Research PhylogeneticBlackwell Publishing Ltd ecology of leaf surface traits in the milkweeds (Asclepias spp.): chemistry, ecophysiology, and insect behavior Anurag A. Agrawal1, Mark Fishbein2, Reinhard Jetter3, Juha-Pekka Salminen4, Jessica B. Goldstein1, Amy E. Freitag1 and Jed P. Sparks1 1Department of Ecology and Evolutionary Biology, Cornell University, Corson Hall, Ithaca, NY 14853-2701, USA; 2Portland State University, Department of Biology, P.O. Box 751, Portland, OR 97207, USA; 3Departments of Botany and Chemistry, University of British Columbia, 3510-6270 University Blvd, Vancouver, BC, V6T 1Z4, Canada; 4Laboratory of Organic Chemistry and Chemical Biology, Department of Chemistry, University of Turku, Turku, FI-20014, Finland Summary Author for correspondence: • The leaf surface is the contact point between plants and the environment and Anurag A. Agrawal plays a crucial role in mediating biotic and abiotic interactions. Here, we took a + Tel: 1 6072544255 phylogenetic approach to investigate the function, trade-offs, and evolution of leaf Email: [email protected] surface traits in the milkweeds (Asclepias). Received: 24 March 2009 • Across 47 species, we found trichome densities of up to 3000 trichomes cm−2 and Accepted: 8 April 2009 epicuticular wax crystals (glaucousness) on 10 species. Glaucous species had a char- acteristic wax composition dominated by very-long-chain aldehydes. New Phytologist (2009) 183: 848–867 • The ancestor of the milkweeds was probably a glaucous species, from which there doi: 10.1111/j.1469-8137.2009.02897.x 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 Key words: adaptation to arid environments, aldehydes, cardenolides, higher maximum photosynthetic rate and lower stomatal density than glabrous spe- cuticular waxes, monarch butterfly cies. Pubescent and glaucous leaf surfaces impeded settling behavior of monarch caterpillar (Danaus plexippus), plant caterpillars and aphids compared with glabrous species, although surface types did defense syndromes, trichomes, water use not show consistent differentiation in secondary chemistry. efficiency. • 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. morphology, formation, chemical composition, and their Introduction functional role as adaptations to habitats and herbivores. The primary function of leaves is to turn light, water and Trichomes have well-documented ecophysiological (e.g. carbon dioxide into chemical energy, and thus leaves are likely Ehleringer & Clark, 1987; Sandquist & Ehleringer, 2003) under strong selection to maximize physiological function and anti-herbivore (e.g. Coley, 1983; Mauricio & Rausher, in a plant’s habitat. However, given that plants are the direct 1997; Haddad & Hicks, 2000) functions. The presence of or indirect source of energy for most animals and many high-density, nonglandular trichomes has been correlated microbes, the leaf surface should also be under strong with plants growing in open, hot, and arid habitats, and in selection to minimize attack by consumers. In this regard, two such situations, trichomes function to shade the leaf surface major leaf surface traits, trichomes (leaf hairs) and epicuticular from ultraviolet (UV) light, reduce the heat load, and reduce wax crystals, may be key plant traits that have the potential to absorbance of solar radiation (Ehleringer et al., 1976). In influence both photosynthesis and resistance to consumption. terms of resistance to herbivores, nonglandular trichomes may These traits have been intensively studied with respect to act as a physical barrier to movement and consumption for 848 New Phytologist (2009) 183: 848–867 © The Authors (2009) 848www.newphytologist.org Journal compilation © New Phytologist (2009) Research 849 many invertebrate herbivores, although various insects have approaches to investigate the defensive ecology of this group adaptations to overcome these effects (Malcolm, 1995; (Agrawal, 2005, 2007; Agrawal & Fishbein, 2006; Agrawal Fordyce & Agrawal, 2001; Agrawal, 2005). et al., 2008). In addition to trichomes, two other traits have Epicuticular waxes are also known to impart both ecophysio- been strongly implicated in resistance to herbivores: cardeno- logical (Schreiber & Riederer, 1996; Oliveira et al., 2003) and lides (toxic steroidal compounds that impact the function of anti-herbivore (Eigenbrode & Espelie, 1995; Müller & Ried- Na+/K+-ATPases) and latex (a sticky, viscous substance that erer, 2005) functions. Epicuticular wax crystals scatter light, exudes upon tissue damage). We previously observed that the giving the plant surface a glaucous (grayish) appearance, limit- amount of defensive latex exuded and trichome density across ing penetration of UV light, as well as photosynthetically 24 species of Asclepias showed correlated evolution (i.e. the active radiation. This may benefit plants growing in open positive correlation was significant after phylogenetic correction) habitats where they receive high light loads (Schreiber & (Agrawal & Fishbein, 2006). We specifically noted exceptions, Riederer, 1996; Oliveira et al., 2003). In addition, wax crystals however, of plants that were high in latex production but free (and also some trichomes) increase water repellency at the leaf of trichomes. Based on visual observations, we predicted that surface, which influences gas exchange and interactions with these species had a thick layer of epicuticular wax. microorganisms (Müller & Riederer, 2005; Brewer & Nuñez, The central goal in this study was to employ phylogenetic 2007). Although wax crystals themselves may promote the information to study the evolutionary history and functional germination of some pathogenic fungi (Mendgen et al., role of plant traits implicated in interactions with the biotic 1996), glaucous leaves are less wettable and therefore more and abiotic environment. As such, we aimed to address long- resistant to many microbes. Further, most invertebrates have standing adaptive hypotheses about convergence in leaf difficulty walking on glaucous surfaces (Eigenbrode & Espelie, surface form and function (Givnish, 1987; Reich et al., 1999; 1995; Eigenbrode et al., 1996; Riedel et al., 2007) and are Ackerly, 2004). We first attempted to categorize 47 Asclepias often deterred by physical or chemical cues in epicuticular species as having glabrous, pubescent, or glaucous leaves. Tri- waxes (Müller & Riederer, 2005). chome density is a quantitative trait that ranges from none Although studies of natural selection on individual species (glabrous) to dense (pubescent), while glaucousness is a discrete can provide direct evidence for the present-day adaptive func- trait – waxes are typically either inconspicuous or uniformly tion of a particular leaf surface trait (e.g. Mauricio & coat leaf surfaces with platelets imparting the appearance of a Rausher, 1997; Sandquist & Ehleringer, 2003), there is also waxy bloom. After we characterized the surface structures using a rich tradition of using comparative approaches to infer longer microscopy, we categorized species by their surface chemistry term evolutionary dynamics and to draw general conclusions (all plants, glaucous or not, have surface waxes). Finally, we regarding the function and adaptive value of such traits (Coley, extended the analysis to potentially correlated variation in 1983; Ehleringer & Clark, 1987; Schreiber & Riederer, 1996; ecophysiological function (photosynthesis, water use efficiency, Markstädter et al., 2000; Oliveira et al., 2003). Historically, stomatal density, and carbon-to-nitrogen ratio), and defense two previous limitations of the comparative approach have investment (insect behavior, cardenolides, and phenolics). reduced its use and impact in studies of plant evolution and For the impact of leaf surface variation on insect behavior, we adaptation. First, phylogenetic information has frequently examined two common specialist herbivores of Asclepias spp., not been available or fully integrated into the study of plant monarch butterflies (Danaus plexippus) and milkweed aphids adaptations. Such phylogenetic information allows us to (Aphis nerii). Thus, our goal was to take a multidisciplinary recognize sets of species that have independently (or conver- approach to understanding the ecology of species that have gently) evolved particular traits. Much stronger evidence for both diverged and converged in their habitat use, leaf surface an adaptive function can be extracted from the ecological traits, and biotic interactions (Chapin et al., 1993; Agrawal & comparison of these species than if convergent species were Fishbein, 2006). not compared (Armbruster, 1997; Wilson et al., 2006). Sec- ondly, only rarely have transitions to multiple phenotypic Materials and Methods states been studied simultaneously in the same lineage. For example, repeated transitions from glabrous (smooth) to Natural history glaucous or pubescent leaves in the same genus might reveal the ecological function of these divergent surface traits. Thus, The milkweeds (Asclepias spp.) are a monophyletic genus multi-trait comparisons in the context of phylogenetic infor- comprising