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.

Defense mutualisms enhance plant diversification.

The ability of plants to form mutualistic relationships with animal defenders has long been suspected to influence their evolutionary success, both by decreasing extinction risk and by increasing opportunity for speciation through an expanded realized niche. Nonetheless, the hypothesis that defense mutualisms consistently enhance plant diversification across lineages has not been well tested due to a lack of phenotypic and phylogenetic information. Using a global analysis, we show that the >100 vascular plant families in which species have evolved extrafloral nectaries (EFNs), sugar-secreting organs that recruit arthropod mutualists, have twofold higher diversification rates than families that lack species with EFNs. Zooming in on six distantly related plant clades, trait-dependent diversification models confirmed the tendency for lineages with EFNs to display increased rates of diversification. These results were consistent across methodological approaches. Inference using reversible-jump Markov chain Monte Carlo (MCMC) to model the placement and number of rate shifts revealed that high net diversification rates in EFN clades were driven by an increased number of positive rate shifts following EFN evolution compared with sister clades, suggesting that EFNs may be indirect facilitators of diversification. Our replicated analysis indicates that defense mutualisms put lineages on a path toward increased diversification rates within and between clades, and is concordant with the hypothesis that mutualistic interactions with animals can have an impact on deep macroevolutionary patterns and enhance plant diversity.

Re-evaluating the costs and limits of adaptive phenotypic plasticity.

When the optimal phenotype differs among environments, adaptive phenotypic plasticity can evolve unless constraints impede such evolution. Costs and limits of plasticity have been proposed as important constraints on the evolution of plasticity, yet confusion exists over their distinction. We attempt to clarify these concepts by reviewing their categorization and measurement, highlighting how costs and limits are defined in different currencies (and may describe the same phenomenon). Conclusions from studies that measure the costs of plasticity have been equivocal, but we caution that these conclusions may be premature owing to a potentially common correlation between environment-specific trait values and the magnitude of trait plasticities (i.e. multi-collinearity) that results in imprecise and/or biased estimates of the costs. Meanwhile, our understanding of the limits of plasticity, and how they may be underlain by the costs of plasticity, is still in its infancy. Based on our re-evaluation of these constraints, we discuss areas for future research.

Ecological genetics of an induced plant defense against herbivores: additive genetic variance and costs of phenotypic plasticity.

Adaptive phenotypic plasticity in chemical defense is thought to play a major role in plant-herbivore interactions. We investigated genetic variation for inducibility of defensive traits in wild radish plants and asked if the evolution of induction is constrained by costs of phenotypic plasticity. In a greenhouse experiment using paternal half-sibling families, we show additive genetic variation for plasticity in glucosinolate concentration. Genetic variation for glucosinolates was not detected in undamaged plants, but was significant following herbivory by a specialist herbivore, Pieris rapae. On average, damaged plants had 55% higher concentrations of glucosinolates compared to controls. In addition, we found significant narrow-sense heritabilities for leaf size, trichome number, flowering phenology, and lifetime fruit production. In a second experiment, we found evidence of genetic variation in induced plant resistance to P. rapae. Although overall there was little evidence for genetic correlations between the defensive and life-history traits we measured, we show that more plastic families had lower fitness than less plastic families in the absence of herbivory (i.e., evidence for genetic costs of plasticity). Thus, there is genetic variation for induction of defense in wild radish, and the evolution of inducibility may be constrained by costs of plasticity.

COSTS OF INDUCED RESPONSES AND TOLERANCE TO HERBIVORY IN MALE AND FEMALE FITNESS COMPONENTS OF WILD RADISH.

Theory predicts that plant defensive traits are costly due to trade-offs between allocation to defense and growth and reproduction. Most previous studies of costs of plant defense focused on female fitness costs of constitutively expressed defenses. Consideration of alternative plant strategies, such as induced defenses and tolerance to herbivory, and multiple types of costs, including allocation to male reproductive function, may increase our ability to detect costs of plant defense against herbivores. In this study we measured male and female reproductive costs associated with induced responses and tolerance to herbivory in annual wild radish plants (Raphanus raphanistrum). We induced resistance in the plants by subjecting them to herbivory by Pieris rapae caterpillars. We also induced resistance in plants without leaf tissue removal using a natural chemical elicitor, jasmonic acid; in addition, we removed leaf tissue without inducing plant responses using manual clipping. Induced responses included increased concentrations of indole glucosinolates, which are putative defense compounds. Induced responses, in the absence of leaf tissue removal, reduced plant fitness when five fitness components were considered together; costs of induction were individually detected for time to first flower and number of pollen grains produced per flower. In this system, induced responses appear to impose a cost, although this cost may not have been detected had we only quantified the traditionally measured fitness components, growth and seed production. In the absence of induced responses, 50% leaf tissue removal, reduced plant fitness in three out of the five fitness components measured. Induced responses to herbivory and leaf tissue removal had additive effects on plant fitness. Although plant sibships varied greatly (49-136%) in their level of tolerance to herbivory, costs of tolerance were not detected, as we did not find a negative association between the ability to compensate for damage and plant fitness in the absence of damage. We suggest that consideration of alternative plant defense strategies and multiple costs will result in a broader understanding of the evolutionary ecology of plant defense.