Amy P. Hastings

Insect Herbivores Drive Real-Time Ecological and Evolutionary Change in Plant Populations

Plant Anti-Insect Armaments Because individual plants are unable to relocate, they are subject to extreme selection by the insects feeding upon them. One means by which plants suppress herbivory is to produce toxic compounds to deter feeding (see the Perspective by Hare). Agrawal et al. (p. 113) compared pesticide–treated or untreated evening primroses. Over 5 years of pesticide treatment, the production of defensive chemicals in the fruit reduced and flowering times shifted, and the primroses competitive ability against dandelions improved. Züst et al. (p. 116) examined large-scale geographic patterns in a polymorphic chemical defense locus in the model plant Arabidopsis thaliana and found that it is matched by changes in the relative abundance of two specialist aphids. Thus, herbivory has strong and immediate effects on the local genotypic composition of plants and traits associated with herbivore resistance. Protecting the common evening primrose from being eaten by insects alters its phenotype in only five growing seasons. Insect herbivores are hypothesized to be major factors affecting the ecology and evolution of plants. We tested this prediction by suppressing insects in replicated field populations of a native plant, Oenothera biennis, which reduced seed predation, altered interspecific competitive dynamics, and resulted in rapid evolutionary divergence. Comparative genotyping and phenotyping of nearly 12,000 O. biennis individuals revealed that in plots protected from insects, resistance to herbivores declined through time owing to changes in flowering time and lower defensive ellagitannins in fruits, whereas plant competitive ability increased. This independent real-time evolution of plant resistance and competitive ability in the field resulted from the relaxation of direct selective effects of insects on plant defense and through indirect effects due to reduced herbivory on plant competitors.

Evidence for adaptive radiation from a phylogenetic study of plant defenses

One signature of adaptive radiation is a high level of trait change early during the diversification process and a plateau toward the end of the radiation. Although the study of the tempo of evolution has historically been the domain of paleontologists, recently developed phylogenetic tools allow for the rigorous examination of trait evolution in a tremendous diversity of organisms. Enemy-driven adaptive radiation was a key prediction of Ehrlich and Ravens coevolutionary hypothesis [Ehrlich PR, Raven PH (1964) Evolution 18:586–608], yet has remained largely untested. Here we examine patterns of trait evolution in 51 North American milkweed species (Asclepias), using maximum likelihood methods. We study 7 traits of the milkweeds, ranging from seed size and foliar physiological traits to defense traits (cardenolides, latex, and trichomes) previously shown to impact herbivores, including the monarch butterfly. We compare the fit of simple random-walk models of trait evolution to models that incorporate stabilizing selection (Ornstein-Ulenbeck process), as well as time-varying rates of trait evolution. Early bursts of trait evolution were implicated for 2 traits, while stabilizing selection was implicated for several others. We further modeled the relationship between trait change and species diversification while allowing rates of trait evolution to vary during the radiation. Species-rich lineages underwent a proportionately greater decline in latex and cardenolides relative to species-poor lineages, and the rate of trait change was most rapid early in the radiation. An interpretation of this result is that reduced investment in defensive traits accelerated diversification, and disproportionately so, early in the adaptive radiation of milkweeds.

A Field Experiment Demonstrating Plant Life-History Evolution and Its Eco-Evolutionary Feedback to Seed Predator Populations

The extent to which evolutionary change occurs in a predictable manner under field conditions and how evolutionary changes feed back to influence ecological dynamics are fundamental, yet unresolved, questions. To address these issues, we established eight replicate populations of native common evening primrose (Oenothera biennis). Each population was planted with 18 genotypes in identical frequency. By tracking genotype frequencies with microsatellite DNA markers over the subsequent three years (up to three generations, ≈5,000 genotyped plants), we show rapid and consistent evolution of two heritable plant life-history traits (shorter life span and later flowering time). This rapid evolution was only partially the result of differential seed production; genotypic variation in seed germination also contributed to the observed evolutionary response. Since evening primrose genotypes exhibited heritable variation for resistance to insect herbivores, which was related to flowering time, we predicted that evolutionary changes in genotype frequencies would feed back to influence populations of a seed predator moth that specializes on O. biennis. By the conclusion of the experiment, variation in the genotypic composition among our eight replicate field populations was highly predictive of moth abundance. These results demonstrate how rapid evolution in field populations of a native plant can influence ecological interactions.

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.

Rapid evolution of plethodontid modulating factor, a hypervariable salamander courtship pheromone, is driven by positive selection.

Sexual communication in plethodontid salamanders is mediated by a proteinaceous pheromone that a male delivers to a female during courtship, boosting her receptivity. The pheromone consists of three proteins from three unrelated protein families. These proteins are among a small group of pheromones known to affect female receptivity in vertebrates. Previously, we showed that the genes of two of these proteins (PRF and SPF) are prone to incessant evolution driven by positive selection, presumably as a consequence of coevolution with female receptors. In this report, we focus on the evolution of the third pheromone protein gene family, plethodontid modulating factor (PMF), to determine whether it shows the same pattern of diversification. We used RT-PCR in mental gland cDNA to survey PMF sequences from three genera of plethodontid salamanders (27 spp.) to measure rates of evolution, level of gene diversification, modes of selection, and types of amino acid substitution. Like PRF and SPF, PMF is produced by a multigene family characterized by gene duplication and high levels of polymorphism. PMF evolution is rapid, incessant, and driven by positive selection. PMF is more extreme in these dimensions than both PRF and SPF. Nestled within this extraordinary variation, however, is a signature of purifying selection, acting to preserve important structural and biochemical features of the PMF protein (i.e., secretion signal, cysteine residues, and pI). Although a pattern of persistent diversification exists at the molecular level, the morphological and behavioral aspects of the pheromone delivery system show evolutionary stasis over millions of years.

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.

Evolution of Plant Growth and Defense in a Continental Introduction

Substantial research has addressed adaptation of nonnative biota to novel environments, yet surprisingly little work has integrated population genetic structure and the mechanisms underlying phenotypic differentiation in ecologically important traits. We report on studies of the common milkweed Asclepias syriaca, which was introduced from North America to Europe over the past 400 years and which lacks most of its specialized herbivores in the introduced range. Using 10 populations from each continent grown in a common environment, we identified several growth and defense traits that have diverged, despite low neutral genetic differentiation between continents. We next developed a Bayesian modeling approach to account for relationships between molecular and phenotypic differences, confirming that continental trait differentiation was greater than expected from neutral genetic differentiation. We found evidence that growth-related traits adaptively diverged within and between continents. Inducible defenses triggered by monarch butterfly herbivory were substantially reduced in European populations, and this reduction in inducibility was concordant with altered phytohormonal dynamics, reduced plant growth, and a trade-off with constitutive investment. Freedom from the community of native and specialized herbivores may have favored constitutive over induced defense. Our replicated analysis of plant growth and defense, including phenotypically plastic traits, suggests adaptive evolution following a continental introduction.

The importance of plant genotype and contemporary evolution for terrestrial ecosystem processes

Plant genetic variation and evolutionary dynamics are predicted to impact ecosystem processes but these effects are poorly understood. Here we test the hypothesis that plant genotype and contemporary evolution influence the flux of energy and nutrients through soil, which then feedback to affect seedling performance in subsequent generations. We conducted a multiyear field evolution experiment using the native biennial plant Oenothera biennis. This experiment was coupled with experimental assays to address our hypothesis and quantify the relative importance of evolutionary and ecological factors on multiple ecosystem processes. Plant genotype, contemporary evolution, spatial variation, and herbivory affected ecosystem processes (e.g., leaf decay, soil respiration, seedling performance, N cycling), but their relative importance varied between specific ecosystem variables. Insect herbivory and evolution also contributed to a feedback that affected seedling biomass of O. biennis in the next generation. Our results show that heritable variation among plant genotypes can be an important factor affecting local ecosystem processes, and while effects of contemporary evolution were detectable and sometimes strong, they were often contingent on other ecological, factors.

Tests of the coupled expression of latex and cardenolide plant defense in common milkweed (Asclepias syriaca)

The coexpression of plant resistance traits suggests the hypothesis that they may have complementary functions in defense against herbivory. To address the extent to which defensive traits are necessarily coupled in plants grown under various conditions, we focused on latex and cardenolides, two potent defenses of milkweeds. We measured defenses across ontogenetic stages, different biotic and abiotic environments, and across genetic families of the common milkweed Asclepias syriaca. We first addressed the extent to which foliar cardenolides are derived from latex because latex actively flows through canals in leaves. We rinsed latex out of shredded leaves, which had no impact on foliar cardenolides, suggesting cardenolides are allocated to leaves independently of latex. Accordingly, there is potential for independent expression of the two traits. We next followed a cohort of plants from germination over three years; expression of both latex exudation and cardenolides increased annually, with the exception of a second year dip in cardenolides. Damage by monarch caterpillars induced ≈50% increases of both latex and cardenolides, with the former occurring rapidly within a day and the latter taking five days of herbivory; these responses were preceded by an earlier peak of the signaling hormones jasmonic acid and abscisic acid. Endogenous jasmonic acid showed an instantaneous positive correlation with latex exudation and foliar cardenolides. Under drought stress, latex and cardenolide expression were reversed, with water stress suppressing latex exudation, but nearly doubling cardenolide concentrations. These drought effects were not driven by phytohormones in the expected manner, as jasmonic acid was unaffected, salicylic acid was strongly suppressed, and abscisic acid tripled in response to drought. Finally, a meta-analysis of four previously published field studies representing 85 genetic families of A. syriaca revealed no evidence for a genetic correlation between latex exudation and foliar cardenolide concentrations. The same lack of a correlation was observed across 22 populations of A. syriaca when grown in a common environment. Thus, the two most important defensive traits of milkweeds, although often coexpressed, can become uncoupled during some ontogenetic stages, under some biotic and abiotic conditions, and there is no evidence that they evolve together.

Phylogeny of the plant genus Pachypodium (Apocynaceae)

Background. The genus Pachypodium contains 21 species of succulent, generally spinescent shrubs and trees found in southern Africa and Madagascar. Pachypodium has diversified mostly into arid and semi-arid habitats of Madagascar, and has been cited as an example of a plant group that links the highly diverse arid-adapted floras of Africa and Madagascar. However, a lack of knowledge about phylogenetic relationships within the genus has prevented testing of this and other hypotheses about the group. Methodology/Principal Findings. We use DNA sequence data from the nuclear ribosomal ITS and chloroplast trnL-F region for all 21 Pachypodium species to reconstruct evolutionary relationships within the genus. We compare phylogenetic results to previous taxonomic classifications and geography. Results support three infrageneric taxa from the most recent classification of Pachypodium, and suggest that a group of African species (P. namaquanum, P. succulentum and P. bispinosum) may deserve taxonomic recognition as an infrageneric taxon. However, our results do not resolve relationships among major African and Malagasy lineages of the genus. Conclusions/Significance. We present the first molecular phylogenetic analysis of Pachypodium. Our work has revealed five distinct lineages, most of which correspond to groups recognized in past taxonomic classifications. Our work also suggests that there is a complex biogeographic relationship between Pachypodium of Africa and Madagascar.