protozoan parasite (Ophryocystis elektroscirrha). Milkweeds contain toxic secondary chemicals called cardenolides, higher concentrations of which are associated with reduced parasite growth. Previous work showed that declines in foliar cardenolides caused by aphid attack render monarch caterpillars more susceptible to infection. Here, we ask whether cardenolide induction by monarchs increases monarch resistance to disease. We subjected the high-cardenolide milkweed A. curassavica and the low-cardenolide A. syriaca to caterpillar grazing, and reared infected and uninfected caterpillars on these plants. As expected, monarchs suffered less parasite growth and disease when reared on A. curassavica than on A. syriaca. We also found that herbivory increased cardenolide concentrations in A. curassavica, but not A. syriaca. However, cardenolide induction in A. curassavica was insufficient to influence monarch resistance to the parasite. Our results suggest that interspecific variation in cardenolide concentration is a more important driver of parasite defense than plasticity via induced defenses in this tri-trophic system.

Experimental evidence shows no fractionation of strontium isotopes ((87)Sr/(86)Sr) among soil, plants, and herbivores: implications for tracking wildlife and forensic science.

Strontium isotopes ((87)Sr/(86)Sr) can be useful biological markers for a wide range of forensic science applications, including wildlife tracking. However, one of the main advantages of using (87)Sr/(86)Sr values, that there is no fractionation from geological bedrock sources through the food web, also happens to be a critical assumption that has never been tested experimentally. We test this assumption by measuring (87)Sr/(86)Sr values across three trophic levels in a controlled greenhouse experiment. Adult monarch butterflies were raised on obligate larval host milkweed plants that were, in turn, grown on seven different soil types collected across Canada. We found no significant differences between (87)Sr/(86)Sr values in leachable Sr from soil minerals, organic soil, milkweed leaves, and monarch butterfly wings. Our results suggest that strontium isoscapes developed from (87)Sr/(86)Sr values in bedrock or soil may serve as a reliable biological marker in forensic science for a range of taxa and across large geographic areas.

The ecology and evolution of animal medication: genetically fixed response versus phenotypic plasticity.

Animal medication against parasites can occur either as a genetically fixed (constitutive) or phenotypically plastic (induced) behavior. Taking the tritrophic interaction between the monarch butterfly Danaus plexippus, its protozoan parasite Ophryocystis elektroscirrha, and its food plant Asclepias spp. as a test case, we develop a game-theory model to identify the epidemiological (parasite prevalence and virulence) and environmental (plant toxicity and abundance) conditions that predict the evolution of genetically fixed versus phenotypically plastic forms of medication. Our model shows that the relative benefits (the antiparasitic properties of medicinal food) and costs (side effects of medicine, the costs of searching for medicine, and the costs of plasticity itself) crucially determine whether medication is genetically fixed or phenotypically plastic. Our model suggests that animals evolve phenotypic plasticity when parasite risk (a combination of virulence and prevalence and thus a measure of the strength of parasite-mediated selection) is relatively low to moderately high and genetically fixed medication when parasite risk becomes very high. The latter occurs because at high parasite risk, the costs of plasticity are outweighed by the benefits of medication. Our model provides a simple and general framework to study the conditions that drive the evolution of alternative forms of animal medication.

Specificity of herbivore-induced hormonal signaling and defensive traits in five closely related milkweeds (Asclepias spp.).

Despite the recognition that phytohormonal signaling mediates induced responses to herbivory, we still have little understanding of how such signaling varies among closely related species and may generate herbivore-specific induced responses. We studied closely related milkweeds (Asclepias) to link: 1) plant damage by two specialist chewing herbivores (milkweed leaf beetles Labidomera clivicolis and monarch caterpillars Danaus plexippus); 2) production of the phytohormones jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA); 3) induction of defensive cardenolides and latex; and 4) impacts on Danaus caterpillars. We first show that A. syriaca exhibits induced resistance following monarch herbivory (i.e., reduced monarch growth on previously damaged plants), while the defensively dissimilar A. tuberosa does not. We next worked with a broader group of five Asclepias, including these two species, that are highly divergent in defensive traits yet from the same clade. Three of the five species showed herbivore-induced changes in cardenolides, while induced latex was found in four species. Among the phytohormones, JA and ABA showed specific responses (although they generally increased) to insect species and among the plant species. In contrast, SA responses were consistent among plant and herbivore species, showing a decline following herbivore attack. Jasmonic acid showed a positive quantitative relationship only with latex, and this was strongest in plants damaged by D. plexippus. Although phytohormones showed qualitative tradeoffs (i.e., treatments that enhanced JA reduced SA), the few significant individual plant-level correlations among hormones were positive, and these were strongest between JA and ABA in monarch damaged plants. We conclude that: 1) latex exudation is positively associated with endogenous JA levels, even among low-latex species; 2) correlations among milkweed hormones are generally positive, although herbivore damage induces a divergence (tradeoff) between JA and SA; 3) induction of cardenolides and latex are not necessarily physiologically linked; and 4) even very closely related species show highly divergent induction, with some species showing strong defenses, hormonally-mediated induction, and impacts on herbivores, while other milkweed species apparently use alternative strategies to cope with insect attack.

Genetic variation in plant volatile emission does not result in differential attraction of natural enemies in the field.

Volatile organic chemical (VOC) emission by plants may serve as an adaptive plant defense by attracting the natural enemies of herbivores. For plant VOC emission to evolve as an adaptive defense, plants must show genetic variability for the trait. To date, such variability has been investigated primarily in agricultural systems, yet relatively little is known about genetic variation in VOCs emitted by natural populations of native plants. Here, we investigate intraspecific variation in constitutive and herbivore-induced plant VOC emission using the native common milkweed plant (Asclepias syriaca) and its monarch caterpillar herbivore (Danaus plexippus) in complementary field and common garden greenhouse experiments. In addition, we used a common garden field experiment to gauge natural enemy attraction to milkweed VOCs induced by monarch damage. We found evidence of genetic variation in the total constitutive and induced concentrations of VOCs and the composition of VOC blends emitted by milkweed plants. However, all milkweed genotypes responded similarly to induction by monarchs in terms of their relative change in VOC concentration and blend. Natural enemies attacked decoy caterpillars more frequently on damaged than on undamaged milkweed, and natural enemy visitation was associated with higher total VOC concentrations and with VOC blend. Thus, we present evidence that induced VOCs emitted by milkweed may function as a defense against herbivores. However, plant genotypes were equally attractive to natural enemies. Although milkweed genotypes diverge phenotypically in their VOC concentrations and blends, they converge into similar phenotypes with regard to magnitude of induction and enemy attraction.

A genetically-based latitudinal cline in the emission of herbivore-induced plant volatile organic compounds.

The existence of predictable latitudinal variation in plant defense against herbivores remains controversial. A prevailing view holds that higher levels of plant defense evolve at low latitudes compared to high latitudes as an adaptive plant response to higher herbivore pressure on low-latitude plants. To date, this prediction has not been examined with respect to volatile organic compounds (VOCs) that many plants emit, often thus attracting the natural enemies of herbivores. Here, we compared genetically-based constitutive and herbivore-induced aboveground vegetative VOC emissions from plants originating across a gradient of more than 10° of latitude (>1,500 km). We collected headspace VOCs from Asclepias syriaca (common milkweed) originating from 20 populations across its natural range and grown in a common garden near the range center. Feeding by specialist Danaus plexippus (monarch) larvae induced VOCs, and field environmental conditions (temperature, light, and humidity) also influenced emissions. Monarch damage increased plant VOC concentrations and altered VOC blends. We found that genetically-based induced VOC emissions varied with the latitude of plant population origin, although the pattern followed the reverse of that predicted-induced VOC concentration increased with increasing latitude. This pattern appeared to be driven by a greater induction of sesquiterpenoids at higher latitudes. In contrast, constitutive VOC emission did not vary systematically with latitude, and the induction of green leafy volatiles declined with latitude. Our results do not support the prevailing view that plant defense is greater at lower than at higher latitudes. That the pattern holds only for herbivore-induced VOC emission, and not constitutive emission, suggests that latitudinal variation in VOCs is not a simple adaptive response to climatic factors.

Attenuation of the jasmonate burst, plant defensive traits, and resistance to specialist monarch caterpillars on shaded common milkweed (Asclepias syriaca).

Plant responses to herbivory and light competition are often in opposing directions, posing a potential conflict for plants experiencing both stresses. For sun-adapted species, growing in shade typically makes plants more constitutively susceptible to herbivores via reduced structural and chemical resistance traits. Nonetheless, the impact of light environment on induced resistance has been less well-studied, especially in field experiments that link physiological mechanisms to ecological outcomes. Accordingly, we studied induced resistance of common milkweed (Asclepias syriaca, a sun-adapted plant), and linked hormonal responses, resistance traits, and performance of specialist monarch caterpillars (Danaus plexippus) in varying light environments. In natural populations, plants growing under forest-edge shade showed reduced levels of resistance traits (lower leaf toughness, cardenolides, and trichomes) and enhanced light-capture traits (higher specific leaf area, larger leaves, and lower carbon-to-nitrogen ratio) compared to paired plants in full sun. In a field experiment repeated over two years, only milkweeds growing in full sun exhibited induced resistance to monarchs, whereas plants growing in shade were constitutively more susceptible and did not induce resistance. In a more controlled field experiment, plant hormones were higher in the sun (jasmonic acid, salicylic acid, abscisic acid, indole acidic acid) and were induced by herbivory (jasmonic acid and abscisic acid). In particular, the jasmonate burst following herbivory was halved in plants raised in shaded habitats, and this correspondingly reduced latex induction (but not cardenolide induction). Thus, we provide a mechanistic basis for the attenuation of induced plant resistance in low resource environments. Additionally, there appears to be specificity in these interactions, with light-mediated impacts on jasmonate-induction being stronger for latex exudation than cardenolides.

Latitudinal patterns in plant defense: evolution of cardenolides, their toxicity and induction following herbivory.

Attempts over the past 50 years to explain variation in the abundance, distribution and diversity of plant secondary compounds gave rise to theories of plant defense. Remarkably, few phylogenetically robust tests of these long-standing theories have been conducted. Using >50 species of milkweed (Asclepias spp.), we show that variation among plant species in the induction of toxic cardenolides is explained by latitude, with higher inducibility evolving more frequently at lower latitudes. We also found that: (1) the production of cardenolides showed positive-correlated evolution with the diversity of cardenolides, (2) greater cardenolide investment by a species is accompanied by an increase in an estimate of toxicity (measured as chemical polarity) and (3) instead of trading off, constitutive and induced cardenolides were positively correlated. Analyses of root and shoot cardenolides showed concordant patterns. Thus, milkweed species from lower latitudes are better defended with higher inducibility, greater diversity and added toxicity of cardenolides.