3D-surface MALDI mass spectrometry imaging for visualising plant defensive cardiac glycosides in Asclepias curassavica.

Mass spectrometry-based imaging (MSI) has emerged as a promising method for spatial metabolomics in plant science. Several ionisation techniques have shown great potential for the spatially resolved analysis of metabolites in plant tissue. However, limitations in technology and methodology limited the molecular information for irregular 3D surfaces with resolutions on the micrometre scale. Here, we used atmospheric-pressure 3D-surface matrix-assisted laser desorption/ionisation mass spectrometry imaging (3D-surface MALDI MSI) to investigate plant chemical defence at the topographic molecular level for the model system Asclepias curassavica. Upon mechanical damage (simulating herbivore attacks) of native A. curassavica leaves, the surface of the leaves varies up to 700 µm, and cardiac glycosides (cardenolides) and other defence metabolites were exclusively detected in damaged leaf tissue but not in different regions of the same leaf. Our results indicated an increased latex flow rate towards the point of damage leading to an accumulation of defence substances in the affected area. While the concentration of cardiac glycosides showed no differences between 10 and 300 min after wounding, cardiac glycosides decreased after 24 h. The employed autofocusing AP-SMALDI MSI system provides a significant technological advancement for the visualisation of individual molecule species on irregular 3D surfaces such as native plant leaves. Our study demonstrates the enormous potential of this method in the field of plant science including primary metabolism and molecular mechanisms of plant responses to abiotic and biotic stress and symbiotic relationships.

Invasive paper wasp turns urban pollinator gardens into ecological traps for monarch butterfly larvae.

Invasive species can be particularly disruptive when they intersect with organisms of conservation concern. Stabilizing the declining eastern migratory population of monarch butterflies (Danaus plexippus) is projected to require extensive habitat restoration across multiple land use sectors including metropolitan areas. Numerous conservation programs encourage urban citizens to plant gardens with milkweeds, the obligate larval host plants of the monarch. Here, we show that predation by Polistes dominula, an invasive paper wasp that is particularly abundant in urban settings, can turn such sites into ecological traps for monarch larvae. Polistes dominula was the predominant paper wasp seen foraging in central Kentucky pollinator gardens. In 120 observed encounters with monarch larvae on milkweeds in gardens, most second to fourth instars were killed, whereas most fifth instars escaped by thrashing or dropping. The wasps bit and carried off second instars whole, whereas third and fourth instar kills were first gutted, then processed and carried away piecemeal. Predation on sentinel larvae was much higher in urban gardens than in rural settings. The wasps exploited ornamental butterfly "hibernation boxes" in pollinator gardens as nesting habitat. Polistes dominula is an under-recognized predator that may diminish the urban sector's contributions to monarch habitat restoration.

The demographic effects of functional traits: an integral projection model approach reveals population-level consequences of reproduction-defence trade-offs.

Quantitatively linking individual variation in functional traits to demography is a necessary step to advance our understanding of trait-based ecological processes. We constructed a population model for Asclepias syriaca to identify how functional traits affect vital rates and population growth and whether trade-offs in chemical defence and demography alter population growth. Plants with higher foliar cardenolides had lower fibre, cellulose and lignin levels, as well as decreased sexual and clonal reproduction. Average cardenolide concentrations had the strongest effect on population growth. In both the sexual and clonal pathway, the trade-off between reproduction and defence affected population growth. We found that both increasing the mean of the distribution of individual plant values for cardenolides and herbivory decreased population growth. However, increasing the variance in both defence and herbivory increased population growth. Functional traits can impact population growth and quantifying individual-level variation in traits should be included in assessments of population-level processes.

Germination niche breadth varies inconsistently among three Asclepias congeners along a latitudinal gradient.

Species responses to climate change will be primarily driven by their environmental tolerance range, or niche breadth, with the expectation that broad niches will increase resilience. Niche breadth is expected to be larger in more heterogeneous environments and moderated by life history. Niche breadth also varies across life stages. Therefore, the life stage with the narrowest niche may serve as the best predictor of climatic vulnerability. To investigate the relationship between niche breadth, climate and life stage we identify germination niche breadth for dormant and non-dormant seeds in multiple populations of three milkweed (Asclepias) species. Complementary trials evaluated germination under conditions simulating historic and predicted future climate by varying cold-moist stratification temperature, length and incubation temperature. Germination niche breadth was derived from germination evenness across treatments (Levins Bn ), with stratified seeds considered less dormant than non-stratified seeds. Germination response varies significantly among species, populations and treatments. Cold-moist stratification ≥4 weeks (1-3 °C) followed by incubation at 25/15 °C+ achieves peak germination for most populations. Germination niche breadth significantly expands following stratification and interacts significantly with latitude of origin. Interestingly, two species display a positive relationship between niche breadth and latitude, while the third presents a concave quadratic relationship. Germination niche breadth significantly varies by species, latitude and population, suggesting an interaction between source climate, life history and site-specific factors. Results contribute to our understanding of inter- and intraspecific variation in germination, underscore the role of dormancy in germination niche breadth, and have implications for prioritising and conserving species under climate change.

Toxicity of Milkweed Leaves and Latex: Chromatographic Quantification Versus Biological Activity of Cardenolides in 16 Asclepias Species.

Cardenolides are classically studied steroidal defenses in chemical ecology and plant-herbivore coevolution. Although milkweed plants (Asclepias spp.) produce up to 200 structurally different cardenolides, all compounds seemingly share the same well-characterized mode of action, inhibition of the ubiquitous Na+/K+ ATPase in animal cells. Over their evolutionary radiation, milkweeds show a quantitative decline of cardenolide production and diversity. This reduction is contrary to coevolutionary predictions and could represent a cost-saving strategy, i.e. production of fewer but more toxic cardenolides. Here we test this hypothesis by tandem cardenolide quantification using HPLC (UV absorption of the unsaturated lactone) and a pharmacological assay (in vitro inhibition of a sensitive Na+/K+ ATPase) in a comparative study of 16 species of Asclepias. We contrast cardenolide concentrations in leaf tissue to the subset of cardenolides present in exuding latex. Results from the two quantification methods were strongly correlated, but the enzymatic assay revealed that milkweed cardenolide mixtures often cause stronger inhibition than equal amounts of a non-milkweed reference cardenolide, ouabain. Cardenolide concentrations in latex and leaves were positively correlated across species, yet latex caused 27% stronger enzyme inhibition than equimolar amounts of leaf cardenolides. Using a novel multiple regression approach, we found three highly potent cardenolides (identified as calactin, calotropin, and voruscharin) to be primarily responsible for the increased pharmacological activity of milkweed cardenolide mixtures. However, contrary to an expected trade-off between concentration and toxicity, later-diverging milkweeds had the lowest amounts of these potent cardenolides, perhaps indicating an evolutionary response to milkweed's diverse community of specialist cardenolide-sequestering insect herbivores.

Asclepias Syriaca (Common Milkweed) flowering date shift in response to climate change.

The consequences of altered flowering dates due to climate change can be severe, especially for plants that rely on coordinated flower and pollinator emergence for reproduction. The plant Asclepias syriaca (Common Milkweed) relies on pollinators for movement of its pollen and evidence suggests that it has recently been declining. Given these factors and this plant's importance as a host species for the declining Danaus plexippus (Monarch Butterfly), it is critical to determine if its flowering is being modified by climate change. As a first step to answering this question I quantified the relationship between climate and flowering date for A. syriaca using data from the USA National Phenology Network repository and the National Oceanic and Atmospheric Administration. I found that temperatures were higher than they had been historically (1895-2010) and mean flowering dates occurred earlier with higher temperatures. Additionally, there is a significant negative interactive effect of temperature and year on flowering date indicating that from 2011 through 2016 higher temperatures are correlated with increasingly earlier flowering dates. The change in flowering appears to be symmetrical in regards to the flowering time distribution, in that along with the mean, both maximum and minimum flowering dates are occurring earlier, as well. There is no evidence that earlier flowering is due to earlier initial growth or results in later fruit ripening. Consequences of this shift in flowering can only be speculated upon at this point, but due to the ecological importance of A. syriaca and its susceptibility to phenological mismatch, they should be considered when developing conservation plans for A. syriaca and the organisms for which it is a host.

Long- and short-term responses of Asclepias species differ in respect to fire, grazing, and nutrient addition.

PREMISE OF THE STUDY: The tallgrass prairie ecosystem has experienced a dramatic reduction over the past 150 yr. This reduction has impacted the abundance of native grassland species, including milkweeds (Asclepias). METHODS: We used two long-term (27 yr) data sets to examine how fire, grazing, and nutrient addition shape milkweed abundance in tallgrass prairie. We compared these results to those of a greenhouse experiment that varied nutrient levels in the absence of competition, herbivory, and mutualistic relationships. KEY RESULTS: Asclepias species exhibited broad patterns in response to burning regimes that did not include grazing, but experienced more species-specific patterns in other combinations. Asclepias syriaca was the only species to increase in abundance in plots that included burning and nutrient addition. In the greenhouse we found that nitrogen significantly increased biomass, while no effect of phosphorus was detected. CONCLUSIONS: These results indicate that A. syriaca will do best in settings with high nutrient loads, low competition, and no grazers. These characteristics define a small portion of the tallgrass prairie but exemplify modern agricultural settings, which have replaced prairies. However, other milkweeds examined did not share this pattern, which indicates that milkweed species will respond differently when exposed to agricultural settings, with some less able to cope with land conversion to pasture or row-crop agriculture.

Japanese beetles' feeding on milkweed flowers may compromise efforts to restore monarch butterfly habitat.

The eastern North American migratory population of monarch butterflies (Danaus plexippus) is in serious decline. Habitat restoration, including adding millions of host plants to compensate for loss of milkweed in US cropland, is a key part of the international conservation strategy to return this iconic butterfly to sustainable status. We report here that Popillia japonica, a polyphagous, invasive beetle, aggregates and feeds on flowers of Asclepias syriaca, the monarch's most important larval food plant, reducing fruiting and seed set by >90% and extensively damaging milkweed umbels in the field. The beetle's ongoing incursion into the monarch's key breeding grounds in the US Midwest is likely to limit pollination and outcrossing of wild and planted milkweeds, reducing their capacity to colonize new areas via seeds. Popillia japonica represents a previously undocumented threat to milkweeds that should be considered in models for monarch habitat restoration.

Trait differences in responses to arbuscular mycorrhizal fungi are stronger and more consistent than fixed differences among populations of Asclepias speciosa.

PREMISE OF THE STUDY: Arbuscular mycorrhizal (AM) fungi can promote plant growth and reproduction, but other plant physiological traits or traits that provide defense against herbivores can also be affected by AM fungi. However, whether responses of different traits to AM fungi are correlated and whether these relationships vary among plants from different populations are unresolved. METHODS: In a common garden experiment, we grew Asclepias speciosa plants from seed collected from populations found along an environmental gradient with and without AM fungi to assess whether the responses of six growth and defense traits to AM fungi are correlated. KEY RESULTS: Although there was strong genetic differentiation in mean trait values among populations, AM fungi consistently increased expression of most growth and defense traits across all populations. Responses of biomass and root to shoot ratio to AM fungi were positively correlated, suggesting that plants that are more responsive to AM fungi allocated more biomass belowground. Responses of biomass and trichome density to AM fungi were negatively correlated, indicating a trade-off in responsiveness between a growth and defensive trait. CONCLUSIONS: Our results suggest that while there is substantial population differentiation in many traits of A. speciosa, populations respond similarly to AM fungi, and both positive and negative correlations among trait responses occur.

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.

Fitness costs of animal medication: antiparasitic plant chemicals reduce fitness of monarch butterfly hosts.

The emerging field of ecological immunology demonstrates that allocation by hosts to immune defence against parasites is constrained by the costs of those defences. However, the costs of non-immunological defences, which are important alternatives to canonical immune systems, are less well characterized. Estimating such costs is essential for our understanding of the ecology and evolution of alternative host defence strategies. Many animals have evolved medication behaviours, whereby they use antiparasitic compounds from their environment to protect themselves or their kin from parasitism. Documenting the costs of medication behaviours is complicated by natural variation in the medicinal components of diets and their covariance with other dietary components, such as macronutrients. In the current study, we explore the costs of the usage of antiparasitic compounds in monarch butterflies (Danaus plexippus), using natural variation in concentrations of antiparasitic compounds among plants. Upon infection by their specialist protozoan parasite Ophryocystis elektroscirrha, monarch butterflies can selectively oviposit on milkweed with high foliar concentrations of cardenolides, secondary chemicals that reduce parasite growth. Here, we show that these antiparasitic cardenolides can also impose significant costs on both uninfected and infected butterflies. Among eight milkweed species that vary substantially in their foliar cardenolide concentration and composition, we observed the opposing effects of cardenolides on monarch fitness traits. While high foliar cardenolide concentrations increased the tolerance of monarch butterflies to infection, they reduced the survival rate of caterpillars to adulthood. Additionally, although non-polar cardenolide compounds decreased the spore load of infected butterflies, they also reduced the life span of uninfected butterflies, resulting in a hump-shaped curve between cardenolide non-polarity and the life span of infected butterflies. Overall, our results suggest that the use of antiparasitic compounds carries substantial costs, which could constrain host investment in medication behaviours.

Different rates of defense evolution and niche preferences in clonal and nonclonal milkweeds (Asclepias spp.).

Given the dual role of many plant traits to tolerate both herbivore attack and abiotic stress, the climatic niche of a species should be integrated into the study of plant defense strategies. Here we investigate the impact of plant reproductive strategy and components of species' climatic niche on the rate of chemical defense evolution in the milkweeds using a common garden experiment of 49 species. We found that across Asclepias species, clonal reproduction repeatedly evolved in lower temperature conditions, in species generally producing low concentrations of a toxic defense (cardenolides). Additionally, we found that rates of cardenolide evolution were lower for clonal than for nonclonal species. We thus conclude that because the clonal strategy is based on survival, long generation times, and is associated with tolerance of herbivory, it may be an alternative to toxicity in colder ecosystems. Taken together, these results indicate that the rate of chemical defense evolution is influenced by the intersection of life-history strategy and climatic niches into which plants radiate.

Disease ecology across soil boundaries: effects of below-ground fungi on above-ground host-parasite interactions.

Host-parasite interactions are subject to strong trait-mediated indirect effects from other species. However, it remains unexplored whether such indirect effects may occur across soil boundaries and connect spatially isolated organisms. Here, we demonstrate that, by changing plant (milkweed Asclepias sp.) traits, arbuscular mycorrhizal fungi (AMF) significantly affect interactions between a herbivore (the monarch butterfly Danaus plexippus) and its protozoan parasite (Ophryocystis elektroscirrha), which represents an interaction across four biological kingdoms. In our experiment, AMF affected parasite virulence, host resistance and host tolerance to the parasite. These effects were dependent on both the density of AMF and the identity of milkweed species: AMF indirectly increased disease in monarchs reared on some species, while alleviating disease in monarchs reared on other species. The species-specificity was driven largely by the effects of AMF on both plant primary (phosphorus) and secondary (cardenolides; toxins in milkweeds) traits. Our study demonstrates that trait-mediated indirect effects in disease ecology are extensive, such that below-ground interactions between AMF and plant roots can alter host-parasite interactions above ground. In general, soil biota may play an underappreciated role in the ecology of many terrestrial host-parasite systems.

Self-pollination rate and floral-display size in Asclepias syriaca (Common Milkweed) with regard to floral-visitor taxa.

BACKGROUND: Animals fertilize thousands of angiosperm species whose floral-display sizes can significantly influence pollinator behavior and plant reproductive success. Many studies have measured the interactions among pollinator behavior, floral-display size, and plant reproductive success, but few studies have been able to separate the effects of pollinator behavior and post-pollination processes on angiosperm sexual reproduction. In this study, we utilized the highly self-incompatible pollinium-pollination system of Asclepias syriaca (Common Milkweed) to quantify how insect visitors influenced male reproductive success measured as pollen removal, female reproductive success measured as pollen deposition, and self-pollination rate. We also determined how floral-display size impacts both visitor behavior and self-pollination rate. RESULTS: Four insect taxonomic orders visited A. syriaca: Coleoptera, Diptera, Hymenoptera, and Lepidoptera. We focused on three groups of visitor taxa within two orders (Hymenoptera and Lepidoptera) with sample sizes large enough for quantitative analysis: Apis mellifera (Western Honey Bee), Bombus spp. (bumble bees) and lepidopterans (butterflies and moths). Qualitatively, lepidopterans had the highest pollinator importance values, but the large variability in the lepidopteran data precluded meaningful interpretation of much of their behavior. The introduced A. mellifera was the most effective and most important diurnal pollinator with regard to both pollen removal and pollen deposition. However, when considering the self-incompatibility of A. syriaca, A. mellifera was not the most important pollinator because of its high self-pollination rate as compared to Bombus spp. Additionally, the rate of self-pollination increased more rapidly with the number of flowers per inflorescence in A. mellifera than in the native Bombus spp. CONCLUSIONS: Apis mellifera's high rate of self-pollination may have significant negative effects on both male and female reproductive successes in A. syriaca, causing different selection on floral-display size than native pollinators.

Milkweed (Gentianales: Apocynaceae): a farmscape resource for increasing parasitism of stink bugs (Hemiptera: Pentatomidae) and providing nectar to insect pollinators and monarch butterflies.

In peanut-cotton farmscapes in Georgia, the stink bugs Nezara viridula (L.) and Chinavia hilaris (Say) (Hemiptera: Pentatomidae) and the leaffooted bug, Leptoglossus phyllopus (L.) (Hemiptera: Coreidae), disperse at crop-to-crop interfaces to feed on bolls in cotton. The main objective of this study was to determine whether insecticide-free tropical milkweed (Asclepias curassavica L.), a nectar-producing plant, can increase parasitism of these bugs by Trichopoda pennipes (F.) (Diptera: Tachinidae) and provide nectar to monarch butterflies and insect pollinators in these farmscapes. Peanut-cotton plots with and without flowering milkweed plants were established in 2009 and 2010. Adult T. pennipes, monarch butterflies, honey bees, and native insect pollinators readily fed on floral nectar of milkweed. Monarch larvae feeding on milkweed vegetation successfully developed into pupae. In 2009, N. viridula was the primary host of T. pennipes in cotton, and parasitism of this pest by the parasitoid was significantly higher in milkweed cotton (61.6%) than in control cotton (13.3%). In 2010, parasitism of N. viridula, C. hilaris, and L. phyllopus by T. pennipes was significantly higher in milkweed cotton (24.0%) than in control cotton (1.1%). For both years of the study, these treatment differences were not owing to a response by the parasitoid to differences in host density, because density of hosts was not significantly different between treatments. In conclusion, incorporation of milkweed in peanut-cotton plots increased stink bug parasitism in cotton and provided nectar to insect pollinators and monarch butterflies.

Generalised pollination systems for three invasive milkweeds in Australia.

Because most plants require pollinator visits for seed production, the ability of an introduced plant species to establish pollinator relationships in a new ecosystem may have a central role in determining its success or failure as an invader. We investigated the pollination ecology of three milkweed species - Asclepias curassavica, Gomphocarpus fruticosus and G. physocarpus - in their invaded range in southeast Queensland, Australia. The complex floral morphology of milkweeds has often been interpreted as a general trend towards specialised pollination requirements. Based on this interpretation, invasion by milkweeds contradicts the expectation than plant species with specialised pollination systems are less likely to become invasive that those with more generalised pollination requirements. However, observations of flower visitors in natural populations of the three study species revealed that their pollination systems are essentially specialised at the taxonomic level of the order, but generalised at the species level. Specifically, pollinators of the two Gomphocarpus species included various species of Hymenoptera (particularly vespid wasps), while pollinators of A. curassavica were primarily Lepidoptera (particularly nymphalid butterflies). Pollinators of all three species are rewarded with copious amounts of highly concentrated nectar. It is likely that successful invasion by these three milkweed species is attributable, at least in part, to their generalised pollinator requirements. The results of this study are discussed in terms of how data from the native range may be useful in predicting pollination success of species in a new environment.

Pollinator effectiveness varies with experimental shifts in flowering time.

The earlier flowering times exhibited by many plant species are a conspicuous sign of climate change. Altered phenologies have caused concern that species could suffer population declines if they flower at times when effective pollinators are unavailable. For two perennial wildflowers, Tradescantia ohiensis and Asclepias incarnata, we used an experimental approach to explore how changing phenology affects the taxonomic composition of the pollinator assemblage and the effectiveness of individual pollinator taxa. After finding in the previous year that fruit set varied with flowering time, we manipulated flowering onset in greenhouses, placed plants in the field over the span of five weeks, and measured pollinator effectiveness as the number of seeds produced after a single visit to a flower. The average effectiveness of pollinators and the expected rates of pollination success were lower for plants of both species flowering earlier than for plants flowering at historical times, suggesting there could be reproductive costs to earlier flowering. Whereas for A. incarnata, differences in average seed set among weeks were due primarily to changes in the composition of the pollinator assemblage, the differences for T. ohiensis were driven by the combined effects of compositional changes and increases over time in the effectiveness of some pollinator taxa. Both species face the possibility of temporal mismatch between the availability of the most effective pollinators and the onset of flowering, and changes in the effectiveness of individual pollinator taxa through time may add an unexpected element to the reproductive consequences of such mismatches.

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.