Floral function: effects of traits on pollinators, male and female pollination success, and female fitness across three species of milkweeds (Asclepias).

PREMISE OF THE STUDY: Central questions in plant reproductive ecology are whether the functions of floral traits in hermaphrodites create conflict between sexes that could slow evolution, and whether individual floral traits function in pollinator attraction, efficiency, or both. We studied how floral traits affect pollinator visitation and efficiency, and how they affect male and female function and female fitness within and across three Asclepias species that differ in floral morphology. METHODS: Using separate multiple regressions, we regressed pollen removal, deposition, and fruit number onto six floral traits. We also used path analyses integrating these variables with pollinator visitation data for two of the species to further explore floral function and its effects on fruit production. KEY RESULTS: Most traits affected male pollination success only, and these effects often differed between species. The exception was increased slit length, which increased pollinia insertion in two of the species. There were no interspecific differences in the effects of the traits on female pollination success. All traits except horn reach affected pollination efficiency in at least one species, and horn reach and two hood dimensions were the only traits to affect pollinator attraction, but in just one species. CONCLUSIONS: Traits tended to function in only one sex, and more traits affected function through pollinator efficiency than through attraction. There was no significant link between female pollination success and female fitness in any of the three species; this pattern is consistent with fruit production not being limited by pollen deposition.

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.

Navigating the tip of the genomic iceberg: Next-generation sequencing for plant systematics.

PREMISE OF THE STUDY: Just as Sanger sequencing did more than 20 years ago, next-generation sequencing (NGS) is poised to revolutionize plant systematics. By combining multiplexing approaches with NGS throughput, systematists may no longer need to choose between more taxa or more characters. Here we describe a genome skimming (shallow sequencing) approach for plant systematics. METHODS: Through simulations, we evaluated optimal sequencing depth and performance of single-end and paired-end short read sequences for assembly of nuclear ribosomal DNA (rDNA) and plastomes and addressed the effect of divergence on reference-guided plastome assembly. We also used simulations to identify potential phylogenetic markers from low-copy nuclear loci at different sequencing depths. We demonstrated the utility of genome skimming through phylogenetic analysis of the Sonoran Desert clade (SDC) of Asclepias (Apocynaceae). KEY RESULTS: Paired-end reads performed better than single-end reads. Minimum sequencing depths for high quality rDNA and plastome assemblies were 40× and 30×, respectively. Divergence from the reference significantly affected plastome assembly, but relatively similar references are available for most seed plants. Deeper rDNA sequencing is necessary to characterize intragenomic polymorphism. The low-copy fraction of the nuclear genome was readily surveyed, even at low sequencing depths. Nearly 160000 bp of sequence from three organelles provided evidence of phylogenetic incongruence in the SDC. CONCLUSIONS: Adoption of NGS will facilitate progress in plant systematics, as whole plastome and rDNA cistrons, partial mitochondrial genomes, and low-copy nuclear markers can now be efficiently obtained for molecular phylogenetics studies.

Evolution of specialization: a phylogenetic study of host range in the red milkweed beetle (Tetraopes tetraophthalmus).

Specialization is common in most lineages of insect herbivores, one of the most diverse groups of organisms on earth. To address how and why specialization is maintained over evolutionary time, we hypothesized that plant defense and other ecological attributes of potential host plants would predict the performance of a specialist root-feeding herbivore (the red milkweed beetle, Tetraopes tetraophthalmus). Using a comparative phylogenetic and functional trait approach, we assessed the determinants of insect host range across 18 species of Asclepias. Larval survivorship decreased with increasing phylogenetic distance from the true host, Asclepias syriaca, suggesting that adaptation to plant traits drives specialization. Among several root traits measured, only cardenolides (toxic defense chemicals) correlated with larval survival, and cardenolides also explained the phylogenetic distance effect in phylogenetically controlled multiple regression analyses. Additionally, milkweed species having a known association with other Tetraopes beetles were better hosts than species lacking Tetraopes herbivores, and milkweeds with specific leaf area values (a trait related to leaf function and habitat affiliation) similar to those of A. syriaca were better hosts than species having divergent values. We thus conclude that phylogenetic distance is an integrated measure of phenotypic and ecological attributes of Asclepias species, especially defensive cardenolides, which can be used to explain specialization and constraints on host shifts over evolutionary time.

Host plant species affects virulence in monarch butterfly parasites.

1. Studies have considered how intrinsic host and parasite properties determine parasite virulence, but have largely ignored the role of extrinsic ecological factors in its expression. 2. We studied how parasite genotype and host plant species interact to determine virulence of the protozoan parasite Ophryocystis elektroscirrha (McLaughlin & Myers 1970) in the monarch butterfly Danaus plexippus L. We infected monarch larvae with one of four parasite genotypes and reared them on two milkweed species that differed in their levels of cardenolides: toxic chemicals involved in predator defence. 3. Parasite infection, replication and virulence were affected strongly by host plant species. While uninfected monarchs lived equally long on both plant species, infected monarchs suffered a greater reduction in their life spans (55% vs. 30%) on the low-cardenolide vs. the high-cardenolide host plant. These life span differences resulted from different levels of parasite replication in monarchs reared on the two plant species. 4. The virulence rank order of parasite genotypes was unaffected by host plant species, suggesting that host plant species affected parasite genotypes similarly, rather than through complex plant species-parasite genotype interactions. 5. Our results demonstrate that host ecology importantly affects parasite virulence, with implications for host-parasite dynamics in natural populations.

Herbicidal effects on nontarget vegetation: investigating the limitations of current pesticide registration guidelines.

The impact of herbicide exposure on nontarget vegetation within agroecosystems has sparked extensive research that revealed that current pesticide registration guidelines may be inadequate at predicting the effects of herbicides on wild plants and habitats. This study extends the current interest by presenting three experiments highlighting some of the limitations to current phytotoxicity testing guidelines. Several crops and wild plant species were grown under greenhouse conditions following standard protocol for phytotoxicity testing. Plants were sprayed with five different herbicides at the four- to six-leaf stage, and biomass was recorded at 28 d after spray. Results showed that current regulatory protocol will likely underestimate herbicide phytotoxicity if testing does not include data for the complete tank-mix formulation. The present study also showed that the range in herbicide sensitivity among cultivars of the same crop can be quite extensive and that, depending on the cultivar included in a risk assessment, conclusions regarding the phytotoxicity of any given herbicide may differ. Although no significant differences in sensitivity were found between crops and related wild species, results revealed that current guidelines are too rigid in terms of species selection. Considering the variability among crop cultivars, coupled with the ecological importance and the ease of germination of many noncrop plant species, pesticide regulatory guidelines would be improved if wild species were included in testing. Findings of the present study indicate that current pesticide regulatory guidelines require modifications to ensure a more accurate assessment of herbicide effects on nontarget plant species.

Plant defense syndromes.

Given that a plant's defensive strategy against herbivory is never likely to be a single trait, we develop the concept of plant defense syndromes, where association with specific ecological interactions can result in convergence on suites of covarying defensive traits. Defense syndromes can be studied within communities of diverse plant species as well as within clades of closely related species. In either case, theory predicts that plant defense traits can consistently covary across species, due to shared evolutionary ancestry or due to adaptive convergence. We examined potential defense syndromes in 24 species of milkweeds (Asclepias spp.) in a field experiment. Employing phylogenetically independent contrasts, we found few correlations between seven defensive traits, no bivariate trade-offs, and notable positive correlations between trichome density and latex production, and between C:N ratio and leaf toughness. We then used a hierarchical cluster analysis to produce a phenogram of defense trait similarity among the 24 species. This analysis revealed three distinct clusters of species. The defense syndromes of these species clusters are associated with either low nutritional quality or a balance of higher nutritional quality coupled with physical or chemical defenses. The phenogram based on defense traits was not congruent, however, with a molecular phylogeny of the group, suggesting convergence on defense syndromes. Finally, we examined the performance of monarch butterfly caterpillars on the 24 milkweed species in the field; monarch growth and survival did not differ on plants in the three syndromes, although multiple regression revealed that leaf trichomes and toughness significantly reduced caterpillar growth. The discovery of convergent plant defense syndromes can be used as a framework to ask questions about how abiotic environments, communities of herbivores, and biogeography are associated with particular defense strategies of plants.

Density-dependent reduction and induction of milkweed cardenolides by a sucking insect herbivore.

The effect of aphid population size on host-plant chemical defense expression and the effect of plant defense on aphid population dynamics were investigated in a milkweed-specialist herbivore system. Density effects of the aposematic oleander aphid, Aphis nerii, on cardenolide expression were measured in two milkweed species, Asclepias curassavica and A. incarnata. These plants vary in constitutive chemical investment with high mean cardenolide concentration in A. curassavica and low to zero in A. incarnata. The second objective was to determine whether cardenolide expression in these two host plants impacts mean A. nerii colony biomass (mg) and density. Cardenolide concentration (microgram/g) of A. curassavica in both aphid-treated leaves and opposite, herbivore-free leaves decreased initially in comparison with aphid-free controls, and then increased significantly with A. nerii density. Thus, A. curassavica responds to aphid herbivory initially with density-dependent phytochemical reduction, followed by induction of cardenolides to concentrations above aphid-free controls. In addition, mean cardenolide concentration of aphid-treated leaves was significantly higher than that of opposite, herbivore-free leaves. Therefore, A. curassavica induction is strongest in herbivore-damage tissue. Conversely, A. incarnata exhibited no such chemical response to aphid herbivory. Furthermore, neither host plant responded chemically to herbivore feeding duration time (days) or to the interaction between herbivore initial density and feeding duration time. There were also no significant differences in mean colony biomass or population density of A. nerii reared on high cardenolide (A. curassavica) and low cardenolide (A. incarnata) hosts.