Convergent evolution of cardiac-glycoside resistance in predators and parasites of milkweed herbivores.

The community of plant-feeding insects (herbivores) that specialize on milkweeds (Apocynaceae) form a remarkable example of convergent evolution across levels of biological organization1. In response to toxic cardiac glycosides produced by these plants, the monarch butterfly (Danaus plexippus) and other specialist herbivores have evolved parallel substitutions in the alpha subunit (ATPA) of the Na+/K+-ATPase. These substitutions render the pump insensitive to cardiac glycosides2,3, allowing the monarch and other specialists, from aphids to beetles, to sequester cardiac glycosides, which in turn provide defense against attacks by enemies from the third trophic level4. The evolution of 'target-site-insensitivity' substitutions in these herbivores poses a fundamental biological question: have predators and parasitoids that feed on cardiac-glycoside-sequestering insects also evolved Na+/K+-ATPases that are similarly insensitive to cardiac glycosides (as predicted by Whiteman and Mooney)5? In other words, can plant toxins cause evolutionary cascades that reach the third trophic level? Here we show that at least four enemies of the monarch and other milkweed herbivores have indeed evolved amino-acid substitutions associated with target-site insensitivity to cardiac glycosides. These attackers represent four major animal clades, implicating cardiac glycosides as keystone molecules6 and establishing ATPalpha, which encodes ATPA, as a keystone gene with effects that reverberate within ecological communities7.

Transcriptomics of monarch butterflies (Danaus plexippus) reveals that toxic host plants alter expression of detoxification genes and down-regulate a small number of immune genes.

Herbivorous insects have evolved many mechanisms to overcome plant chemical defences, including detoxification and sequestration. Herbivores may also use toxic plants to reduce parasite infection. Plant toxins could directly interfere with parasites or could enhance endogenous immunity. Alternatively, plant toxins could favour down-regulation of endogenous immunity by providing an alternative (exogenous) defence against parasitism. However, studies on genomewide transcriptomic responses to plant defences and the interplay between plant toxicity and parasite infection remain rare. Monarch butterflies (Danaus plexippus) are specialist herbivores of milkweeds (Asclepias spp.), which contain toxic cardenolides. Monarchs have adapted to cardenolides through multiple resistance mechanisms and can sequester cardenolides to defend against bird predators. In addition, high-cardenolide milkweeds confer monarch resistance to a specialist protozoan parasite (Ophryocystis elektroscirrha). We used this system to study the interplay between the effects of plant toxicity and parasite infection on global gene expression. We compared transcriptional profiles between parasite-infected and uninfected monarch larvae reared on two milkweed species. Our results demonstrate that monarch differentially express several hundred genes when feeding on A. curassavica and A. incarnata, two species that differ substantially in cardenolide concentrations. These differentially expressed genes include genes within multiple families of canonical insect detoxification genes, suggesting that they play a role in monarch toxin resistance and sequestration. Interestingly, we found little transcriptional response to infection. However, parasite growth was reduced in monarchs reared on A. curassavica, and in these monarchs, several immune genes were down-regulated, consistent with the hypothesis that medicinal plants can reduce reliance on endogenous immunity.

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.

Rapid In Situ Analysis of Plant Emission for Disease Diagnosis Using a Portable Gas Chromatography Device.

We developed and applied a fully automated portable gas chromatography (GC) device for rapid and in situ analysis of plant volatile organic compounds (VOCs) to examine plant health status. A total of 42 emission samples were collected over a period of 5 days from 10 milkweed ( Asclepias syriaca) plants, half of which were infested by aphids. Thirty-five VOC peaks were separated and detected in 8 min. An algorithm based on machine learning, principal component analysis, and linear discriminant analysis was developed to evaluate the GC results. We found that our device and algorithm are able to distinguish between the undamaged control and the aphid-infested milkweeds with an overall accuracy of 90-100% within 48-72 h of the attack. Such rapid in situ detection of insect attack attests to the great potential of VOC monitoring in plant health management.

Growth zone segmentation in the milkweed bug Oncopeltus fasciatus sheds light on the evolution of insect segmentation.

BACKGROUND: One of the best studied developmental processes is the Drosophila segmentation cascade. However, this cascade is generally considered to be highly derived and unusual, with segments being patterned simultaneously, rather than the ancestral sequential segmentation mode. We present a detailed analysis of the segmentation cascade of the milkweed bug Oncopletus fasciatus, an insect with a more primitive segmentation mode, as a comparison to Drosophila, with the aim of reconstructing the evolution of insect segmentation modes. RESULTS: We document the expression of 12 genes, representing different phases in the segmentation process. Using double staining we reconstruct the spatio-temporal relationships among these genes. We then show knock-down phenotypes of representative genes in order to uncover their roles and position in the cascade. CONCLUSIONS: We conclude that sequential segmentation in the Oncopeltus germband includes three slightly overlapping phases: Primary pair-rule genes generate the first segmental gene expression in the anterior growth zone. This pattern is carried anteriorly by a series of secondary pair-rule genes, expressed in the transition between the growth zone and the segmented germband. Segment polarity genes are expressed in the segmented germband with conserved relationships. Unlike most holometabolous insects, this process generates a single-segment periodicity, and does not have a double-segment pattern at any stage. We suggest that the evolutionary transition to double-segment patterning lies in mutually exclusive expression patterns of secondary pair-rule genes. The fact that many aspects of the putative Oncopeltus segmentation network are similar to those of Drosophila, is consistent with a simple transition between sequential and simultaneous segmentation.

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.

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.

Interpreting surveys to estimate the size of the monarch butterfly population: Pitfalls and prospects.

To assess the change in the size of the eastern North American monarch butterfly summer population, studies have used long-term data sets of counts of adult butterflies or eggs per milkweed stem. Despite the observed decline in the monarch population as measured at overwintering sites in Mexico, these studies found no decline in summer counts in the Midwest, the core of the summer breeding range, leading to a suggestion that the cause of the monarch population decline is not the loss of Midwest agricultural milkweeds but increased mortality during the fall migration. Using these counts to estimate population size, however, does not account for the shift of monarch activity from agricultural fields to non-agricultural sites over the past 20 years, as a result of the loss of agricultural milkweeds due to the near-ubiquitous use of glyphosate herbicides. We present the counter-hypotheses that the proportion of the monarch population present in non-agricultural habitats, where counts are made, has increased and that counts reflect both population size and the proportion of the population observed. We use data on the historical change in the proportion of milkweeds, and thus monarch activity, in agricultural fields and non-agricultural habitats to show why using counts can produce misleading conclusions about population size. We then separate out the shifting proportion effect from the counts to estimate the population size and show that these corrected summer monarch counts show a decline over time and are correlated with the size of the overwintering population. In addition, we present evidence against the hypothesis of increased mortality during migration. The milkweed limitation hypothesis for monarch decline remains supported and conservation efforts focusing on adding milkweeds to the landscape in the summer breeding region have a sound scientific basis.

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.

Environmental Persistence Influences Infection Dynamics for a Butterfly Pathogen.

Many pathogens, including those infecting insects, are transmitted via dormant stages shed into the environment, where they must persist until encountering a susceptible host. Understanding how abiotic conditions influence environmental persistence and how these factors influence pathogen spread are crucial for predicting patterns of infection risk. Here, we explored the consequences of environmental transmission for infection dynamics of a debilitating protozoan parasite (Ophryocystis elektroscirrha) that infects monarch butterflies (Danaus plexippus). We first conducted an experiment to observe the persistence of protozoan spores exposed to natural conditions. Experimental results showed that, contrary to our expectations, pathogen doses maintained high infectivity even after 16 days in the environment, although pathogens did yield infections with lower parasite loads after environmental exposure. Because pathogen longevity exceeded the time span of our experiment, we developed a mechanistic model to better explore environmental persistence for this host-pathogen system. Model analysis showed that, in general, longer spore persistence led to higher infection prevalence and slightly smaller monarch population sizes. The model indicated that typical parasite doses shed onto milkweed plants must remain viable for a minimum of 3 weeks for prevalence to increase during the summer-breeding season, and for 11 weeks or longer to match levels of infection commonly reported from the wild, assuming moderate values for parasite shedding rate. Our findings showed that transmission stages of this butterfly pathogen are long-lived and indicated that this is a necessary condition for the protozoan to persist in local monarch populations. This study provides a modeling framework for future work examining the dynamics of an ecologically important pathogen in an iconic insect.

Milkweed butterfly resistance to plant toxins is linked to sequestration, not coping with a toxic diet.

Insect resistance to plant toxins is widely assumed to have evolved in response to using defended plants as a dietary resource. We tested this hypothesis in the milkweed butterflies (Danaini) which have progressively evolved higher levels of resistance to cardenolide toxins based on amino acid substitutions of their cellular sodium-potassium pump (Na(+)/K(+)-ATPase). Using chemical, physiological and caterpillar growth assays on diverse milkweeds (Asclepias spp.) and isolated cardenolides, we show that resistant Na(+)/K(+)-ATPases are not necessary to cope with dietary cardenolides. By contrast, sequestration of cardenolides in the body (as a defence against predators) is associated with the three levels of Na(+)/K(+)-ATPase resistance. To estimate the potential physiological burden of cardenolide sequestration without Na(+)/K(+)-ATPase adaptations, we applied haemolymph of sequestering species on isolated Na(+)/K(+)-ATPase of sequestering and non-sequestering species. Haemolymph cardenolides dramatically impair non-adapted Na(+)/K(+)-ATPase, but had systematically reduced effects on Na(+)/K(+)-ATPase of sequestering species. Our data indicate that major adaptations to plant toxins may be evolutionarily linked to sequestration, and may not necessarily be a means to eat toxic plants. Na(+)/K(+)-ATPase adaptations thus were a potential mechanism through which predators spurred the coevolutionary arms race between plants and insects.

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.

Selection of Reference Genes for RT-qPCR Analysis in the Monarch Butterfly, Danaus plexippus (L.), a Migrating Bio-Indicator.

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a powerful technique to quantify gene expression. To facilitate gene expression study and obtain accurate results, normalization relative to stably expressed reference genes is crucial. The monarch butterfly, Danaus plexippus (L.), is one of the most recognized insect species for its spectacular annual migration across North America. Besides its great voyages, D. plexippus has drawn attention to its role as a bio-indicator, ranging from genetically modified organisms (GMOs) to natural ecosystems. In this study, nine reference genes from D. plexippus genome were selected as the candidate reference genes. The expression profiles of these candidates under various biotic and abiotic conditions were evaluated using the four readily available computational programs, BestKeeper, Normfinder, geNorm, and ΔCt method, respectively. Moreover, RefFinder, a web-based computational platform integrating the four above mentioned algorisms, provided a comprehensive ranking of the stability of these reference genes. As a result, a suite of reference genes were recommended for each experimental condition. Specifically, elongation factor 1α (EF1A) and ribosomal protein 49 (RP49) were the most stable reference genes, respectively, under biotic (development, tissue, and sex) and abiotic (photoperiod, temperature, and dietary RNAi) conditions. With the recent release of a 273-million base pair draft genome, results from this study allow us to establish a standardized RT-qPCR analysis and lay a foundation for the subsequent genomic and functional genomic research in D. plexippus, a major bio-indicator and an emerging model for migratory animals.

Climate change may alter breeding ground distributions of eastern migratory monarchs (Danaus plexippus) via range expansion of Asclepias host plants.

Climate change can profoundly alter species' distributions due to changes in temperature, precipitation, or seasonality. Migratory monarch butterflies (Danaus plexippus) may be particularly susceptible to climate-driven changes in host plant abundance or reduced overwintering habitat. For example, climate change may significantly reduce the availability of overwintering habitat by restricting the amount of area with suitable microclimate conditions. However, potential effects of climate change on monarch northward migrations remain largely unknown, particularly with respect to their milkweed (Asclepias spp.) host plants. Given that monarchs largely depend on the genus Asclepias as larval host plants, the effects of climate change on monarch northward migrations will most likely be mediated by climate change effects on Asclepias. Here, I used MaxEnt species distribution modeling to assess potential changes in Asclepias and monarch distributions under moderate and severe climate change scenarios. First, Asclepias distributions were projected to extend northward throughout much of Canada despite considerable variability in the environmental drivers of each individual species. Second, Asclepias distributions were an important predictor of current monarch distributions, indicating that monarchs may be constrained as much by the availability of Asclepias host plants as environmental variables per se. Accordingly, modeling future distributions of monarchs, and indeed any tightly coupled plant-insect system, should incorporate the effects of climate change on host plant distributions. Finally, MaxEnt predictions of Asclepias and monarch distributions were remarkably consistent among general circulation models. Nearly all models predicted that the current monarch summer breeding range will become slightly less suitable for Asclepias and monarchs in the future. Asclepias, and consequently monarchs, should therefore undergo expanded northern range limits in summer months while encountering reduced habitat suitability throughout the northern migration.

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.

Stepwise evolution of resistance to toxic cardenolides via genetic substitutions in the Na+/K+ -ATPase of milkweed butterflies (lepidoptera: Danaini).

Despite the monarch butterfly (Danaus plexippus) being famous for its adaptations to the defensive traits of its milkweed host plants, little is known about the macroevolution of these traits. Unlike most other animal species, monarchs are largely insensitive to cardenolides, because their target site, the sodium pump (Na(+)/K(+) -ATPase), has evolved amino acid substitutions that reduce cardenolide binding (so-called target site insensitivity, TSI). Because many, but not all, species of milkweed butterflies (Danaini) are associated with cardenolide-containing host plants, we analyzed 16 species, representing all phylogenetic lineages of milkweed butterflies, for the occurrence of TSI by sequence analyses of the Na(+)/K(+) -ATPase gene and by enzymatic assays with extracted Na(+)/K(+) -ATPase. Here we report that sensitivity to cardenolides was reduced in a stepwise manner during the macroevolution of milkweed butterflies. Strikingly, not all Danaini typically consuming cardenolides showed TSI, but rather TSI was more strongly associated with sequestration of toxic cardenolides. Thus, the interplay between bottom-up selection by plant compounds and top-down selection by natural enemies can explain the evolutionary sequence of adaptations to these toxins.

Experimental examination of intraspecific density-dependent competition during the breeding period in monarch butterflies (Danaus plexippus).

A central goal of population ecology is to identify the factors that regulate population growth. Monarch butterflies (Danaus plexippus) in eastern North America re-colonize the breeding range over several generations that result in population densities that vary across space and time during the breeding season. We used laboratory experiments to measure the strength of density-dependent intraspecific competition on egg laying rate and larval survival and then applied our results to density estimates of wild monarch populations to model the strength of density dependence during the breeding season. Egg laying rates did not change with density but larvae at high densities were smaller, had lower survival, and weighed less as adults compared to lower densities. Using mean larval densities from field surveys resulted in conservative estimates of density-dependent population reduction that varied between breeding regions and different phases of the breeding season. Our results suggest the highest levels of population reduction due to density-dependent intraspecific competition occur early in the breeding season in the southern portion of the breeding range. However, we also found that the strength of density dependence could be almost five times higher depending on how many life-stages were used as part of field estimates. Our study is the first to link experimental results of a density-dependent reduction in vital rates to observed monarch densities in the wild and show that the effects of density dependent competition in monarchs varies across space and time, providing valuable information for developing robust, year-round population models in this migratory organism.

Behavioural resistance against a protozoan parasite in the monarch butterfly.

1. As parasites can dramatically reduce the fitness of their hosts, there should be strong selection for hosts to evolve and maintain defence mechanisms against their parasites. One way in which hosts may protect themselves against parasitism is through altered behaviours, but such defences have been much less studied than other forms of parasite resistance. 2. We studied whether monarch butterflies (Danaus plexippus L.) use altered behaviours to protect themselves and their offspring against the protozoan parasite Ophryocystis elektroscirrha (McLaughlin & Myers (1970), Journal of Protozoology, 17, p. 300). In particular, we studied whether (i) monarch larvae can avoid contact with infectious parasite spores; (ii) infected larvae preferentially consume therapeutic food plants when given a choice or increase the intake of such plants in the absence of choice; and (iii) infected female butterflies preferentially lay their eggs on medicinal plants that make their offspring less sick. 3. We found that monarch larvae were unable to avoid infectious parasite spores. Larvae were also not able to preferentially feed on therapeutic food plants or increase the ingestion of such plants. However, infected female butterflies preferentially laid their eggs on food plants that reduce parasite growth in their offspring. 4. Our results suggest that animals may use altered behaviours as a protection against parasites and that such behaviours may be limited to a single stage in the host-parasite life cycle. Our results also suggest that animals may use altered behaviours to protect their offspring instead of themselves. Thus, our study indicates that an inclusive fitness approach should be adopted to study behavioural defences against parasites.

Evidence for trans-generational medication in nature.

Parasites pose a serious threat to host fitness, and natural selection should favour host traits that reduce infection or disease symptoms. Here, we provide the first evidence of trans-generational medication, in which animals actively use medicine to mitigate disease in their offspring. We studied monarch butterflies and their virulent protozoan parasites, and found that neither caterpillars nor adult butterflies could cure themselves of disease. Instead, adult butterflies preferentially laid their eggs on toxic plants that reduced parasite growth and disease in their offspring caterpillars. It has often been suggested that sick animals may use medication to cure themselves of disease, but evidence for the use of medication in nature has so far been scarce. Our results provide evidence that infected animals may indeed use medicine as a defence against parasites, and that such medication may target an individual's offspring rather than the individual itself.

Phylogenetic ecology of leaf surface traits in the milkweeds (Asclepias spp.): chemistry, ecophysiology, and insect behavior.

The leaf surface is the contact point between plants and the environment and plays a crucial role in mediating biotic and abiotic interactions. Here, we took a phylogenetic approach to investigate the function, trade-offs, and evolution of leaf surface traits in the milkweeds (Asclepias). Across 47 species, we found trichome densities of up to 3000 trichomes cm(-2) and epicuticular wax crystals (glaucousness) on 10 species. Glaucous species had a characteristic wax composition dominated by very-long-chain aldehydes. The ancestor of the milkweeds was probably a glaucous species, from which there have been several independent origins of glabrous and pubescent types. Trichomes and wax crystals showed negatively correlated evolution, with both surface types showing an affinity for arid habitats. Pubescent and glaucous milkweeds had a higher maximum photosynthetic rate and lower stomatal density than glabrous species. Pubescent and glaucous leaf surfaces impeded settling behavior of monarch caterpillars and aphids compared with glabrous species, although surface types did not show consistent differentiation in secondary chemistry. We hypothesize that pubescence and glaucousness have evolved as alternative mechanisms with similar functions. The glaucous type, however, appears to be ancestral, lost repeatedly, and never regained; we propose that trichomes are a more evolutionarily titratable strategy.