Copyright by Catalina Estrada 2009

Catalina Estrada

2009

The Dissertation committee for Catalina Estrada certifies that this is the approved version of the following dissertation:

Sexual Behavior, Intraspecific Signaling and the Evolution of Mimicry

among Closely Related Species

Committee:

______Lawrence E. Gilbert, Supervisor

______Molly Cummings

______Jennifer Brodbelt

______Michael Singer

______Ulrich G. Mueller

Sexual Behavior, Intraspecific Signaling and the Evolution of Mimicry

among Closely Related Species

Catalina Estrada, M.S.

Dissertation

Presented to the Faculty of the Graduate School of

The University of Texas at Austin

in Partial Fulfillment

of the Requirements

for the Degree of

Doctor of Philosophy

The University of Texas at Austin

May, 2009 Dedicación

Para Maya, Samraat, nuestra familia alrededor del mundo y el resto de las mariposas.

Acknowledgements

I am forever in debt to many people that helped make this work possible, and who were my inspiration, support and family during these constantly challenging grad school years. I want to thank my supervisor and friend Larry Gilbert for his advice and help in this project, and for allowing me to use and enjoy his small patches of tropical rain forest in Austin. His dedication and passion for nature and for education has inspired me as his student and teaching assistant. Thanks to Molly Cummings, Jennifer Brodbelt, Mike Singer and Ulrich Mueller, professors in my thesis committee, who guided the progress of this work from early ideas to the completion of the dissertation. Many professors, lecturers and fellow grad students were very influential in my academic development during these years. I want to thank in particular to Ulrich Muller, Mike Ryan, and Mike Singer because their fascinating courses help focus my research interests.

This work would not have been possible without the support of an amazing team of collaborators. I would like to express my deep and sincere gratitude to Professor Stefan Schulz and his graduate student Selma Yildizhan from the Institute of Organic Chemistry, Technische Universität Braunschweig. I learned so much from them about chemistry and chemical ecology, and was inspired by their talent and hospitability. Jamtsho and Tony Alexander assisted me in numerous ways to keep the butterflies and plants happy in the greenhouses in Patterson Laboratory and Brackenridge Field Laboratory. A wonderful team of undergraduates also help me with data collection and maintenance of greenhouses. In particular, I thank Mary Owens, Angie Martίnez, Siona Marout, Ariani Wartenberg, Kathryn Busby and Colin Strickland. Also a big thanks to Cesar Rodriguez-Saona from the Department of Extension Specialists at Rutgers, The State University of New Jersey, for his help with data collection in projects that follow up this dissertation research. Finally, thanks to Anna-Karin Borg- Karlson and Johan Andersson from the Royal Institute of Technology KTH, Stockholm, Sweden, and Medhi Moini from the Mass Spectrometry Facility, Department of Chemistry and Biochemistry at UT, for advice with sampling methods.

I am deeply grateful to my life partner, Samraat Pawar. He has been my support, advisor and friend. He thought me with his example to never forget the essential reasons that motivate my interest and love for my research. Thanks to my family in Colombia and India; without their encouragement and loving support I would not be able to finish this work. Thank you sweet Maya, you came to remind me what amazing things can happen in life.

A fantastic group of colleagues and friends also contributed in many ways to the success of this work and to make my time in Austin a wonderful experience. I particularly want to thank Juanita Choo, Rob Plowes, Evan Economo, Barret Klein, Christian Rabeling, Simone Cappellari, Natalia Biani, Juan Carlos Santos, Sasha Mikheyev, Deepa Agashe, Naiara Pinto, Krushnamegh Kunte, Ximena Bernal, Ron Bocsa and Kendra Brauer.

I am deeply grateful to the staff of Integrative Biology for their constant support. Particularly I want to thank our wonderful graduate coordinator Sandy Monahan, who has always been there with a smile to help us and rescue us from paper work and deadlines! Thanks also to John and Grace Crutchfield for giving BFL the warm and familiar spirit.

This work was funded by the National Science Foundation and the Office of International Science and Engineering under grant No 0608167, Animal Behavior Society, Lepidoptera Research Foundation, the Section of Integrative Biology of the University of Texas at Austin, the Texas Academy of Sciences, the Southwestern Association of Naturalists, and the Private Lands Research Grants-Environmental Science Institute.

Sexual Behavior, Intraspecific Signaling and the Evolution of Mimicry

among Closely Related Species

Catalina Estrada, Ph.D.

The University of Texas at Austin

Supervisor: Lawrence E. Gilbert

Mimicry, an adaptation to deceive, fascinated early naturalist and has been proof of evolution by natural selection since proposed by Henry W. Bates 150 years ago. Yet, despite the abundant theoretical and empirical work that it has inspired, little is known of effects in intra and interspecific communication that might result from resembling phenotypic traits of sympatric species. In this dissertation research I studied sexual behavior and communication in Heliconius, a genus of diverse toxic butterflies with extraordinary convergence in wing coloration, habitat preferences and flight characteristics. Well-known ecological interactions and evolutionary history of Heliconius contrast with a poor understanding of key elements of their sexual behavior and intraspecific communication, which are central for the evolution of mimicry in this genus of butterflies.

This thesis starts with an introduction that, expanding on the ideas above, explains the motivation behind studying sexual communication and behavior in Heliconius. In the subsequent four chapters I report on two aspects of sexual behavior that are presumably connected in these butterflies with the occurrence of mimicry: Pupal mating behavior and antiaphrodisiac pheromones. Pupal mating is a mate-searching strategy wherein males find females when still immature and guard them with the goal of mating at female

vii eclosion. This mating behavior might have influenced the evolution of mimicry as males rely less on commonly used species recognition traits that in mimetic Heliconius are shared with coexisting species. I identified cues males use to find and recognize conspecific immatures, which not only come from the animal themselves but also from the host plant where they are located. Chemical and visual cues are involved in the process of finding partners, but only sex-specific pheromones allow males to identify females before their eclosion. The second aspect of sexual behavior studied in Heliconius involved the identification of a pheromone that, after being transferred to females at mating, renders them unattractive to courting males. Variation in the chemical composition of such antiaphrodisiacs across eleven species in this genus showed that, contrary to my expectations, there is no evidence that mimicry has affected the evolution of this signal. Instead, I found that clade-specific mating systems in these butterflies adequately explain the observed patterns of interspecific variation.

viii

TABLE OF CONTENTS

CHAPTER 1: Introduction 1 CHAPTER 2: Host Plants and Immatures as Mate Searching Cues in Heliconius Butterflies 7 ABSTRACT ...... 7 INTRODUCTION ...... 7 METHODS ...... 10 RESULTS ...... 16 DISCUSSION ...... 19 FIGURES ...... 27 TABLES ...... 33 CHAPTER 3: Pupal Guarding by Male Butterflies: Pre-eclosion Sex Discrimination and Sexual Conflict 39 ABSTRACT ...... 39 INTRODUCTION ...... 39 METHODS ...... 42 RESULTS ...... 47 DISCUSION ...... 51 FIGURES ...... 57 TABLE ...... 63 CHAPTER 4: Interspecific Variation of Abdominal Gland Scents among Heliconius Butterflies (Lepidoptera: Nymphalidae): Effect of Mimicry and Mating System 64 ABSTRACT ...... 64 INTRODUCTION ...... 64 METHODS ...... 69 RESULTS ...... 75 DISCUSSION ...... 78 FIGURES ...... 87 TABLE ...... 94

CHAPTER 5: Antiaphrodisiac Pheromones in Butterflies: Intra and Interspecific Communication in a Community of Mimetic Species 95 ABSTRACT ...... 95 INTRODUCTION ...... 96 METHODS ...... 99 RESULTS ...... 102 DISCUSSION ...... 104 FIGURES ...... 111 TABLE ...... 114 APPENDIXES 115 BIBLIOGRAPHY 123 VITA 144

CHAPTER 1: Introduction

Animals communicate to find and recognize mating partners, aggregate, and transfer information about the environment. Such intraspecific communication, particularly in a sexual context, is crucial for successful reproduction and to maintain the integrity of a species. In nature, the evolution of signals and behaviors are not just driven by intraspecific interactions, but are also constrained by selection pressures from a complex network of species (interspecific interactions), and several abiotic factors (Endler 1993, Zuk & Kolluru 1998). The context in which animal communication systems evolve is then important as the direction and intensity of natural and sexual selection pressures might shape mating signals and preferences in ways that promote speciation and influence species coexistence (Alatalo et al. 1994, Schluter 2001, Ord & Martins 2006). Natural and sexual selection can act in opposite directions if conspecifics prefer signals that also make animals more conspicuous to their natural enemies (Endler 1980, Ryan et al. 1982, Grether 1997, Stuart-Fox et al. 2004, Bernal et al. 2006) or less fit to the environmental conditions they experience (Nielsen & Watt 2000, Ellers & Boggs 2003). Such opposite selective pressures generate diversity and seems to explain the occurrence of intraspecific geographic divergence in signaling systems (Endler 1995), color polymorphism (Nielsen & Watt 2000), signaling strategies (Ryan et al. 1982) and private channels of communication (Cummings et al. 2003).

Alternatively, mate preferences can also evolve in the same direction in which natural selection is shaping mating signals (Jiggins et al. 2001b, Nosil et al. 2002), which occasionally leads to reproductive isolation between populations that occupy contrasting environments (Schluter 2001). One of the best examples of such correlated evolution of signal and preferences has been studied in Heliconius butterflies (Nymphalidae: Heliconiinae). In these insects changes in mimetic color patterns have coevolved with the use of those patterns as mating cues (Jiggins et al. 2001b, Jiggins et al. 2004), probably facilitated by a strong genetic association between both traits (Kronforst et al. 2006). In

addition, reduction in hybrid reproductive success due to intermediate wing phenotypes has resulted in reinforcement on color pattern preferences in areas of interspecific contact, playing a central role in the evolution of reproductive isolation between emergent species (Jiggins et al. 2001b, Naisbit et al. 2001, Kronforst et al. 2007). Heliconius are distributed from Argentina to the southern United States (Brown 1981). They exhibit remarkable intra and interspecific color pattern diversity which, together with habitat preferences and flight characteristics, often converge among species within the genus to form extraordinary examples of mimicry (Brown 1981, Turner 1981, Mallet & Gilbert 1995, Srygley & Ellington 1999, Estrada & Jiggins 2002).

The dual function of wing color patterns (intraspecific communication and defense), although effective to generate diversity in the genus, creates reproductive interference as efficiency in species recognition decreases among coexisting mimetic species (Estrada & Jiggins 2008). Such interference likely persists in nature because the benefits of mimicry as a defense strategy outweigh the costs of interspecific attraction and courtship. However, costs caused by inefficient species recognition, although trivial, could be important and explain some pattern of mimicry observed in Heliconius and other mimetic organisms. For example, in Heliconius convergence in warning coloration has normally happened between members of distant clades (Eltringham 1916, Gilbert 1991). This suggest that perhaps this defense strategy evolves more readily between distantly related species that use additional species recognition cues and are unlikely to hybridize, reducing costs of interspecific attraction to waste of energy but not gametes (Mallet et al. 2007, Estrada & Jiggins 2008). Moreover, with more than two species sharing warning colorations, cost of reproductive interference could be added up as more heterospecific similar individuals coexist. This additive effect might provide an explanation for the lack of mimetic convergence among local unpalatable species (Brower et al. 1963), still an unresolved paradox in the evolution of Müllerian mimicry (mimicry between toxic species) (Mallet & Gilbert 1995, Joron & Mallet 1998).

My dissertation research has been focused on two aspects of the sexual behavior of Heliconius butterflies that might reduce signaling confusion among co-mimic species in the face of wing color pattern similarities. In chapters 2 and 3, I report results of studies on a mating system that provide alternative cues for mate recognition and could have facilitated the evolution of intrageneric mimicry in Heliconius, pupal mating. Chapters 4 and 5 are an account of the research on evolution of chemical signals in these butterflies, testing whether intraspecific communication has been influenced by interaction among co-mimetic Heliconius and predators.

1. Mate searching behavior and recognition during pupal mating in Heliconius butterflies (chapters 2 and 3)

Around half of the species in the genus Heliconius exhibit a unique mating system where males find pupae on (or close to) larval host plants, and mate with females as they emerge. We call this strategy ‘pupal mating’ (Gilbert 1976, Deinert et al. 1994). Males of these species are not exclusively attracted to pupae but also initiate courtship toward females encountered in flight, although the proportions of matings using each strategy are still unknown for natural populations. Heliconius species are host plant specialists and larvae of most species feed on one or few Passiflora (Passifloraceae) species within each locality along their geographic range (Benson et al. 1975, Gilbert 1991). Therefore, the use of plants as mating places could account for a reduction of interspecific interference, particularly with teneral females, presumably facilitating the evolution of intrageneric mimicry in Heliconius (Gilbert 1991). Using chemical analysis and behavioral assays, we found that males use volatiles released by host plants and caterpillars to find potential mates and probably learn the location of that site for future visits. Such mating cues are produced by plants soon after any tissue damage (e.g. caterpillars feeding or mechanical damage) and are the same regardless of the species of caterpillar feeding. Therefore, although host plants seem to play a major role in male searching behavior, species recognition at this stage is most probably achieved by other

cues from the caterpillars. We also found that males are able to estimate the maturity and sex of pupae using primarily short-range sex specific pheromones. We identified these pheromones for one species, and experimentally tested their effect in male behavior toward pupae.

Our main motivation in studying pupal mating in Heliconius charithonia was to understand the mechanism by which males find and recognize mates when looking for immature butterflies. These are key aspects in evaluating the role that this mating system might have had in the formation of butterfly communities and evolution of intrageneric mimicry in Heliconius butterflies. However finding and recognizing mates occur repeatedly during pupal mating and involve several developmental stages. Our research has unraveled part of the signaling involved in this system and thus connections with the overall implications of pupal mating in the ecology and evolution of these butterflies can not be done yet. Therefore, although we discuss the implication of this mating system in the evolution of mimicry, this has not been the main focus of chapters. Chapter 2, for example, concentrates on the role of host plants in the mating behavior of butterflies, a factor so far neglected in the literature, despite some evidence of the importance of this resource in searching behavior and evolution of phytophagous insects. Chapter 3, on the other hand, is written as a contribution of our understanding of signaling during precopulatory mate guarding, a male strategy to compete for females which include pupal mating behavior. Here we also explore the possibility that cues used by males to identify pupal sex and maturity can provide clues about the occurrence of sexual conflict in Heliconius species with this mating strategy.

2. Antiaphrodisiacs and chemical warning signals from abdominal glands of Heliconius (chapters 4 and 5)

During courtship, mated and virgin butterfly females adopt a posture in which their wings are horizontally spread and their abdomens lifted, exposing abdominal glands

4 toward males. At this time, males assess their receptivity to mate and stop courting those females that appear unreceptive. Interest in mating is signaled by females through behavior as well as chemical signals from abdominal glands which have been previously supplied by males during mating (antiaphrodisiac pheromones) (Gilbert 1976, Schulz et al. 2008). In Heliconius, females have a strong post-mating odor detectable by humans, which besides the antiaphrodisiac function, have also been postulated to serve as warning signal of these butterflies toxicity (Eltringham 1925, Ross et al. 2001). Given the high incidence of interspecific attraction between species that have converged in wing warning coloration and use those in species recognition (Estrada & Jiggins 2008), we hypothesized that antiaphrodisiac pheromones should converge between co-mimics to reduce harassment from males of either species. Similar convergence could be expected for aposematic odors as that would increase the resemblance of co-mimic species to predators (Moore et al. 1990). Results of chemical analyses of abdominal glands for eleven Heliconius species showed a high variation in their chemical composition with similarities between species that are in some cases congruent with the phylogeny of the group. Antiaphrodisiac pheromones were the same in only one pair of co-mimic species, but this similarity most likely resulted from the conservation of ancestral volatile compounds rather that from convergence of signals due to mimicry. Although we did not find any support for our hypothesis, behavioral assays showed that males respond to pheromones released by other Heliconius, indicating that cross-species communication might happen despite signal differences, and suggesting that convergence in signals has perhaps not been necessary.

It is still uncertain which species recognition cues operate in addition to wing coloration in Heliconius, or whether the variation of such cues across species can explain the patterns of mimicry observed in this genus of butterflies. Although this work did not answer these questions, our studies in chemical communication and sexual behavior have contributed in a significant way to guide future research in this direction. First, for pupal mating species, we found evidence that species recognition cues in immatures, if any, will operate at short distance only and thus are available after males have found suitable

5 pupation habitats using their larval host plant cues. We propose that larval coloration, pupal cuticular odors, and host plant cues might be used by males together or in isolation for mate recognition at different states of pupal mating. Second, observations of courtship behavior from our experiments with antiaphrodisiacs suggest the presence of species specific pheromones in females which operate at short range and after attraction by wing coloration. This has been suggested before (Boppré 1984, Estrada & Jiggins 2008), but here we found that such chemical signals are not released through the abdominal glands during courtship displays.

CHAPTER 2: Host Plants and Immatures as Mate Searching Cues in Heliconius Butterflies

ABSTRACT The study of interactions between phytophagous insects and their host plants extends beyond the understanding of how insects deal with plant chemical defenses. Sexual communication and behaviors of herbivores are often integrated in a variety of ways with their hosts, as those might influence the timing and location of reproduction or can provide clues to find partners. While butterfly evolution has been closely linked to the use of larval host plants, the role of such resources in their sexual behavior has been poorly studied. We conducted series of choice experiments in insectaries in order to determine the role of cues from host plants in mate searching behavior of Heliconius charithonia butterflies. In this species males specialize in searching for pupae that they then guard until eclosion to copulate with the eclosing females (pupal mating). Host plants, particularly in the presence of larvae, attracted males, likely cued by volatiles released by their feeding. Males visited plants with such cues more often than undamaged plants and displayed searching behavior up and down the plant and in front of larvae, suggesting that odors signal the location of potential partners. Although males were attracted by common odors released after plant tissue damage, heterospecific caterpillars made plants generally less attractive. We propose that larval specific phenotypes possibly facilitate species recognition at those early stages. Overall our results suggest that host plants might play an important role in the sexual behavior of butterflies as it has been demonstrated for other phytophagous insects.

INTRODUCTION One of the most critical challenges for sexually reproducing animals is finding and recognizing potential mates. Males, in particular, exhibit searching and mating tactics that help them increase their reproductive success by maximizing fertilization rates (Bush 1969, Parker 1978). Because it is unlikely that females are distributed randomly in their

7 environment, a diverse range of mate-locating strategies have evolved that increase access of males to receptive females. In insects such strategies typically fall between two extremes. At one end, males actively explore through the environment to locate females, while at the other, males perch individually or in groups and wait for females to arrive or they attract them using aggregation or sexual pheromones (Parker 1978, Thornhill & Alcock 1983). In both cases males concentrate efforts in areas with attractive resources (oviposition, feeding, sunspots or emergence places) or landmarks where chances of finding receptive females are highest. For example, several species of phytophagous insects meet, court and mate on or close to their host plants (Landolt & Phillips 1997, Reddy & Guerrero 2004). Such plants are good encounter places as females are likely looking for feeding or oviposition places. Moreover, for species where females are receptive shortly after emerging, and pupation happen close, the probability of finding recently eclosed virgins increase (Thornhill & Alcock 1983, Rutowski 1991, Landolt & Phillips 1997).

Similarly to other phytophagous insects, in some species of butterflies mate locating behavior is linked to their larval host plants (Scott 1975a, Rutowski 1991). Rutowski (1991) found that in 10 of 44 species (9 genera, 4 families) with known mating behavior host plants play a role in their search for mates. Such role range from males choosing to perch or patrol in areas where host plants are abundant (Courtney & Parker 1985, Rutowski & Gilchrist 1988), to those that focus their attention to the plant themselves were they seek for females in some cases even before their eclosion (Borch & Schmid 1973, Gilbert 1976, Elgar & Pierce 1988). Several studies have shown that adaptation to new hosts plants have an important role in butterfly diversification (Ehrlich & Raven 1964, Braby & Trueman 2006, Weingartner et al. 2006, Wheat et al. 2007, Peña & Wahlberg 2008). However, although this has been explained mainly as a consequences of coevolution of larval feeding and plant chemical defenses (Ehrlich & Raven 1964), and there is no strong evidence that speciation in butterflies has been a consequence of the interaction of this resource and their mating systems (Nice & Shapiro 2001, Nice et al. 2002), it is possible that incorporating plant and plant habitat cues in addition to wing

coloration while searching for mates could also have significant implications in the evolution this group of insects (Gilbert 1978).

Sexual behavior of insect herbivores and their host plants are connected in numerous ways (Cocroft et al. 2008). Male mate searching can be greatly influenced by plant chemical and/or visual characteristics (Prokopy & Owens 1983, Landolt & Phillips 1997, Reddy & Guerrero 2004). In several orders of insects host plant volatiles released after larval or female feeding can be used as mate finding cues (Ruther et al. 2000, Tooker et al. 2002, Linn et al. 2003, Ginzel & Hanks 2005, Moayeri et al. 2007) or might enhance attractiveness of aggregation or sex pheromones (Light et al. 1993, Ruther et al. 2001, Deng et al. 2004, Soroker et al. 2004, Yang et al. 2004). Yet, while males of some phytophagous species are attracted to plant cues in laboratory and field assays little is known of the capability of males to discriminate and select host plants (Landolt & Phillips 1997). In the case of butterflies, cues for mate search involving host plants have not been investigated despite the importance of these resources in mating behavior and their potential driving adaptative radiations in this group of insects (Gilbert 1978, Dennis & Shreeve 1988, Gilbert 1991).

Here we investigated the importance of host plants in male searching behavior of Heliconius butterflies (Nymphalidae: Heliconiinae). In these insects habitat and host species partitioning are observed in groups of sympatric species whose larvae feed exclusively on plants in the genus Passiflora (Passifloracea) (Benson 1978, Gilbert 1991, Estrada & Jiggins 2002). Similar to other butterflies (Gilbert & Singer 1975), in some Heliconius oviposition preferences, rather than constrains in larval development (Smiley 1978) have determined the patterns of host plant use. This case of “ecological monophagy” has been explained in part by their male searching and mating behaviors (Gilbert 1978, 1991). Males in the genus establish home ranges where they trap-line habitat patches with adult resources and visit host plants likely looking for mates (Ehrlich & Gilbert 1973, Brown & Benson 1977, Mallet & Jackson 1980, Mallet & Gilbert 1995). Moreover, in about half of the species in the genus Heliconius males specialize in

searching for pupae that then they guard until eclosion, at which point emerging females are mated (pupal mating) (Gilbert 1976, Deinert et al. 1994). Females are believed to normally mate once (Gilbert 1976, Ehrlich & Ehrlich 1978, Boggs 1990), but males mate multiple times creating a strong biased operational sex ratio (Emlen & Oring 1977). Pupal mating behavior evolved once in Heliconius and appears to occur in all species within a monophyletic clade in the genus (18 spp.) (Lee et al. 1992, Beltrán et al. 2007). In this mating system location of potential partners happens several times as males are known to find and visit pupae throughout their development but are able to identify their sex using chemical signals released only few days before eclosion (Deinert 2003, chapter 3). The role of host plants seems to be central in finding and probably recognizing conspecific pupae given that long range pupal pheromones have not been found and that isolated pupae are less likely to be detected by males in insectaries (chapter 3). Moreover, males occasionally sit and try to mate eclosing butterflies regardless of the species or gender when pupae from other Heliconius species are transferred to their host plants (Gilbert 1991).

We first conducted experiments designed to test the hypothesis that host plants play an important role in mate locating behavior in the pupal mating species Heliconius charithonia (Linnaeus). Attraction of males to host plants with different treatments was measured in a greenhouse population in order to identify the origin, kind and specificity of cues used by males to find pupae. Second, we summarized from the literature on mate searching behavior species of butterflies where host plant seems to be a key element for finding females.

METHODS

Butterfly and plant rearing

Bioassays were done using Heliconius charithonia vazquezae from a population originated from adults and larvae collected around Austin (Texas, USA) and supplemented periodically with wild-caught individuals. Butterfly stocks were maintained in 4 x 6 m Lord and Burnham greenhouses at the University of Texas at Austin at about 32°C and high humidity where they breed freely using Passiflora biflora and P. lutea. Adults were fed ad libitum with sugar and honey water solution (10%) and with pollen (Gilbert 1972, Dupland Pianka et al 1977) and nectar from Psiguria spp., Psychotria poeppigiana and Lantana camara flowers. White-colored larvae pupate on or close to host plants and eclose as adult after 9 to12 days depending on ambient temperature. Pupae are typically light brown but the cuticle become translucent, and wings and adult body colors are revealed about 24 hours before eclosion. Passiflora lutea is the main host plant of H. charithonia in Central Texas which is also used by other Heliconiini such as Agraulis vanilla and Dryas iulia. For experiments we collected P. lutea from natural populations along Waller Creek at the campus of University of Texas in Austin, potted them and kept them isolated from female butterflies prior to the experiments.

Greenhouse assays: general methodology

Test on the role of larval host plant in mate searching behavior of H. charithonia males were carried out from May to August 2005-2007. We conducted simultaneous choice experiments using a 22 x 10x 10 m sub-compartment of a 22 x 22 x 10 m greenhouse at Brackenridge Field Laboratory at University of Texas (Austin). Butterflies were kept in semi-natural, horizontally and vertically heterogeneous habitat. Thus, in addition to larval host plants for the experimental butterflies, the greenhouse possessed an array of tropical trees, understory monocots, and large vines as well as Psiguria spp., Psychotria poeppigiana, Cnidosculos multilobus and Lantana camara that provide nectar and pollen. Temperature fluctuated daily from about 20 to 32 oC and Light/dark periods followed natural cycles as walls and roof are made with a clear polycarbonate panels partially covered with 50% shade cloth. Exhaust fans across the southeastern side of the

greenhouse draw outside air over an evaporative surface extending the entire length of the northwestern side allowing for a unidirectional flow of cool humid air perpendicular to the long axis of the compartment (from NW to SE) during warm parts of the day when butterflies are active. Experiments were designed to answer four questions: 1) Are host plants or particular immature stages on those plants attractive to males? 2) which cues (visual or olfactory) do males use to find future mating opportunities?, 3) Are cues produced by host plants, immatures or both, and 4) At what point in the process of locating future mating sites does species recognition occurs in this pupal mating system? To answer each question we performed 10 to 19 experimental replicates wherein a specific potential searching cue (treatment) was paired with a control. Experiments consisted of the introduction of P. lutea in the greenhouse with a resident population of H. charithonia males. Depending on the question, such plants were set intact or carried different types of damage (see below and table 2.1).

Before each test we introduced 13 to 15 unmated males to the greenhouse from our breeding population that were allowed to habituate to the place for about 20 hours. Only males more than five days old were used. Body and forewing length were measured for each individual with a caliper to the closest 0.01 mm and wings were marked with a color code using Sharpie® permanent markers so individual recognition was possible. Males were used no more than twice but individuals were exchanged between different experiments. Thus typically, males without previous experience in the experimental greenhouse, beyond the habituation period, accounted for about half of the tested group. No females or immatures were present in the experimental arena during the period the experiments were carried out.

We set control and treatment P. lutea plants in the greenhouse at the start of each experiment. They were located in places where butterflies had not encountered other plants previously and at equivalent distances from pollen and nectar sources. Test Passiflora were placed more than 5 meters apart from each other and their position inside

the greenhouse was changed between consecutive experiments to minimize the effect of the plant location on results. Passiflora lutea chosen from a pool of 20 individuals were matched in size and rotated between tests as much as possible. We videotaped experimental plants for 5 hours (from 9:00 to 16:00 hours) and registered the number and identity of males that approached the plant, the time they spend visiting, and the behaviors exhibited. We consider it to be an approach if a male diverted his flight path and approached any part of the plant. Males attracted to plants exhibited three main characteristic behaviors: 1) they hovered in front of one small section of the plant, 2) they hovered near the immature or a damaged part of the plant, or 3) they flew up and down the plant and surroundings in a conspicuous ‘searching’ mode. In some cases a combination of behaviors was displayed in the same visit. Video cameras were set on tripods attached to step ladders such that the field of view included the whole focal plant plus a similar area around this. This allowed us to register the total number of butterflies that flew by the plant without approaching, information used to control for the effect of plant location and butterfly activity during the test. Videos were digitalized using Windows Movie Maker version 5.1 and visiting time was measure at the closest second using this program.

Greenhouse assays: specific tests

Table 2.1 summarizes experiments we carried out to answer the four questions mentioned above. We first examined whether males were searching for host plants alone or whether they were attracted to a particular immature stage present in the plant. We paired undamaged P. lutea with plants carrying a fifth instar larva, a prepupa or a young pupa. Prepupae are larvae at the end of fifth instar that have ceased feeding and have changed coloration slightly before suspending themselves for pupation. To decrease the probability of confounding results in male attraction due to release of plant volatiles after tissue damage, plants used as undamaged plant treatments or those carrying prepupae or pupae had never been exposed to herbivory before the experiment. In contrast, plants

13 with larvae were often reused for this treatment, although larvae were removed between tests and plants were protected from herbivory for several days prior to the following experiment. The sex of immatures used was determined after experiments but not considered for analysis since males in another pupal mating species are apparently unable to distinguish the sex of the pupae until few days before eclosion (Deinert 2003).

In the first set of experiments we found that males visited plants with larvae more often than they did to other treatments. In the second set of experiments we asked whether such behavior had been triggered by, olfactory cues from larvae or their interaction with the plant, visual cues from larvae, or a combination of both. For each test we set three P. lutea plants in the greenhouse. The first plant had two H. charithonia fifth instar larvae. We assumed this situation provided both, olfactory and visual cues from plant and larvae thus we used it as a control to compare data from the other two treatments. The second plant had two artificial models of a fifth instar caterpillar. Models were made following those in Papaj and Newsom (2005). Briefly, a recently killed fifth instar larva served as a mold made with silicon rubber (Platsil Gel-10, Politek). Once the silicone cured, the larva was removed and the space was filled with translucent silicone rubber that was then dried at room temperature. Models were painted with water colors and larvae setae were imitated using pieces of black minutien (Austerlitz insect pins 0.15 mm). We matched the color dominating the larval body with the one in the model using a spectrophotometer Ocean Optics Base 32, Version 1.03.0 (Fig. 2.1). Wavelengths from 300 to700 nm were measured relative to a white reflectance standard in caterpillars and models. Original data was standardized by dividing each value by the maximum reflectance for the whole spectrum. The wavelength of the maximum reflectance and the point in the curve where the reflectance is changing faster are commonly used measures to describe and compare reflectance spectra (Keyser & Hill 1999, Sheldon et al. 1999). Models were set in wire and attached to plants on the tops of leaves. Plants from this treatment lacked odors from either larvae or damaged tissue when compared to control. Finally, our third treatment was a plant with two concealed live larvae allowed to eat. Larvae and one of the branches of the plant were isolated inside a green net and hidden

behind the foliage. Plants from this treatment lacked visual cues from larvae when compared to controls as we assumed that odors escaped the net but larvae could not be seen by males.

The third set of experiments attempted to find whether attractive chemical compounds (suggested by the experiments above) were released by host plants, larvae or both. Once again each tests consisted of two treatment and a control P. lutea plants. As a control we used a plant with two concealed larvae that were allowed to eat, thus the treatment had both larval and host plant odors. In a second plant a small section of one leaf, proportional to this eaten by two fifth instar caterpillars, was cut every hour using scissors. This treatment lacked odors from larvae, or larva-plant interaction when compared with control plants. Finally, the third plant was set similarly to controls but larvae concealed inside the net were not allowed to eat, therefore this treatment lacked odors from plant-damaged tissue when compared to control.

Our final set of experiments aimed to determine whether H. charithonia males use species-specific cues in their searching behavior for immatures. For each test we set two plants, one P. lutea with two fifth instar larvae of H. charithonia and one with two larvae of Agraulis vanilla (Fig. 2.2). This species normally coexist and share host with H. charithonia in natural communities.

Statistic analysis

The number of visits received by each experimental plant was standardized by the general activity of butterflies observed that day, measured as the total number of butterflies captured in the videos in all treatments. These proportions were arsin-square- root transformed and the mean number of visits compared with a paired t-test or a general linear model (GLM) in experiments with two and three plant treatments respectively. Analyses with GLM were done with ‘number of visits’ as dependent variable, and ‘treatment’ (control and experimental host plants) and ‘test’ (replicates) as fixed factors,

15 thus comparable to a two-way ANOVA without replication (Sokal & Rohlf 1969). The average time males spent in each visit was square-root transformed and analyzed in a similar way as number of visits. Post hoc pair-wise comparisons were done with Tukey’s HDS test. Data was analyzed using SPSS (16.0.1 for Windows).

Literature review

Sources of information of male searching behavior include ISI Web of Science, Google Scholar, Journal of the Lepidopterist’s Society, Journal of the Research on Lepidoptera, Scientific Electronic Literature Online (SciELO) and regional entomology journals available online.

RESULTS Heliconius charithonia males varied considerably in the level of general activity and searching behaviors exhibited within and among experiments. Some groups or individual males were in general seen more often searching around plants while other exhibited primarily patrolling behaviors and engaged in long male-male chases. Due to this variability, data of plant visitation was standardized by dividing the number of visits to a plant by the total number of butterflies captured in videos in that particular day. Results from analyses done this way were more conservative but in all cases similar to those obtained with visitation raw data. Therefore here we only report analyses done with standardized data. In general, a low proportion of males within a tested group approached plants, and often the same individuals visited them repeatedly throughout the duration of the trial. We counted each visit as a discrete event and assumed males were independently influenced every time they flew by the plant and were attracted by specific cues. However similar results could be obtained if such cues trigger learning of the plant location and produced an increase of foraging behavior around the area. It should be noted also that when data of all experiments were pooled together males that approached

plants had on average longer wing length than those that did not approach, although the difference was very small (approaching males = 43.48 ± 1.97 mm, other males = 42.33 ± 2.79 mm, t-test, df =253, P < 0.002). Experienced and inexperienced males did not differ in their responses to our treatments (χ2 test, P = 0.661, N=311)

Experiment 1. Males that flew close to experimental Passiflora lutea were attracted more often and for longer time those plants with larvae vs. undamaged plants or those with non-feeding immature stages (prepupae and pupae) (Fig. 2.3, table 2.2). Individual males visited the same plant repeatedly during the course of a test but on average more males were attracted to plants with larvae than to plants with other immatures or controls (Fig. 2.3A, χ2 = 13.69, df = 1, P < 0.001, χ2 = 0.48, df = 1, P = 0.49, χ2 = 0.44, df = 1, P = 0.51 for comparisons between control and plant with larvae, control and plant with prepupae and control and plant with pupae respectively). In most visits to control plants or those with prepupae and pupae males approached the foliage and left within few seconds (61%, 45%, 68% and 100% for control and plants with larvae, prepupae and pupae respectively). In contrast, plants with larvae more often elicited searching behavior that consisted of males flying up and down the plant and surroundings (37%, 47%, 22%, 24% for controls, larvae, prepupae and pupae treatments respectively). Males clearly recognized the presence of immatures as they hovered in front of them often for several seconds; this behavior was, however, more frequently seen in treatments with larvae than in those with prepupae or pupae (52%, 26%, 12% for plants with larvae, prepupae and pupae respectively).

Experiment 2. In the second set of experiments we investigated whether host plants with larvae caused arrestment of males due to 1) olfactory cues from larvae (and/or their interaction with the plant), 2) visual cues from larvae, or 3) a combination of both. Plants with silicone models, lacking odors from larvae or damaged tissue, elicited fewer and shorter visits compared to plants with live larvae, regardless of whether they were visible to or concealed from males (Fig. 2.4, table 2.3). Significant differences were found in the relative number of visits between the three treatments although Tukey’s post hoc pair-

ways comparisons were not significant (P = 0.06 and P = 0.95 for comparisons between control and visual, and control and olfactory cues respectively). On the other hand, post hoc comparisons showed a significant difference in average visit duration between control and plants with silicone models (P = 0.015) but not between control and plants with concealed larvae (P = 0.997), suggesting that olfactory cues alone can attract males flying close by. In fact most visitors to plants with exposed (control) and hidden larvae, presumably in presence of attractive odors, exhibited searching behaviors around the plant (71%) while fewer males searched when only visual cues from the silicone larva models were present (24%). Likewise results in experiment 1, males hovered in front of live larvae in most visits (69%), and, at least in some cases, in front of models (35%). Although we tried to match the base coloration of the model to this in a live animal, the former lacked ultraviolet (UV) marks present in some fifth instar H. charithonia (Figs 2.1, 2.5, table 2.4). Given that in some visits males hovered in front of our models it is possible that the lack of UV might have accounted for the lower attraction of this treatment to males. More individual males were attracted to plants with live larvae than to those with the silicone model, although the differences were not significant (Fig. 2.4A, χ2 = 2.77, df = 1, P = 0.09 and χ2 = 0.6, df = 1, P = 0.44 for comparisons between control and plants with model or concealed larvae respectively).

Experiment 3. Here we asked whether olfactory cues that caused arrestment in males were produced by the plant, the larvae or a product of their interaction. Male responses toward plants damaged with scissors, exposed to herbivory or those carrying non-feeding larvae did not differ. While duration of visits to undamaged plants with non feeding larvae were slightly shorter, we did not find significant difference in the relative number or duration of visits (Fig. 2.6, table 2.5), neither in the number of individual males attracted to plants across treatments (Fig. 2.6A, χ2 = 1.12, df = 1, P = 0.29 and χ2 = 0.42, df = 1, P = 0.52 for comparisons between control and plants cut with scissors or with larvae respectively). Cues that arrested males to plants appear to be released by both plants with damaged tissue and larvae.

Experiment 4. When we exposed males to plants with H. charithonia or A. vanilla larvae, they were more likely to visit and spend time searching those with their conspecific larvae, although the duration of visits was not significantly different between treatments (Fig. 2.7, table 2.6). Similarly, almost twice the number of males responded to plants with conspecifics than to those with A. vanilla (Fig. 2.7A, χ2 = 5.96, df = 1, P = 0.02). However once attracted to the plant males in both treatments displayed a comparable high proportion of searching behavior up and down the foliage (62%).

Summary of species that utilize host plants during mate searching

Surveying the literature we found that, besides Heliconius, 66 species (39 genera/ 5 families) search and mate in habitats containing their host plants (Appendix 1). Although many of those simply intensify their search in habitats with abundant hosts, in about half of the species males seems to actively seek around and above the foliage or perch exclusively on the plant itself (Table 2.7).

DISCUSSION Female butterflies typically locate plants to lay their eggs using a combination of volatiles and visual cues such as leaf shape and presence of new shoots (Rausher 1978, Papaj 1986, Feeny et al. 1989, Mackay & Jones 1989, Berenbaum & Feeny 2008). Our results show that plants play an important role in mate locating behavior of Heliconius, and that, like females, males are using visual and chemical cues to incorporate plants in their searching strategies. Searching patterns in insects are determined by the spatial distribution of their resources and multiple cues and criteria are necessary to narrow their search from a suitable habitat to the resource itself (Papaj & Prokopy 1989, Bell 1990). These experiments do not allow us to establish how males find the habitat of their host plants, however, our observations suggest that once males are in the area, visual and chemical cues from the plant probably serve to focus their search. Undamaged plants

were visited occasionally and often males were attracted to them when new shoots were exposed. Furthermore, the chances of finding and searching around plants increased by about three folds in the presence of chemical cues released by larvae or damaged tissue. Such higher number of approaches was the product of both, more individual males being attracted, and more visits performed by the same males. This suggests that odors probably not only signal the potential to find pupae, but also might trigger learning of location of that site for future visits to pupae that will soon form. There is compelling evidence of learning in insects (Dukas 2008a). Butterflies learn to associate colors or shapes with rewards (Rausher 1978, Papaj & Prokopy 1989, Weiss 1997, Weiss & Papaj 2003). Heliconius, in particular, maintain home ranges which probably result from a sophisticated spatial memory (Turner 1971, Ehrlich & Gilbert 1973). Their ability to remember locations is suggested by the establishment of site specific nocturnal roosting (Turner 1971, Mallet 1986, Mallet & Gilbert 1995) and regular patterns of visits to adult resources (Gilbert 1991). This behavior might also allow the incorporation of host plants to periodic patrolling until meristems are suitable for oviposition (Ehrlich & Gilbert 1973) or until mature pupae on those plants are found and ready to be guarded. Males visit plants regularly to access pupal developmental state and later wait until female eclosion (Deinert 2003), hence learning the location of such resources is a key factor in this mating behavior. Experiments in natural habitats (L. Mendoza-Cuenca, personal communication), and observation in our insectaries have shown that pupae are rarely found and females eclose unguarded when pupae are artificially suspended on Passiflora within few days of eclosion. This confirms the lack of long-range attraction pheromones in pupa (Chapter 3) and suggests that male’s early experience with host plants and immatures play an important role in pupal mating.

Plants release a wide range of organic volatiles as the result of their interaction with herbivores causing different behavioral responses in phytophagous insects and their natural enemies (Turlings et al. 1990, Dicke & van Loon 2000, Kessler & Baldwin 2002). Although many volatiles have been identified in leaves and fruits of Passiflora (Dhawan et al. 2004), results from our experiments here and chemical analysis (C. Estrada and C.

Rodriguez-Saona, unpublished data) provide evidence of the potential role of green-leaf volatiles in Heliconius mate searching behavior. Green-leaf volatiles are six-carbon alcohols, aldehydes and acetates commonly release at low rates from uninfested plants and at larger amounts soon after herbivory or tissue damage (Visser et al. 1979, Dicke & van Loon 2000, Kessler & Baldwin 2002). Such patterns of emission agree with behaviors found in our tests. First there was a rise in visitation rates from undamaged plant to those with any damaged tissue. Second, males were equally attracted to plants damaged with scissors than to those carrying caterpillars. Odors from caterpillars also arrested males to host plants, but preliminary chemical analysis indicate that the volatiles they release are similar to those emitted by the plant (Estrada C. and C. Rodriguez-Saona, unpublished data). Males never landed and tasted plants as females often do when choosing oviposition places (Rausher 1978) suggesting that compounds with lower volatility perceived only by contact might not be involved. Finally, although green-leaf volatiles are more likely involved in mate searching behavior of these butterflies, the role of additional cues such as terpenoids or other herbivore-induced plant odors cannot be ruled out.

If males respond to ubiquitous chemical released after tissue damage it could be expected that any herbivore feeding on their host plant could trigger similar reactions. Although some exceptions occur, different herbivores cause only minor variations in the volatile blend produced by a species of plant (Turlings et al. 1995, Geervliet et al. 1997). Results from experiments with plants carrying heterospecific larvae are thus intriguing as we found that males were in general less attracted to plants with Agraulis vanillae than to those with their conspecific larvae. Consequently, in these butterflies is possible that specificity when searching for mates is due to additional cues independent of the host plant. Larval coloration and odors are likely candidates. Males clearly recognized the presence of immatures of either species and hovered over them for several seconds, even in the case of silicone models. Once attracted by host odors the presence of heterospecic larvae could fail to elicit learning of plant location or trigger frequent visitation to the area. No evidence of major differences in volatiles released by larvae from both species

has been found (Estrada C. and Rodriguez-Saona C., unpublished data). In contrast, Heliconiini late instar caterpillars are brightly colored and quite diverse patterns occur across species in the group, particularly among those that exhibit pupal mating (Brown 1981, Mallet & Gilbert 1995). Such colorations likely functions to advertise larval toxicity to predators, and, although few cases of mimetic convergence have been proposed, they could also provide species-specific information to searching males (Brown & Benson 1977). Signals that have evolved in a defense context are often also used in intraspecific signaling (Summers et al. 1999, Weller et al. 1999, Jiggins et al. 2001b, Nosil et al. 2002). For example, females of the pipevine swallowtail butterfly, cued by conspecific larval warning colorations, avoid oviposition on occupied plants likely reducing competition for their offspring (Papaj & Newsom 2005).

Similar to other Heliconius, local populations of H. charithonia are often monophagous, although they use more than twenty Passiflora species throughout their range of distribution from Peru to southern United States (Comstock & Brown 1950, Benson et al. 1975, Beccaloni et al. 2008). Their host plants belong to different subgenera (Plectostemma and Granadilla) (Yockteng & Nadot 2004) and exhibit a wide diversity of leaf shapes. Furthermore, as many as 20 species of Heliconiini can be encountered in local communities, including several species of pupal maters (Brown 1979, Gilbert 1991). Such diversity is often correlated to the amount of Passiflora species available and coexistence of butterflies is the result of resource partitioning by Passiflora species, habitat and part of the plant utilized (Benson 1978, Mallet & Gilbert 1995). Phytophagous insects typically use specific ratios of common compounds, rather than specie-specific chemicals for host plant attraction and recognition (Bruce et al. 2005). Therefore, if as implied in our experiments, plant volatiles play a key role in male mate searching behavior of Heliconius, we could expect that similar blends are produced by host plants used by a butterfly species across its geographic range and that they are different from those released by other Passiflora in the same locality. Alternatively, if volatile mixtures vary in some other ways, local populations of males could learn to associate the visual and chemical characteristics from a particular Passiflora species with

the presence of conspecific larvae or pupae and concentrate their search only in those species in their home range. A combination of both hypotheses is also likely given our results from tests with A. vanillae and the general complexity of coexisting species of butterflies and plants across their geographic range. Experience can modify preferences for food or oviposition choices in phytophagous insects (Papaj & Prokopy 1989, but see Parmesan et al. 1995). For example, associative learning between host chemicals and leaf shape have been observed in the context of oviposition for the swallowtail butterfly Battus philenor, allowing this species to adapt to seasonal changes in their hosts relative abundances (Papaj 1986). Learning to associate plant and conspecific immature stimuli would have a selective advantage in mate searching behavior as males could track female oviposition preferences across their geographic range.

Mate searching, species recognition and mate choice in butterflies commonly involve the use of wing colour patterns (eg. Wiernasz 1995, Fordyce et al. 2002, Costanzo & Monteiro 2007, Papke et al. 2007). In Heliconius, in particular, male mate preferences have coevolved with changes in mimetic colorations (Jiggins et al. 2004, Kronforst et al. 2006), and pre and postmating isolation among closely related species is driven in part by differences in these traits (Jiggins et al. 2001b, Naisbit et al. 2001, Kronforst et al. 2006, Mavarez et al. 2006). This is also true for species that exhibit pupal mating (Crane 1955, Estrada & Jiggins 2008). Males of these species search for and court adult females and evidence suggest there is male polymorphism in mating strategies with patrollers and pupal mater males coexisting within populations (Hernández & Benson 1998, Mendoza-Cuenca & Macías-Ordó nez 2005). They also exhibit communal roosting, were visual cues from wing coloration seem to be important (Mallet & Gilbert 1995). Intrageneric Müllerian mimicry in this group of butterflies is common and often involves pair of species in different phylogenetic clades, (Eltringham 1916, Gilbert 1991, Beltrán et al. 2007). As species converge in their colour patterns due to mimicry, and those patterns are also used in mate recognition, interspecific sexual attraction is common and might impose a cost to mimicry due to signal confusion (Estrada & Jiggins 2008). Although it is not known what proportion of mating in the wild occurs on pupae, field

studies suggest that this is the predominant mating system for pupal mating species (Deinert 2003; L. Mendoza-Cuenca personal communication). It is thus possible that a switch from searching for mates using primarily wing coloration to searching for pupation places using plant and immature cues in a proportion of males in the population may reduce costs of harassment between co-mimics. Furthermore, employing alternative signals to wing patterns during mate choice may imply that in pupal mating species novel colorations originated by mutations might have better chances of persisting in the population. This would contribute to the diversification of warning coloration in species with pupal mating some of which presumably become models other Heliconius (non- pupal mating) mimic (Mallet 1999, Flanagan et al. 2004). Overall the occurrence of this mating system appear to have had significant implications in the evolution of mimicry in these greatly diverse group of butterflies (Gilbert 1991).

Role of host plants in the mate searching behavior of butterflies.

Similar to Heliconius, several species of butterflies utilize host plants and host plant habitats to find mates as these places are good predictor of eclosion sites of females (Scott 1974, Rutowski 1991, Wiklund 2003). From the list of species where host plant seem to play a key role in sexual behavior (Table 2.7) only in two cases the cues that males use in this task have been suggested. Anthocharis cardamines (Pieridae) males patrol for females in forest edges and are occasionally attracted to host by their white flowers where they find females and mate (Table 2.7). The authors however, considered visits to flowers as accidents due to male appeal for white, the dominant color of females of this species (Wiklund & Ahrberg 1978). In another example, males of Jalmenus evagoras (Lycaenidae) seems to be are attracted to their host Acacia by the presence of mutualistic ants that attend larvae and pupae of this species. However whether or not males use cues from the plant itself have not been examined (Elgar & Pierce 1988). The amount of species whose mate searching behavior is associated with their host suggest that at least in some butterflies males have the ability to find and recognize host plants

and they do so as a way to find partners. The ability to find plants by males although poorly recognized as a mate searching strategy is common in other contexts among Lepidoptera. Males of Ithomiine and Danaine butterflies and Arctiidae moths harvest alkaloids from withered tissue of particular plant species which they use as defenses as well as precursors for pheromones (Pliske et al. 1976, Weller et al. 1999). Attraction to plants seems to be mediated by volatiles derived from chemical transformations of the alkaloids (Pliske et al. 1976, Birch et al. 1990). Plant volatiles are also cues that enhance attraction to female sex pheromone in several moth species (Emelianov et al. 2001, Deng et al. 2004, Yang et al. 2004). Finally, males are oriented to nectar and pollen sources using flower coloration as well as odors (Weiss 1997, Andersson & Dobson 2003b, Cunningham et al. 2006).

The interaction between host plants and the sexual behavior of phytophagous insects might drive ecological and evolutionary processes with important outcomes in the diversification of these groups of organism (Drès & Mallet 2002, Cocroft et al. 2008). First, insects with low population sizes that mate in their host plants could evolve host plant specialization (Gilbert 1978, Futuyma & Moreno 1988, Jaenike 1990) which in turn can originate habitat differentiation likely affecting other aspects of their ecology and behavior such as escape from enemies, thermoregulation, or dispersion (Gilbert 1984, Dennis & Shreeve 1988, Dennis et al. 2004). Second, host plant fidelity, defined by Feder (1998) as the tendency of phytophagous insects to mate and oviposite in the host plant, facilitates ecological speciation by promoting assortative mating increasing reproductive isolation (Schluter 2001, Funk & Nosil 2008). Although several herbivorous insects seem to have diverged genetically due to such mechanism (Feder 1998, Funk 1998, Via 1999, Cocroft et al. 2008), the evidence for this happening in butterflies is so far inconclusive. Speciation in at least two genera of lycaenid butterflies, Lycaeides and Mitoura, has been proposed to be the result of host shifts associated to host fidelity (Nice & Shapiro 2001, Nice et al. 2002, Forister 2005, Gompert et al. 2006). Species feeding in different host plants exhibit behavioral and morphological differences suggesting perhaps that preferences for different plants might have contributed to their diversification. However,

25 genetic differentiation has not being found and authors proposed that host shifts might have happened recently and chosen neutral molecular markers failed to detect variations originated in that short time (Nice & Shapiro 2001, Nice et al. 2002). Another lycaenid genus, Euphilotes, known to oviposite, feed and mate on flowers of its larval host plant Eurigonun, exhibit a complex array of species, geographic races and sympatric host races distributed across western United States (Pratt 1994). Evolution in this group seems to be in part the result of host shifts to plants with different blooming patterns and facilitated by a sedentary behavior of adults around their host plant (Pratt 1994). In contrast to these lycaenids, there is no evidence that host plant shifts initiate speciation in Heliconius (Jiggins et al. 1997), instead, divergence on wing warning patterns and adaptation to local mimicry rings have been the major causes of diversification in the genus (Mallet et al. 1998, Jiggins et al. 2001b). Nevertheless, oviposition preferences and host fidelity seems to be necessary to explain plant specialization and thus the coexistence of multiple congeneric species that characterize Heliconius communities (Gilbert 1991, Jiggins et al. 1997, Gilbert 2003).

Adaptation to feed in new hosts is widely recognized as an important driver in butterfly diversification (Ehrlich & Raven 1964, Braby & Trueman 2006, Mullen 2006, Weingartner et al. 2006, Wheat et al. 2007, Peña & Wahlberg 2008). It is then likely that using host plants as mating places has the potential to facilitate such process as it has been demonstrated in other phytophagous insects. Certainly, mate searching strategies can reduce hybridization in butterflies as suggested for groups of riodinids whose perching location and timing are used as part of the reproductive isolation mechanism among congeneric sympatric species (Callaghan 1982, Hall 2005). Measuring the extend of host fidelity in butterflies and the implications of this behavior in their diversification is then important, particularly in tropical species from which we have little information of mate searching behavior and where population densities are lower and diets more specialized than in temperate regions (Currie & Fritz 1993, Price et al. 1995, Coley & Barone 1996, but see Novotny et al. 2006, Dyer et al. 2007).

FIGURES

A 1

0.8

B 0.6

0.4

0.2

Reflectance % / Maximun reflectance reflectance % / Maximun Reflectance 0 300 400 500 600 700 Wavelenght (nm)

Figure 2.1. A. Reflectance spectra of base color in body of Heliconius charithonia fifth instar larvae (grey) and silicone rubber model (black). B. Live (left) and silicone rubber model (right) fifth instar larvae.

Figure 2.2. Fifth instar Agraulis vanillae larvae

A 0.16 * B 16 * 14 0.12

) 12 s visits (

of 0.08 10 8 duration

Number 6 0.04 Visit 4

2 0 C L C Pp C P 0 (16) (44) (8) (12) (9) (13) C L C Pp C P

Figure 2.3. Standardized number of visits (A) and average visit duration (B) of Heliconius charithonia males to Passiflora lutea plants in experiment 1 (Mean ± 1SE, N = 19 for experiments with larvae, and N= 10 for experiments with prepupae and pupae). Treatments include undamaged P. lutea (C = controls) paired to plants with fifth instar larvae (L), prepupae (Pp) or pupae (P). Asterisk (*) indicate significant differences (P < 0.05, paired t-test). Numbers in parenthesis indicate the overall number of males that visited plants in each treatment.

A B 0.12 a a 40 a

0.1 35

30 a 0.08 (s)

visits

25 of 0.06 20 a b duration 0.04 15 Number Visit 0.02 10 5 0 Control Visual Olfactory 0 Control Visual Olfactory (21) (11) (27)

Figure 2.4. Standardized number of visits (A) and average visit duration (B) of Heliconius charithonia males to Passiflora lutea plants in experiment 2 (Mean ± 1SE, N = 12). Treatments include plants with larvae (control), silicone model larvae (visual), and concealed larvae (olfactory). Bars labeled with different letter are significantly different (P < 0.05 Tukey’s HDS post hoc analysis). Numbers in parenthesis indicate the overall number of males that visited plants in each treatment.

A B C D

Figure 2.5. Heliconius charithonia larvae under visible (A) and ultraviolet (UV) light in their dorsal (B), lateral (C) and ventral (D) positions. White patterns in pictures B to D correspond to UV reflectance. Pictures were taken with a high-resolution digital video camera with UV sensitivity (Sony XCD-Sx900U, Edmund Industrial Optics), using a 380 nm filter. Gain and Shutter were kept constant at 2228 and 2043 respectively. Each larva was chilled and then set next to a white reflectance standard on a black surface and under two sources of black light. The positions of the camera as well as light sources were maintained constant for all pictures. Each larva was photographed in its dorsal, lateral and ventral side and pictures were saved as bmp files using the software Sony XCD- SX900 (version 1.1.0.7 2002).

A 0.12 B 16 14 0.1

) 12 s (

0.08

visits 10

of 0.06 8 duration 6 0.04 Number Visit 4 0.02 2

0 0 Control Scissors Larvae Control Scissors Larvae damage damage (38) (29) (32)

Figure 2.6. Standardized number of visits (A) and average visit duration (B) of Heliconius charithonia males to Passiflora lutea plants in experiment 3 (Mean ± 1SE, N = 15). Treatments include olfactory cues from larvae feeding on host plant (control), scissors damage, and larvae. No significant different were found among treatments (P > 0.05, ANOVA table 2.5). Numbers in parenthesis indicate the overall number of males that visited plants in each treatment.

A 0.2 B * 25

0.15 20

visits (s)

of 15 0.1 10 duration 0.05 Number 5 Visit

0 0 H. charithonia A. vanillae H. charithonia A. vanillae (46) (26)

Figure 2.7. Standardized number of visits (A) and average visit duration (B) of Heliconius charithonia males to Passiflora lutea plants in experiment 4 (Mean ± 1SE, N = 15). Treatments include plants with H. charithonia and plants with Agraulis vanilla larvae. Asterisk (*) indicate significant differences (P < 0.05, paired t-test). Numbers in parenthesis indicate the overall number of males that visited plants in each treatment.

TABLES

Table 2.1. Experimental design to find cues involved in discovery and recognition of mates in pupal mating species Heliconius charithonia.

Experiment objective Control Treatment N 1. To examine whether Undamaged host plant Fifth instar larvae on host 19 host plant or immature plant stages are attractive to Undamaged host plant Prepupae on host plant 10 males Undamaged host plant Young pupae on host plant 10

2. To examine the use Exposed larvae on host 1. Silicone larvae model on 12 of visual and chemical plant host plant (remove damage cues to find host plants. (visual and olfactory cues tissue and larvae odors) from larvae and damaged 2. Concealed larvae on host tissue) plant (remove visual cues from larvae)

3. To examine origin of Concealed fifth instar 1. Host plant material cut 15 cues larvae allowed to eat with scissors (remove odors (odors of larvae and host from larvae) plant) 2. Concealed fifth instar larvae on host plant not allowed to eat (remove odors of damaged tissue)

4. To test for species Conspecific larvae Heterospecific larvae 15 recognition cues during (Agraulis vanilla) mate searching

Table 2.2 Results of paired t-test for experiments 1. Treatments include undamaged Passiflora lutea (control) paired to plants with fifth instar larvae, prepupae or pupae of H. charithonia.

Control vs. larvae Control vs. prepupae Control vs. pupae

df t P* df t P* df t P*

a) Average number of visits (standardized)

18 5.197 < 0.001 9 0.695 0.505 9 0.532 0.607

b) Average visit duration

18 3.474 0.003 9 0.253 0.806 9 1.259 0.240

*Reported P values are for two-tailed t-test. Values in bold represent significant effect.

Table 2.3. Results of Two-way ANOVA without replications for experiment 2. Treatments include Passiflora lutea plants with larvae (control), silicone model larvae and concealed larvae.

Source df MS F P*

a) Average number of visits (standardized)

Tests 11 0.036 1.480 0.208 Treatments 2 0.083 3.446 0.050 Error 22 0.024

b) Average visit duration

Tests 11 4.525 1.515 0.196 Treatments 2 19.801 6.631 0.006 Error 22 2.986

*Reported P values are for two-tailed F-test. Values in bold represent significant effect.

Table 2.4. Analysis of UV reflectance in pictures of H. charithonia fifth instar larvae. Using Photoshop (Adobe System Incorporated, version 8.0), male and female pictures were compared using two criteria. First we measured percentage of the body with UV marks. The number of pixels with UV was estimated by selecting a UV mark with the eyedropper tool mark and using the histogram function in the RBG channel. We compared the amount of pixels with UV reflectance to the total number of pixels of the larva. Second, we measured percentage of intensity of UV reflectance compared with the maximum intensity reflected by the white standard. The mean luminosity of the larval area with UV marks was calculated using the histogram function in the luminosity channel. Mean ± standard deviation are given. No significant differences were found across measures between sexes (Mann-Whitney U Tests, P > 0.05).

Position (sample size) % body with UV % intensity of UV marks reflectance

Female Dorsal (10) 30.31 ± 16.92 32.25 ± 8.07 Lateral (6) 18.03 ± 7.52 36.76 ± 10.57 Ventral (3) 14.65 ± 3.39 29.11 ± 7.46

Male Dorsal (7) 29.68 ± 13.67 36.62 ± 10.04 Lateral (7) 24.55 ± 16 31.48 ± 6.18 Ventral (4) 11.03 ± 7.56 24.25 ± 3.38

Table 2.5. Results of Two-way ANOVA without replications for experiment 3. Treatments include olfactory cues from larvae feeding on the host plant, Passiflora lutea (control), damaged tissue using scissors and larvae.

Source df MS F P*

a) Average number of visits (standardized)

Tests 14 0.034 2.391 0.024 Treatments 2 0.015 1.095 0.348 Error 28 0.014

b) Average visit duration

Tests 14 4.460 2.057 0.051 Treatments 2 3.603 1.662 0.208 Error 22 2.168

*Reported P values are for two-tailed F-test. Values in bold represent significant effect.

Table 2.6. Results of paired t-test for experiment 4. Treatments include plants with H. charithonia and plants with Agraulis vanilla larvae.

df t P*

a) Average number of visits (standardized)

14 2.307 0.037

b) Average visit duration

14 1.545 0.145

*Reported P values are for two-tailed t-test. Values in bold represent significant effect.

Table 2.7. Butterfly species with mate locating behavior involving host plants.

Butterfly species1,2 Host plant3 Searching strategy Ref.4 Lycaenidae Brephidium exilis Atriplex spp. Patrol around and close to 1 branches of plant Callophrys muiri (Mitoura Cupressus macnabiana Males form leks close to plant 2 muiri) C. nelsoni (Mitoura Calocedrus decurrens Males form leks close to plant 2, 14 nelsoni) Euphilotes battoides Eriogonun Patrol on and between plants 1,3 E. enoptes (Philotes Eriogonun Patrol on and between plants 1,3 enoptes) E. rita Eriogonun Patrol on and between plants 1,3 Habrodais crysalus Quercus chrysolepsis Patrol around and close to 1 branches of plant H. grunus Quercus chrysolepsis Patrol around and close to 1 branches of plant Hypaurotis crysalus Quercus gambellii Patrol on and around plant 1 Jalmenus evagoras1 Acacia Patrol on and around plant. 4 Pupal mating. Phaeostrymon alcestis Sapindus drummondi Patrol on and around plant 1 Plebejus idas (Lycaeides Fabaceae Patrol on and around plant 15 idas) P. melissa (Lycaeides Fabaceae Patrol on and around plant 15 melissa) Satyrium fulginosa Lupines Perch on and around plant 1 Nymphalidae Anartia jatrophae Bacopa monnieri Perch, males defend territories 5 always containing host plant Asterocampa leilia1 Celtis pallida Perch on or around plant 6 Eueides aliphera Passiflora Perch, males defend territories 7 always containing host plant Heliconius 18 spp. Passiflora Patrol on and around plant. 8 Pupal mating Pieridae Anthocharis cardamines1 Several crucifers Patrol edges of forest and 9 around plant Neophasia menapia Patrol around and close to 1 branches of plant Pieris rapae crucivora Brassica oleracea Patrol around and close to 10 (planted) branches of plant Papilionidae Ornithoptera priamus Aristolochia tagala Patrol, males attracted to pupa 11 caelestis on host plant Papilio glaucus Prunus, Patrol around and close to 16

Fraxinus,Liriodendrum branches of plant P. xuthus Citrus, Poncirus, Patrol around and close to 17 Zanthoxylum branches of plant Parnassius clodius Patrol on and around host plant 1 Hesperiidae Erynnis brizo Quercus gambellii Perch and patrol on and around 12 plant E. martialis Ceanothus fendleri Perch and patrol on and around 12 plant E. pacuvius Ceanothus fendleri Perch and patrol on and around 12 plant Ochlodes yuma Phragmites communis Perch on and around plant 1, 13 Riodinidae Apodemia nais Ceanothus fendleri Perch and patrol on and around 1, 12 plant 1 Species included in (Rutowski 1991) 2 When butterfly scientific name changed since reported in references, the more recent name appears first and old name is given in parenthesis. We used here Brower (2008) taxonomic nomenclature. 3 Host plant species reported in references. 4 References: 1, Scott (1975a) ; 2, Nice and Shapiro (2001), 3, Pratt (1994) ; 4, Elgar and Pierce (1988) ; 5, Lederhouse et al. (1992); 6, Rutowski and Gilchrist (1988); 7, Benson et al. (1989) ; 8, Gilbert (1991) ; 9, Wiklund and Ahrberg (1978) ; 10, Hirota and Obara (2000) ; 11, Borch and Schmid (1973) ; 12, Scott (1982) ; 13 Scott et al. (1977) . 14 Forister (2005) ; 15. Nice et al. (2002) . 16, Lederhouse (1995) . 17. Yamashita (1995) .

CHAPTER 3: Pupal Guarding by Male Butterflies: Pre-eclosion Sex Discrimination and Sexual Conflict

ABSTRACT Sexual selection in the form of male-male competition has led to the evolution of a wide range of mate searching and mating behaviors in animals. In few species, selection pressures for gaining access to receptive females has resulted in males developing the ability to find females before they are sexually mature and associating with them until mating. This behavior, known as precopulatory mate guarding, increases reproductive success in males but might cause reduction of fitness in females, resulting in potential for sexual conflict. Here we characterized precopulatory mate guarding behavior in the butterfly Heliconius charithonia and investigated visual and chemical cues involved in recognition of immature females. Chemical analysis and behavioral tests allow us to conclude that males of this species recognize female pupae by using sex specific chemical cues that act at short range at the end of pupal development. Odors released from pupae were identical in both sexes except for the volatiles linalool and linalool oxide produced respectively by male and female pupae. Visual cues, although important to locate and recognize pupae are likely not important for sex recognition. Based on the chemicals used in sex recognition by males, we propose that H. charithonia females have control over the production of this signal, indicating that there is no sexual conflict even in absence of direct female mate choice probably.

INTRODUCTION Sexual selection in the form of intrasexual competition for mates has lead to the evolution of a wide range of mate searching and mating behaviors in animals (Emlen & Oring 1977, Thornhill & Alcock 1983, Clutton-Brock & Parker 1992). Reproductive fitness in males is typically determined by the number of matings they obtain, together with the reproductive value of their partners (Trivers 1972). Consequently, selective

pressures for gaining access to receptive females before other males do has resulted in adaptations that improve mate location such as early maturation or well-developed sensory and motor abilities (Andersson & Iwasa 1996). Competition for mates has in some species selected for the ability to locate, and ocassionally establish permanent associations with females no yet sexually mature (Thornhill & Alcock 1983, Ridley 1990). Remaining close or attaching themselves to unreceptive females give males a competitive advantage over other males increasing their chances of mating (Parker 1970). Variations of this behavior have evolved several times (Ridley 1983) and have been studied under names like precopulatory mate guarding in species of aquatic crustaceans, salmon, frogs and ants (Ridley 1983, Jormalainen 1998, Foitzik et al. 2002, Morbey 2002), pupal attendance in mosquitoes (Provost & Haeger 1967, Slooten & Lambert 1983, Conner & Itagaki 1984), cohabitation of males and juvenile females in spiders (Jackson 1986, Fahey & Elgar 1997), and pupal mating in butterflies (Gilbert 1976, 1984, Deinert 2003).

Precopulatory mate guarding has evolved as a male time investment strategy when fitness gained by spending time guarding an unreceptive female is higher than this gained by continuing searching for potential mates (Parker 1974). Theoretical models that examine the stability of precopulatory mate guarding predict that given time-restricted female receptivity (Ridley 1983), and the existence of cues that signal proximity to maturity (Parker 1974), both encounter rates and guarding costs are key factors that affect fitness payoff of males and determine guarding duration (reviewed by Jormalainen 1998).

The benefit to males of mate guarding as a behavior to gain access to females is clear: with highly biased operational sex ratios and very dispersed receptive females, guarding increases their chances of mating (Parker 1974, Ridley 1983). In contrast, the benefits for females are unknown. In some species of crustaceans females are able to resist males and decrease the duration of guarding or delay oviposition until a male they like is attached (Ridley 1983, Jormalainen 1998). In other cases however, females do not have active mate choice as mating occurs with teneral females not yet able to escape or

avoid copulation (spiders and butterflies) (Ridley 1983, Deinert 2003). Such coercing mating may impose costs to females if mating happens with low quality males causing reduction in their fecundity. Thus if females experience direct or indirect costs due to increase mortality, reduction of feeding capabilities or limitations in active female mate choice there is potential of sexual conflict as a consequence of this male searching behavior (Clutton-Brock & Parker 1995, Jormalainen 1998, Arnqvist & Rowe 2005).

A poorly studied component of precopulatory mate guarding is the underlying mechanisms and the evolutionary origin of clues used by males to forecast female receptivity. These aspects are important to understand how this behavior evolved and how it is maintained across a diverse range of organisms. It can also provide clues about the incidence of sexual conflict. For example, clues that help males to assess female maturity could be part of a communication system (signals) where females that benefit with guarding inform males of their oncoming receptivity. In contrast, these cues could be metabolic byproducts of traits that appear during development and close to maturity with little female control (Jormalainen 1998).

In butterflies several species in the genus Heliconius (Nymphalidae: Heliconiinae), and only one species outside the genus, exhibit precopulatory mate guarding by perching on pupae (Gilbert 1976, 1984, Elgar & Pierce 1988). In Heliconius, males search for females by trap-lining habitat patches with larval host plants and in fact some species specialized on searching for immatures which then they visit regularly to assess their developmental state (Gilbert 1976, Deinert et al. 1994, Mallet & Gilbert 1995, chapter 2). At the end of the pupation period males position themselves on the pupae (sit) and wait until they are able to mate with emerging females (pupal mating) (Gilbert 1991, Deinert 2003). In some species males even penetrate the pupal cuticle and are thus able to compete for copulations as the teneral female emerges. Pupal mating behavior evolved once in Heliconius and appears to occur in all species within a monophyletic clade in the genus (18 sp. 42% of the species in the genus) (Lee et al. 1992, Beltrán et al. 2007). Males of the pupal mating clade retain the capacity to search for and

41 court adult females (Crane 1955, Estrada & Jiggins 2008) and some evidence suggests there is male polymorphism in mating strategies with patrollers and pupal mater males coexisting within populations (Hernández & Benson 1998, Mendoza-Cuenca & Macías- Ordó nez 2005). Females are believed to mate only once (Gilbert 1976, Ehrlich & Ehrlich 1978, Boggs 1990), but males mate multiple times creating a strong biased operational sex ratio (Emlen & Oring 1977). Although pupal mating was noted anecdotally by lepidopterists over a century ago (e.g. Edwards 1881, Bellinger 1954, Gilbert 1991), it was not known to modern behavioral ecologists who studied Heliconius (e.g. Crane 1955, Turner 1971) until independently rediscovered by Gilbert (1976). In the last 25 years pupal mating has become a topic of interest to evolutionary ecologist, however, few studies have investigated this mating system in detail (Deinert et al. 1994, Deinert 2003, Mendoza-Cuenca & Macías-Ordó nez 2005), and little is known about the cues used to find and recognize conspecific females.

Here we investigated the pupal mating behavior of Heliconius charithonia (Linnaeus). Our research focused on identifying visual and chemical cues that allow males to estimate maturity and to recognize the sex of pupae, both key elements of precopulatory mate guarding. We discuss such cues found in H. charithonia pupae in reference to what they suggest about the incidence of sexual conflict in this particular mating behavior.

METHODS

Butterfly rearing

From February to May 2007 we studied a population of Heliconius charithonia vazquezae in a 22x10x10 m greenhouse in Brackenridge Field Laboratory (BFL) at University of Texas, Austin. This greenhouse had a climate control system that allowed external variations of temperature and humidity but prevented extreme minimum and

maximum temperatures common in winter or summer. Temperature fluctuated daily from about 15 to 30 oC in mid February and from 20 to 32 oC at the end of May. Light/dark periods followed natural cycles as walls and roof are made with a clear cover. Evaporated cooled air flowes across the facility’s natural habitat of trees, shrubs and vines so that butterflies experience approximately field conditions. We started a population of H. charithonia with adults collected around the field station in summer of 2006 and supplemented periodically with wild-caught individuals. Butterflies were allowed to breed freely but we controlled population size by removing adults to prevent overpopulation and overuse of host plants (c.a. 50 butterflies with a 2:1 male:female ratio). New individuals were marked daily with a serial number written with a permanent marker in their forewing. This, together with frequent census and listing of dead or removed butterflies, served to estimate their residence time in the greenhouse, which was used as an approximation for life span. Body and forewing length were also measured for each individual with a caliper to the closest 0.01 mm. Adults fed ad libitum with sugar water solution (10%) and pollen and nectar from Psiguria tabascensis, P. bignoniacea, Psychotria poeppigiana and Lantana camara flowers.

Although Passiflora affinis and P. lutea are the host plant of local populations of H. charithonia (Austin area, Texas, USA), females laid eggs, and larvae grew successfully, in other Passiflora at the greenhouse (P. biflora, P. auriculata, P. orstedii, P. pittieri and P. talamancensis). Larvae pupate on or close to host plants and eclosed as adult butterflies after 9 to12 days depending on ambient temperature. Pupae are typically light brown but the cuticle become translucent, and wings and adult body colors are revealed about 24 hours before eclosion. Because studies in Heliconius pupal mating suggest that males recognize female pupae only at the last part of the pupal stage (Deinert 2003), here we classified as early pupae those from day 1 to7, and as late pupae those whose cuticle was already translucent. We marked and monitored pupae daily until eclosion to determine whether they have been guarded by males.

Visual analysis

Previous analysis revealed lack of differences in both, morphology and coloration between male and female H. charithonia pupae (C. Estrada, unpublished data). However, butterfly eyes are sensitive to ultraviolet light (UV) (Briscoe & Chittka 2001). Therefore, to determine whether pupae reflect UV light and whether there are differences in patterns that could help males identify female pupae we took pictures using a UV sensitive lens. Pictures were taken with a high-resolution digital video camera with UV sensitivity (Sony XCD-Sx900U, Edmund Industrial Optics), using a 380 nm filter. Gain and Shutter were kept constant at 2228 and 2043 respectively. Each pupa was set next to a white reflectance standard on a black surface and under two sources of black light. The positions of the camera as well as light sources were maintained constant for all pictures. Each pupa was photographed in its dorsal, lateral and ventral sides.

Pictures were saved as bmp files using the software Sony XCD-SX900 (version 1.1.0.7 2002) and analyzed using Photoshop (Adobe System Incorporated, version 8.0). Male and female pupae pictures were compared using two criteria. First we measured the percentage of the body with UV marks. The number of pixels with UV was estimated by selecting a UV mark with the eyedropper tool mark and using the histogram function in the RBG channel. We compared the amount of pixels with UV reflectance to the total number of pixels of the pupa. Second, we measured the percentage of intensity of UV reflectance of pupae compared with the maximum intensity reflected by the white standard. The mean luminosity of the pupal area with UV marks was calculated using the histogram function in the luminosity channel.

The percentage of area cover with UV marks and its luminosity in male and female pupae were compared using the non-parametric Mann-Whitney U Tests in R 2.1.1 (R Development Core Team, 2005).

Chemical analysis

In order to characterize pupal odors we generated two types of samples. First, cuticular chemicals from individual H. charithonia pupae were extracted for 5 minutes with 1.5 ml of methylene chloride at room temperature. The solvent was reduced to about 30 μl using a gentle stream of nitrogen and then kept at -20oC until analysis. Extracts from four late male and six late female pupae were analyzed. Second, we sampled volatiles released by male and female pupae using Solid Phase Microextraction (SPME) with a 65 μm polydimethylsiloxane / divinylbenzene (PDMS/DVB) fiber (Supelco, Sigma-Aldrich Corporation). One pupa was introduced in a 4ml glass vial with a plastic lid and septum and kept at 24oC. Then the SPME was introduced and the fiber exposed. After 5 hours the needle was retracted and immediately analyzed by gas chromatography / mass spectrometry (GC-MS). GC-MS of methylene chloride extracts and SPME were performed with a Hewlett-Packard model 5973 mass selective detector connected to a Hewlett-Packard model 6890 gas chromatograph using a BPX-5 fused silica capillary column (SGE, 25 m x 0.25 mm, 0.25 µm film thickness). Injection was performed in splitless mode (250°C injector temperature) with helium as the carrier gas (constant flow of 1 ml/min). The temperature program started at 50°C, held for 1 minute, and then rose to 320°C with a heating rate of 5°C/min. All compounds were identified by comparison of the mass spectra, gas chromatographic retention index, and retention times with those of authentic reference samples. Chiral analyses were performed using a Supelco Beta- Dex 225 capillary column (30 m x 0.25 mm, 0.25 µm film thickness) in the same GC-MS equipment and a temperature program started at 50°C, held for 1 minute, and then rose to 200°C with a heating rate of 2°C/min.

Quantification of the two main volatiles identified by SPME were carried out using external standardization (Supelco 2001). Calculating the amount of a compounds in head space samples extracted with SPME are challenging both because of the effect of sampling conditions in the extraction, and because differeces in affinity of compounds to the fiber coat (Górecki et al. 1999). We created a calibration curve for each compound by analyzing the head space of solutions with different concentration dissolved in pentadecane. This hydrocarbon was used to simulate volatilization from the hydrophobic

cuticle of the pupal case. Head space of 1µl of 1, 10, 100, or 1000 ng/µl solutions were sampled during 120 minutes in a 4 ml glass vial and under the same conditions as pupae were sampled before (previous tests showed that at about 60 minutes liquid and gas phase solutions were in equilibrium in the vial).

Gas chromatogram peaks from methylene chloride extracts were compared between male and female pupae samples. We included in the analysis compounds present in at least two samples with peak areas higher than 0.1% of the total peak area. The area under the peak, which is proportional to the amount of the compound in the extract, was converted into percentage for each sample. Information for each compound was then relative to the total mixture (compositional data) and thus transformed accordingly before the analysis (Aitchison 1986, Liebig et al. 2000). First, we considered absence of compounds as an artifact of our measuring process and applied the zero replacement 2 technique by Fry et al. (2000). Zero percentages were replaced by τA = δ(M+1)(N-M)/N

and nonzero ones by Wi x τS, where Wi is the percentage of peak i (when Wi > 0), τS = δM(M+1)/N2, M is the number of zeros in an individual sample, N the total number of peaks analyzed, and δ is the maximum rounding error (δ = 0.0001). Then percentage data

were log-ratio transformed following Reyment (1989) formula Zi,j = log((Xi,j/g(Xj)),

where Xi,j is the peak area of compound i in individual j, g(Xj) is the geometric mean of

the area of all peaks for individual j, and Zi,j is the transformed area for peak i in individual j. The number of variables (peaks) to compare between male and female pupae chromatograms was reduced using Principal Component Analysis (PCA). Extracted factors (those with eigenvalues >1) where then compared between sexes using Hotelling’s T square test for multiple variables (STATISTICA, version 8, 2007).

Behavioral assays

Once positioned on pupae males wait until eclosion and only leave due to intense male-male competition or strong disturbances. After few minutes, however, they often

return and sit again or compete with new resident males. We took advantage of this behavior to test potential compounds used as cues of sex identification by males. Males that were sitting on pupae were gently disturbed and those pupae were then left unmanipulated, or perfumed with a test odor. We monitored whether males came back and considered them to have accepted a pupa if they sat and guarded it again. Test where males did not come back within 10 minutes where discharged. The sex of the pupa was determined after eclosion and only experiments done on female pupae were considered for the analysis.

Two compounds were used to perfume pupae. Preliminary tests showed that even after several minutes males rejected pupae to which common solvents (hexane, methylene chloride and mineral oil) were added. Therefore compounds were tested in their pure form. Chemicals were added by touching the pupae with the tip of a 0.5 μl micropipette. Each pupae obtained an average of 30.4 μg (± 20.4 μg, N=20) of the compound. Quantities were calculated in the laboratory by adding the compounds to empty pupal cases and weighted them using an ultra-microbalance (Metter Toledo UMX2 d=0.1 μg) at 29oC and 24% humidity. Chemiclas used were racemic linalool (Fluka) and racemic cis- and trans furanoid linalool oxide (2-methyl-2-vinyl-5-(1- hydroxy-1-methylenthyl) tetrahydrofuran) synthesized following Reiter et al. (2003). Proportion of female pupae accepted after treatment with odors where compared to control pupae using a Fisher’s exact test, performed using the statistical computing program R 2.2.1. (R Development Core Team, 2005).

RESULTS Males motivated to mate repeatedly flew circuits of hosts and non host plants frequently inspecting hanging objects with similar size, shape and color to pupae (e.g. dead leaves or branches, empty pupal cases). Following the encounter with a pupa males would fly very close for about five seconds surrounding it with their antennae. In the case of a female pupa, time spent by investigating males increases about three days before

eclosion. Males sat on and guarded 77% of female pupae and 29% of male pupae (χ2 test, df=1, P < 0.001, N=297 pupae with 1:1 sex ratio). Guarding started in most cases on the afternoon prior to the day of eclosion (71%), but occasionally males sat on pupae up to 10 days before, although for a short time. One or two males (mean 1.5 ± 0.51 males) sat opposite to each other with their heads down, and defended their position against others by opening their wings embracing the pupa. Most guarding males (93%) only sat on while the rest also inserted their abdomen through the cuticle close to the pupal abdominal section (Fig. 3.1); in both cases mating happened at the moment of female eclosion.

Less than half of the males from our greenhouse population that were at least 5 days old were observed guarding pupae throughout the study period (40%, N= 121). Such males were similar in size (mean wing length of guarding and non guarding males was 41.36 and 40.78 mm respectively, T-test, df= 107, P = 0.26), but lived in average about 10 days longer than those never seen guarding pupae (mean live span of guarding and non-guarding males was 26.35 and 17. 59 days respectively, T-test, df= 83, P < 0.001). Furthermore, we observed individual males guarding in average 0.8 pupae during their live at the greenhouse (± 1.5, max = 11) and found a significant correlation between longevity and male number of guarding (Spearman rank order correlation, 0.38, P < 0.05). Because our observations resulted from periodic visits to the greenhouse it is unknown whether males never seen guarding had different mating strategies, as has been suggested (Mendoza-Cuenca & Macías-Ordó nez 2005), or had failed to win a position on the pupae.

Visual analysis

Early pupae exhibit several marks with ultraviolet (UV) reflection distributed across their body (Fig 3.2). Nevertheless, it seems unlikely they are used as cues for female pupae recognition as we did not find any consistent difference in their

distribution, area of the body covered or intensity. UV marks covered about 15% of the pupal body in both sexes (Fig. 3.3A, Mann-Whitney U Tests, U = 13, P = 0.914; U = 13, P = 0.792; U = 16, P = 0.352 for dorsal, lateral and ventral position respectively), and had similar intensity when compared with a white standard (Fig. 3.3B, Mann-Whitney U Tests, U = 7, P = 0.352; U = 15, P = 1; U = 13, P = 0.791 for dorsal, lateral and ventral position respectively). Furthermore, most UV marks disappeared when the pupal cuticle became translucent, although in most cases guarding started before any apparent change in coloration has occurred.

Chemical analysis

Extracts of H. charithonia cuticle contained similar compounds at comparable ratios in male and female pupa. A principal component analysis of 55 peaks produced seven principal components with eigenvalues higher than 1 that explained 97% of the variation. Scatter plots of the first three factors did not separate male from female samples and results from the Hotelling’s T square test also indicated lack of significant differences between sexes (T² = 102.461 F(7,2) = 3.6593, P < 0.231) (Fig. 3.4). The main compounds in the pupal cuticle were saturated straight chain alkanes with lengths between 23 to 33 carbon atoms preferentially odd numbered, 11,15,19 trimethylhentriacontane, octacosanal, octacosanol, and 2, 5 dialkyl tetrahydrofuranes. Minor compounds include tricosene, octacosyl hexadecanate and octadecanoate as well as additional alkenes, aldehydes, and branched alkanes (Fig. 3.4).

Eight compounds were found consistently in the head space of pupae when sampled with SPME (Table 3.1). From those, only two may served to discriminate between sexes; the monoterpene linalool was exclusively found in late male pupae, while linalool oxide (furanoid) was released from late female pupae and found only in traces in early ones. Chiral analysis indicated that female pupae produced a racemic mixture of all four enantiomers of furanoid linalool oxide. During five hours of sampling late pupae

released about 10 ng of linalool or linalool oxide (R2 = 0.991 and R2 = 0.989 coefficient of determination for linalool and linalool oxide respectively) (Fig. 3.5). The former terpens plus linalool oxide acetate were the only compounds found almost exclusively in late pupae and thus indicate the proximity to eclosion. Proportions of other volatiles varied considerably among samples and between sexes, but it is still unknown whether these differences are an artifact of the sampling method, as peaks in our chromatograms were small, or are part of the cues males use to assess the developmental state and sex of pupae. The main compounds detected in both types of samples were similar among pupae raised with different species of Passiflora host plant, which indicate that diet has probably little effect on pupal odors.

Behavioral assays

We performed the bioassay to test sex-specific odors in 91 presumed female pupae from the greenhouse population. Of these, 17 were males and results were not included in the analysis. We did not include 14 more pupae because males sat earlier than two days from eclosion, the pupa died, or the guarding male did not come back within 10 minutes after disturbed. Overall, our analyses were performed in 20 female pupae for each treatment: control (unmanipulated pupae), linalool oxide (female odor) and linalool (male odor). Following disturbance males returned to hover in front of the pupae after about 1.3 minutes (±1.8 minutes, min = 3 s, max = 8.7 minutes), and they accepted and sat on their original pupae after about 2.8 minutes (± 2.5, min = 7.2 s, max = 8.4 minutes). Adding male specific odor (linalool) significantly decreased the chance that a given male would accept his female pupa again. In only in 3 of 20 tests a male resumed sitting on the same female pupae after male odor was experimentally applied. This is a significantly lower proportion than obtained in control tests (14 of 20) (Fisher’s exact test, P = 0.001). In contrast, the percentage of males accepting pupae perfumed with female odor (linalool oxide) (13 of 20) was essentially the same as those accepting controls (Fisher’s exact test P = 1) Fig 3.6).

DISCUSION A key component in the origin and maintenance of precopulatory mate guarding is the ability of males to recognize immature conspecific females and estimate their maturity (Parker 1974). We found that males of Heliconius charithonia are efficient finding and guarding female pupae which they recognize using simple sex-specific chemical compounds released only at the end of pupal development. Although males are probably using a combination of olfactory and visual cues to find pupation sites (chapter 2) at a closer range visual cues rather than long range pheromones seems to be used to locate individual pupae within a zone of search primed by odor. This is suggested by the searching behavior of males, which included close inspection of pupae and similar objects, and the lack of attraction to concealed pupae (C. Estrada & L.E. Gilbert, unpublished data). After location, olfactory cues effective only at short range, rather than visual differences between pupae, are then used to estimate their maturity and sex.

Males were efficient in recognizing female pupae, but a high proportion of males were found also guarding males. Whether these mistakes were due to recognition failure by males or the release of wrong sex-specific cues need to be determined. Although learning in the context of sexual behavior has been demonstrated in insects (Dukas et al. 2006, Dukas 2008b), we did not find any evidence of consistent mistakes by individual males or mistakes related to male experience. For example, males that sat on male pupae were as old as those that sat in female pupae, and for those that sat in both sexes there is no indication that mistakes happened first. High motivation to mate in males, on the other hand, is known to drive failures in discrimination, and courtship or mating attempts toward males, heterospecifics or inappropriate objects is common among insects (Thornhill & Alcock 1983).

Alternatively some male pupae could release female odors. Female mimicry is a competitive mating strategy found in a diverse range of taxa which serve to distract competitors from actual females (Field & Keller 1993, Steiner et al. 2005, Rainey & Grether 2007). In Heliconius however, there are good reasons not to expect female mimicry by males: it seems that the cost of a male pupae attracting guarding (e.g.

51 harassment during eclosion, Gilbert 1976) is higher than the potential benefits of distracting competitors. Furthermore although females can mate immediately after eclosing, males need three to five days to mature. This, together with the fact that this tropical species have continuous generations, makes the prospect of female mimicry very unlikely. Other causes, perhaps related to pupal physiology, could also explain the presence of female odors in males, therefore, at the moment the reasons behind males guarding male pupae are still unknown.

The main compounds used in sex recognition by H. charithonia males were the monoterpenoids linalool and linalool oxide (furanoid), found in male and female pupae respectively. They are common semiochemicals released by leaves in response to insect feeding or by flowers (Turlings et al. 1990, Paré & Tumlinson 1999, Raguso & Pichersky 1999, Andersson et al. 2002), and are known to elicit antennal electrophysiological responses in several insects, including H. charithonia and one congeneric H. melpomene (Andersson & Dobson 2003a, Bruce et al. 2005; C. Estrada and C. Rodriguez-Saona unpublished data). These compounds have also been found in scent mixtures of insects (Bergström & Tengö 1978, Borg-Karlson et al. 2003, El-Sayed 2008). In the swallowtail Papilio polystes (Lepidoptera: Papilionidae), for example, linalool is one of the main volatiles in the male scent, although behavioral tests to elucidate its function have not being done (Omura & Honda 2005). Several male sexual pheromones of phytophagous insects are identical to plant chemicals. In most cases, however, such compounds are attractive to females in all contexts, either if looking for food, oviposition sites or a partner, and it has being propose that such correspondence might be a type of mimicry that increase the chances of males to attract females (Landolt & Phillips 1997). In this case however volatiles have an opposite effect in male butterflies. Linalool and linalool oxide in combination with other floral scents and visual flower cues have been shown to elicit feeding responses in H. melpomene butterflies (Andersson & Dobson 2003b). During pupal mating, however, linalool, in concert with other visual and olfactory cues works to repel H. charithonia males from male pupae. Therefore the attractive or repellent activity of linalool seems to be context-dependent as it has been shown before

for other pheromones in Heliconius (Schulz et al. 2008). This also agrees with observations that signal interaction, and ratios of volatiles rather than particular compounds are important to elicit insect behavioral responses in some species (De Moraes et al. 1998, Raguso 2004, Bruce et al. 2005). Furthermore, in preliminary tests here, and field experiments (L. Mendoza-Cuenca, personal communication) pupae artificially placed on host plants within one day of eclosion where rarely accepted and guarded by males even after they found them. This suggests that not only additional cues but probably also previous learning of pupae location and continuous monitoring are also important factors that play a role in the decision of guarding.

Other volatiles from extracts made with SPME varied considerably among samples and between sexes, although not in a predictive way suggesting they are sex- specific. However, at the moment their contribution helping to discriminate among pupae cannot be ruled out. In particular, the presence of dihydroedulan I preferentially in late male pupae is interesting as this compound occurs in scents males of H. charithonia and other Heliconius transfer to females during mating to make them unattractive to other courting males (antiaphrodisiac pheromones) (Gilbert 1976, chapter 4). It also appears in extracts of abdominal organs from males of one species of milkweed butterflies (Schulz et al. 1993). Yet the behavioral significance of this chemical, which is also part of Passiflora scents (Prestwich et al. 1976), has not been investigated in either of these butterfly’ chemical signals.

Is there sexual conflict in Heliconius due to pupal mating?

During pupal mating females are unable to refuse mating as they could when mature adults, i.e. they cannot adopt common refusal postures or fly away when unreceptive or courted by unwanted males (Wiklund & Ahrberg 1978). Nevertheless, a passive choice still might happen as males compete to each other for a position on the pupa and for mating, and presumably only strong competitor males with good searching

abilities will mate (Deinert et al. 1994). Mating with the winner of such competition might be beneficial for females if these traits are inheritable and their male offspring become also good at competing for mating by waiting on pupae. Furthermore, some research suggest that, in Heliconius as well as in other species with mate guarding, larger males are more likely to evict smaller competitors (Elgar & Pierce 1988, Deinert et al. 1994, Bel-Venner & Venner 2006). Ejaculate size increase with male size (Boggs 1981), therefore mating with bigger males might mean that relatively more male-derived nutrients and defense substances will be transferred to females for egg production and to incorporate in her body (Boggs 1981, Cardoso et al. 2009). In our greenhouse, however, we did not find any difference in male size between males guarding pupae and those never observed doing so, but found that older males were very good competitors for pupae and some of them sat on and mated with several eclosing females. Similar patterns were found in the lycaenid butterfly, Jalmenus evagoras, the only butterfly outside Heliconius known to have precopulatory mate guarding (Elgar & Pierce 1988). In species where male mating success is highly skewed, male success and vigorous courtship displays have been reflected in lower fertilization rates (Warner et al. 1995, Jones 2001, Droney 2003). In butterflies this can also be true given that the size of the ejaculated transfer to females decrease after the first mating (Boggs & Gilbert 1979, Wiklund et al. 1998). Thus, it is possible that mating with wining males could offset the lack of active mate choice if male abilities are inheritable but does not guaranty larger direct benefits to females in term of male-derived nutrients. However adults of Heliconius feed on pollen obtaining amino acids that become a good portion of their reproduction budget (Gilbert 1972, Dunlap-Pianka et al. 1977, O'Brien et al. 2002). Furthermore, species with pupal mating, having monogamous mating system, dependent less on male-derived nutrients particularly after those resources are depleted (Boggs 1990). Thus, in this group of butterflies feeding pollen could mean that females benefit from mate guarding without compromising their reproductive success by the lack of active mate choice. An estimation of the relative fitness of females involved in pupal mating and those that mate as adult is

54 the only definitive test that can determine the cost associated with this mating system and the potential for sexual conflict.

Whether linalool and linalool oxide are signals produced to communicate pupal sex or instead they are cues exploited by searching males with little pupal control could give us a hint to the level of cooperation of females in this mating system (Bradbury & Vehrencamp 1998). Interestingly, these compounds, together with linalool oxide acetate which appeared in both sexes in late pupae, are biosynthetically closely related. All compounds are monoterpenes, the biosynthesis of which especially in plants is well understood (Raguso & Pichersky 1999). Linalool is certainly the biosynthetic precursor to linalool oxide (hence its name). Whether this reaction is mediated enzymatically or nonenzymatically in the pupae is unclear. Finally Linalool oxide can be easily esterified to form linalool oxide acetate. Monoterpenes occasionally provide antimicrobial and antifungal protection in plants (Langenheim 1994, Raguso & Pichersky 1999) or might be used as defense mechanims in insects (Francke & Schulz 1999). Thus, there are three possible scenarios for the occurrence of linalool and linalool oxide in H. charithonia pupae: 1) they are both signals intentionally produced to inform pupal sex; 2) they are both by-products, precursors of enzymatic reactions, or have additional functions (e.g. antifungal) and thus are not signals but only cues males exploit; or 3) only one sex produce a signal by the active accumulation of one compound in the three-step biosynthetic pathway. We have little evidence of which of these alternatives situations apply for H. charithonia although some observations put forward the idea both compounds could be signals. Firstly, in an evolutionary perspective, given that pupa of both sexes produce all the enzymes to synthesize the last product of the pathway (linalool oxide acetate), it seems unlikely that accumulation of one of the compounds would happen if there were strong selection pressures for concealing the sex to potential guarding males. Secondly, neither, linalool or linalool oxide have been found in adult male or female abdominal glands or wing extracts of H. charithonia (chapter 4), although small quantities of linalool have been found in abdominal glands and wings of two Heliconius (S. Schulz unpublished data). This suggests these chemicals are not part of

pheromones used after eclosion for species, sex recognition or other types of intraspecific communication, as was proposed (Gilbert 1976) and commonly happens in mating systems where males locate pre-emergent females by odor and mate at eclosion sites (Bergström & Tengö 1978, Yoshida 1978, Thornhill & Alcock 1983, Steiner et al. 2005).

In summary, this research on pupal mating in H. charithonia showed that single chemicals are enough to distinguish male and female pupae and help males in the decision of which pupae to guard and when. The kind of sex-specific compounds used also suggest that even in the absence of active mate choice female cooperation in this mating system is possible. It however leaves open many questions that might help us understand the effect of pupal mating in the evolution of ecological interactions in this group of butterflies. For instance, the variation of such sex-specific cues across species and their the role along with other cues (e.g. cuticular compounds and host plant information) in species recognition. The lack of species-specificity in pupal cues has been proposed as an important driver of host plant use in this group of butterflies (Gilbert 1991).

FIGURES

Figure 3.1. Males Heliconius charithonia sitting on pupae. A. Position of two males on pupa. B. Male with abdomen inserted through pupal cuticle.

Figure 3.2. Heliconius charithonia pupae under visible (left panels) and ultraviolet (right panels) light in their dorsal (A), ventral (B) and lateral (C) positions. With exception of the bright dots in ventral and lateral positions, white patterns in right pictures correspond to UV reflectance.

A female pupae 20 male pupae

5 Percentage of body Percentage of body with UV marks

0 Dorsal Lateral Ventral B

Percentage of UV intensity 10

0 Dorsal Lateral Ventral

Figure 3.3. Percentage of body patterns with UV reflectance in male and female early pupae (A), and percentage of intensity of such marks when compared with the intensity of UV reflectance of the white standard (B). Dorsal, lateral and ventral refer to the position of pupae when photographed. We did not find significant differences between male and female pupae in these measured parameters (Mann-Whitney U Tests, P > 0.05, N=12).

2.0

1.0

0.0

-1.0 Component 2 Component -2.0 -3.0 -2.0 -1.0 0.0 1.0 2.0 Component 1

Time (m)

Figure 3.4. Example of a total ion gas chromatogram of methylene chloride extract of the cuticle of H. charithonia female pupa. (1) Phytyl acetate, (2) tricosane, (3) pentaconsane, (4) heptacosane, (5) nonacosane, (7) hentriacontane, (10) tritriacontane, (6) octacosanal, (8) octacosanol, (9) 11,15,19 trimethylhentriacontane, (11) 2-hexadecyl-5- tridecyltetrahydrofuran and 2 octadecyl-5-undecyltetrahydrofuran, (12) 2-octadecyl-5- tridecyltetrahydrofuran, (13) highly branched alkanes, (14) wax esters. The insert is a scatter plot of the principal component analysis (60% variation) of the cuticular chemical profile for male (open circles) and females (closed circles).

22 ▲ y = 1.0613x + 14.701 R2 = 0.9914 20

Integrated peak area Integrated peak y = 1.1467x + 12.698 14 R2 = 0.9886 12 1 10 100 1000

Concentration (ng)

Figure 3.5. Calibration curves of linalool (▲) and linalool oxide (●). Open symbols indicate the area under the peak of known concentrations while closed symbols are the peak area of these compounds in pupa head space samples (more than one data point overlap in some closed symbols).

0.8

N.S

0.6

0.4

0.2 ** Proportion pupae acceptedof

0 Control Linalool oxide Linalool

Figure 3.6. Proportion of experiments where H. charitonia males accepted and sat back in pupae after being disturbed. Control refers to unmanipulated pupae, and linalool oxide and linalool refer to pupae where these compounds were added. Linalool oxide was found only in females while linalool is exclusive to male pupae. Proportion of males accepting female pupae after the perfumed treatment were compared to control tests using Fisher’s exact test (** P < 0.01, N.S. not significant). Each proportion is the result of 20 trials.

TABLE

Table 3.1. Volatiles released by pupae of Heliconius charithonia identified using SPMEa,b.

Early Late Early Retention Late female male male female pupae index pupae pupae pupae

Cyclohexanol 2 923 + + +++ ++

α-pinene1 936 +++ Traces ++ +

Branched alkanec 981 ++ ++ ++ ++

Cyclohexyl 1056 ++ ++ +++ + acetate2

Linalool oxide2 1077 Traces ++

Linalool2 1107 +++

Linalool oxide 1280 ++ ++ acetate1

Dihydroedulan I1 1286 +++ + + a Signals represent percentage of the area under the compound peak relative to the largest peak in the gas chromatogram, +: 1% – 10% ++: 10 – 20%, +++ : >20% b Compounds were identified by comparison of mass spectra and gas chromatographic retention index (1) or additionally by comparison with synthetic samples (2). c This compound might be and artifact of the sampling method.

CHAPTER 4: Interspecific Variation of Abdominal Gland Scents among Heliconius Butterflies (Lepidoptera: Nymphalidae): Effect of Mimicry and Mating System

ABSTRACT Evolution of signaling systems is driven by multiple biotic and abiotic selective pressures. Here we examine the patterns of evolution in chemical composition of abdominal scent glands in Heliconius butterflies (Lepidoptera: Nymphalidae). We ask whether the incidence of visual mimicry or clade specific mating system in this genus of toxic butterflies have influenced these chemical signals. Odors produced by males are transferred to females during mating and released from females’ glands with two likely functions: to warn predators of their unpalatability, and to inform courting males of their refusal to mate. Thus if mimicry influences the evolution of these chemical signals we expect them to converge among species with similar warning colorations (co-mimics). This is because such convergence would simultaneously reinforce shared aposematic displays that educate predators and repel males encountering heterospecific co-mimic females. To test this hypothesis we mapped chemicals onto the latest phylogeny of the group and clustered species based on their chemical similarities. We did not find any evidence of the influence of mimicry in the chemical composition of blends from abdominal glands. Neither the complete blends nor compounds with known behavioral function have converged among species with similar wing coloration. Instead, we found differences in the rate of change of chemical composition between to clades that differ in mating system. Blends appeared to have evolved gradually, showing changes congruent with the phylogeny in the pupal mating clade of Heliconius, while in the other, blends have diverged dramatically in composition, away from those of closely related species. Likely selection pressures generating the observed patterns of evolution are discussed.

INTRODUCTION Animals have evolved communication systems that allow them to find and recognize partners, aggregate, and transfer information about the environment.

Understanding general principles governing signal evolution is thus crucial to uncover how signals contribute to shape ecological interactions and facilitate speciation (Bradbury & Vehrencamp 1998, Vet 1999). Animals utilize a great variety of signaling modalities (e.g. static or dynamic visual displays, olfactory, acoustic, seismic, electric), but the use of chemicals is the oldest and more widespread method of communication in nature (Bradbury & Vehrencamp 1998, Vet 1999). A great diversity of chemicals that function in intra and interspecific communication have been described. However a general understanding of how certain compounds become signals and what drives patterns of evolution of this signaling modality is not well known (Cardé & Haynes 2004, Symonds & Elgar 2008). Development of a general theory of chemical communication is hampered first by the paucity of detailed comparative and systematic data on chemical signals and the information they convey. Typically such information is rather unevenly available across species. A second barrier to general theory stems from the fact that message specificity in chemical signaling can be achieved in many ways. Thus, different signals can be produced by changing the chemical composition of a blend, the chirality of certain molecules, or varying the ratio of compounds in the mixture (Roelofs 1995, Cardé & Haynes 2004).

Analysis of variations in chemical composition of pheromones in a phylogenetic context provides the opportunity to explore possible mechanisms promoting the evolution of these signals as has been done for other sensory modalities (Ryan & Rand 1995, McCracken & Sheldon 1997, Sullivan et al. 2000, Päckert et al. 2003, Ord & Martins 2006). Blends can evolve gradually with losses or gains of single components over evolutionary time. This mode of evolution results in closely related species having signals with similar chemical composition and, on the whole, a pheromone blend changing congruently with the phylogeny. In contrast, a lack of phylogenetic signal (Blomberg & Garland 2002), that happen when closely related species have distinct blends, is the outcome of a different mode of pheromone evolution characterized by major changes in composition with new compounds replacing existing ones (Symonds & Elgar 2008). Evidence for both modes has been found. While changes appear to be

65 gradual in aggregation pheromones in Drosophila, attractants released by female tortricid moths, and in odors associated to abdominal hairpencils in milkweed butterflies, major composition shifts are characteristic of aggregation pheromones in bark beetles (Roelofs & Brown 1982, Schulz et al. 1993, Cognato et al. 1997, Roelofs & Rooney 2003, Symonds & Elgar 2004, Symonds & Wertheim 2005). In these examples general patterns of blend composition seem to favor one mode of evolution over the other, but a closer look at the data reveals great diversity on the way blends change even within groups. Variation in the rates of change of pheromone components across species is the outcome, not only of the function of the signal, but also the kind and strength of selective agents affecting local populations and the context in which speciation occurs (e.g. sympatry or allopatry) (Butlin & Trickett 1997, Groot et al. 2006). Clearly, the evolution of chemical signals is influenced by several, often opposing, forces and uncovering the significance of particular selection pressures is the challenge (Symonds & Elgar 2008).

Here we examined patterns of evolution in the chemical composition of abdominal scent glands in Heliconius butterflies (Lepidoptera: Nymphalidae). We investigated whether the incidence of visual mimicry or differences in mating system in the genus have influenced this signaling modality. Heliconius are an extraordinarily diverse group of long-lived, toxic and aposematic butterflies distributed from northern Argentina to southern United States (Brown 1981). They exhibit great intra- and interspecific wing color pattern diversity and show striking convergence onto such patterns forming Müllerian mimicry rings (Brown 1979, Turner 1981). Local communities of these and other closely related passion vine butterflies include up to 20 species often involved in several mimicry complexes (Brown 1979, Gilbert 1991, Mallet & Gilbert 1995, Estrada & Jiggins 2002) which, together with a well resolved phylogeny (Beltrán et al. 2007), makes the Heliconiini clade an excellent model system to study pheromone evolution. Interestingly, mimetic convergence happens more often between two major clades in this genus with distinct mating system and male mate-searching behavior (Gilbert 1991) (Fig. 4.1). The pupal mating clade, that comprises half of the species in the genus, is characterized by species with male precopulatory mate guarding

and females with low remating rates, compared with the other clade. This other clade has species with males that search for adult mates similar to most butterfly species.

Odoriferous compounds are produced in glands located inside two chitinised claspers in the last abdominal segment in males, then transferred to females during mating, and finally released from an extrusible gland of the female located in the last abdominal segment (Eltringham 1925, Emsley 1963, Gilbert 1976, Schulz et al. 2008). Two functions have been attributed to those odors: defense and sexual communication. Comparable with observations in other chemically defended animals (Moore et al. 1990, Eisner & Meinwald 1995, Rowe & Guilford 1999), odors of abdominal glands in Heliconiinae are believed to advertise the toxicity of these butterflies reinforcing warning colorations. Although not yet tested on predators, their protective function has been suggested because females, and occasionally males, release these odors while being handled (Eltringham 1925, Collenette & Talbot 1928), and because some of the components in the mixture are known to work as defensive signals in other insects (Ross et al. 2001, Schulz et al. 2008).

Some of the abdominal compounds are also used in sexual communication (Gilbert 1976, Schulz et al. 2008). Chemical signals that indicate female receptivity to mate are common in animals and in butterflies they are actively released by females during courtship (Happ 1969, Gilbert 1976, Scott 1986, Andersson et al. 2000, Ayasse et al. 2001, Simmons 2001, Johansson & Jones 2007). Female butterflies typically adopt a refusal posture that consists of opening their wings and raising the abdomen toward hovering males (Wiklund & Ahrberg 1978). Courtships toward mated females are often quickly terminated as certain odors released by abdominal glands, called antiaphrodisiac pheromones, render them unattractive (Gilbert 1976, Forsberg & Wiklund 1989, Andersson et al. 2000, Schulz et al. 2008). Studies of Heliconiinae to date have shown that blends from abdominal glands are characterized by a few major volatile compounds along with a complex matrix of less volatile esters (Ockenfels et al. 1998, Ross et al. 2001, Schulz et al. 2007, Schulz et al. 2008). At least for H. melpomene, only volatiles

repulse courting males while the matrix presumably regulates volatile evaporation during female gland exposure (Schulz et al. 2008).

Whatever function these odors have in Heliconius, either signaling conspecifics, predators or both, similar selective forces might drive their evolution. Pheromones like other types of signal are expected to be under strong stabilizing selection as deviations from the main blend or preference might cause reduction in the fitness of producer or receiver (Butlin & Trickett 1997, Groot et al. 2006). Nevertheless, shifts in pheromone blends occur and are product of directional selection originated by several forces including adaptation to new environments (e.g. changes in diet or abiotic factors), or pressures generated by intra- (e.g. sexual selection) or interspecific interactions (e.g. eavesdropping by natural enemies or reproductive interference by sympatric related species) (Stowe et al. 1995, Butlin & Trickett 1997, Higgie et al. 2000, Etges & Jackson 2001, Buckley et al. 2003, Cardé & Haynes 2004, Rundle et al. 2005, Groot et al. 2006). Consequently, if variation across Heliconius species occurs, coexistence with mimetic species might select for convergence of abdominal odors among species sharing the same wing warning coloration. First, if odors, together with coloration are signaling predators, there may be an advantage in signal convergence among mimetic species (co-mimics) because multimodal signaling might be more effective in educating local predators (Moore & Brown 1989, Moore et al. 1990, Rowe & Guilford 1999, Jetz et al. 2001, Siddall & Marples 2008). Visually oriented predators occacionally reject butterflies after testing them (Chai & Srygley 1990). Second, as co-mimic species converge in their wing colour patterns, and those patterns are also used in species recognition (Crane 1955, Jiggins et al. 2001b), interspecific sexual attraction is common and might impose a cost to mimicry due to signal confusion (Estrada & Jiggins 2008). Thus, assuming that such attraction is at least sometimes followed by courtship (e.g. Estrada & Jiggins 2008, chapter 5), antiaphrodisiac signals should converge between co-mimics to reduce inappropriate courtship by males of either co-mimetic species.

In this study we first characterized the chemical composition of abdominal scent glands among Heliconius. Using the latest phylogeny proposed for this group of butterflies (Beltrán et al. 2007), we asked whether variation in chemical composition show signals of pheromone evolution following one of the two models: One in which pheromone evolution has been gradual, i.e. showing strong phylogenetic signal, or one likely under directional selection, characterized by major changes in composition. Second, we analyzed whether variation in blend composition has been driven, in part, by intrageneric mimicry with convergence of signals in species with similar wing color pattern. Third, we discuss other selective forces, such as clade-specific mating system, that might explain the patterns of variation found in those intra and interspecific signals.

METHODS

Butterfly system

The content of abdominal glands was analyzed in ten species of Heliconius. These data was combined with previously published information on an eleventh species (Schulz et al. 2008). Some components of the abdominal blend from H. cydno and H. pachinus have been described earlier (Schulz et al. 2007). These eleven species form four mimicry rings, three of which are typically sympatric in local communities (Mallet & Gilbert 1995). The pair H. melpomene-H.erato (here red, yellow and black) occurs as a series of races that converge in colour pattern across their geographic range (Turner 1981). They coexist with the H. hewitsoni-H. pachinus-H. sara complex (yellow and black) in the Pacific lowlands of Costa Rica and with the pair H. sapho-H. cydno (white and iridescent blue) in other communities of Central and South America. Heliconius hecale and H. ismenius by contrast form Müllerian mimicry complexes with ithomiine butterflies (Nymphalidae: Danainae) across their geographic range. Although the Costa Rican races of these species used here belong on different mimicry rings, races from other parts of their distribution often converge to the same yellow/orange/black pattern

(Brown 1979, DeVries 1987). H. numata also belong to ithomiine mimicry complexes and occasionally converge in parallel with H. hecale onto the same model ithomiine color pattern (Beccaloni 1997). Finally, H. charithonia has been the focus of studies in our laboratory and was included in the analysis to provide an additional comparison. This species does not belong to any mimicry complex with other Heliconius.

Abdominal glands of H. numata from Ecuador were obtained from specimens donated by the Cockrell Butterfly Center, Houston Museum of Natural Science. Other species were maintained in cultures, originated from butterflies collected in Corcovado, Osa Peninsula, and La Selva Biological Station (Costa Rica) and in the case of H. charithonia, from Texas (United States). They were reared in greenhouses at Freiburg (Germany) (H. hecale, H. ismenius, H. erato, H. cydno) and at Austin (Texas-USA) (H. sapho, H. sara, H. pachinus, H. hewitsoni, H. erato) on their host plants in the family Passiflora. Heliconius hewitsoni and H. sapho feed on P. pittieri, H. sara in P. auriculata, H. charithonia in P. biflora, P. lutea and P. lobata, H. pachinus and H. cydno mainly on P. menispermifolia, P. oerstedi, P. vitifolia and P. quadrangularis, H. ismenius on P. quadrangularis and P. serratifolia and H. hecale on P. vitifolia. With the exception of H. sapho, H. hewitsoni and H. sara, the cyanogen sequestering clade (Engler-Chaouat & Gilbert 2007), most Heliconius could be reared on P. biflora when species preferred for oviposition were in short supply. Butterflies had access to sucrose and honey solutions (10%), and flowers of Psiguria spp., Psychotria poeppigiana and Lantana camara as source of pollen. In Austin, greenhouse temperature fluctuated daily from about 20 to 32 oC and Light/dark periods followed natural cycles as walls and roof are made with glass partially covered with 50% shade cloth.

Chemical analysis

Glands from mated and virgin females and claspers from males were dissected from freshly killed butterflies and placed individually in vials with approximately 100 µl

of pentane (Fig. 4.2). The lower tip of the abdomen was also dissected and analyzed to identify compounds found in tissues surrounding the scent glands. Samples were kept at -70°C until analyzed. Three to six butterflies of each sex were analysed individually per species. Only butterflies more than five-day old were used. Pentane extracts were analyzed with gas chromatography- mass spectrometry (GC-MS) with a Hewlett-Packard model 5973 mass selective detector connected to a Hewlett-Packard model 6890 gas chromatograph using a BPX5 fused silica capillary column (SGE, 30 m x 0.25 mm, 0.25 µm film thickness). Injection was performed in splitless mode (250°C injector temperature) with helium as the carrier gas (constant flow of 1 ml/min). The temperature program started at 50°C, was held for 1 minute, and then rose to 320°C with a heating rate of 5°C/min. Compounds were identified by comparison of the mass spectra and retention times with those of authentic reference samples as well as analysis of mass spectral fragmentation patterns.

Results from analysis in H. melpomene have showed that males transfer most compounds from their abdominal glands into females during mating (Schulz 2008). However, the chemical content of females’s glands varied substantially probably due to factors such as the time elapsed since mating and the use of the pheromone while adopting the refusing posture (Andersson et al. 2004). Thus in this study data from females were used only to test whether chemical transference happened in the same way as described for H. melpomene, but further analysis comparing the interspecific variation of gland contents were performed based only on data from males.

The number of compounds present in blends of male’s abdominal glands in the species analyzed was very high (e.g. > 100), similar to results reported for H. melpomene (Schulz et al. 2008). Some intraspecific variation in composition, particularly in minor compounds, also occurred. Thus compounds used in the analysis were restricted to those that appeared in more than one individual male of the species and were not found in the surrounding tissue of the gland or in virgin females. Components were scored as present or absent in the eleven species of Heliconius.

Analysis of chemical composition of abdominal glands

Two indexes were used to estimate the degree of phylogenetic signal in the evolution of abdominal blends and their individual components: the consistency CI = m/s, (Farris 1989) and the retention index RI = (g-s) / (g-m) (Kluge & Farris 1969), where s is the number of evolutionary steps along the phylogeny (length), m is the minimum possible steps, and g is the maximum possible number of changes. In both indexes a value closer to one indicates a higher degree of phylogeny dependence of either a chemical component, or the blend, if a sum of values over all individual components is calculated. Compounds present in only one species (autapomorphies) were excluded from this analysis as they by default have minimum number of steps (s) and tend to inflate CI of the blend (Sanderson & Donoghue 1989). Both indexes were calculated using PAUP* 4.0b10 (Swofford 2002). A randomization test was also calculated to estimate whether CI and RI differed from this obtained from a random distribution of characters (Maddison & Slatkin 1991). Using MESQUITE (Batch Architect Package, reshuffle states within characters) by Madison and Madison (2008) a null distributions of CI and RI was produced by calculating these values from 2,000 matrices created by shuffling data of presence/absence of chemical compounds while keeping the molecular-based phylogeny constant. Two-tailed Z-tests were then used to compare the null distribution with values inferred from our data. The characters were mapped in a phylogeny inferred by mitochondrial (Co and 16S) and nuclear data (Ef1α, dpp, ap and wg) (Beltrán et al. 2007) (Fig. 4.1), pruned to contain only the species studied.

Phylogenetic tools using chemicals as characters were applied to estimate among which species abdominal blends have evolved congruently with a molecular-base phylogeny of the group. Bootstrap analysis on trees constructed by maximum parsimony (MP) using heuristic search with TBR swapping algorithm were performed with 2,000 replicates and confidence level of 50 using PAUP* 4.0b10 (Swofford 2002). Bootstrap values of the resulting consensus tree were then mapped on the molecular-based phylogeny to compare species associations found in both trees. This method allowed

72 recognizing particular groups of species whose pheromones have evolved in a gradual mode and those in which blends have diverge from those in close relatives. If the chemical composition of abdominal glands changed gradually through time, finding consensus trees that resemble closely the inferred phylogeny of the group with high branch supports was expected. In contrast, if mimicry had affected the evolution of the blend, a poor resemblance in both trees together with high branch support in the consensus trees for clades consisting of species with similar wing color pattern was expected. We reported bootstrap percentages of species associations marked with letters a to j in nodes of the pruned phylogeny containing only species studied here (Figs 4.3, 4.4)

With MP two species either sharing or lacking particular compounds could be clustered together if either gains or losses of compounds happened in their closest common ancestor. This analysis can indicate whether the overall evolution of the blend mirrors the phylogenetic history of the species. However, as compounds in a blend produce a scent, it is reasonable to assume that the more compounds are shared between two blends the more similar they are when perceived by a receiver. In other words sharing the lack of a particular compound might not add to their similarity. Thus divergence of abdominal chemical composition among the eleven species was also calculated using Jaccard similarity coefficient (J). With this coefficient similarity is given as a proportion of compounds shared by two species compared with the total amount of compounds in both blends. Similarity was then converted to distance (D = 1- J) and the resulting matrix correlated with a matrix of patristic distances calculated from the branch length of the latest molecular-based phylogeny using PATRISTIC (Fourment & Gibbs 2006). We used the Pearson’s correlation and a Mantel test with 2000 permutations to calculate the significance levels of the correlation. Mantel tests were performed using the statistical computing program R 2.7.1. (Vegan package 1.15-1, R Development Core Team, 2008).

The compounds identified ranged from low molecular mass components with high volatility to those with higher molecular mass, exhibiting lower volatility. Because

we performed analysis of dissected glands instead of collection of volatiles from animals during courtship, several compounds, particularly those of higher molecular masses might not be part of the signal involved in inter or intraspecific communication, especially those of the matrix (Schulz et al. 2008). Nevertheless, these compounds are usually volatile enough to be perceived at least near to the emitter. It is possible that different sets of compounds produced in this gland evolved under different selection pressures. Therefore, separated analyses were performed for all chemical compounds and for different sets of data based on the degree of volatility. Two divisions of the whole data set were done. In the first one all chemicals were divided into two groups (volatiles/no-volatiles A), including compounds with molecular weights below 300 g/mol as volatiles (Bradbury & Vehrencamp 1998). In the second division (volatiles/no- volatiles B) we called volatiles those chemicals with lower molecular weight than weight of compounds dominating virgin female glands, which are odorless for us (c.a. 270 g/mol).

Several chemicals present in abdominal glands are biosynthetically related and thus their presence in a blend is not independent from each other. This could bias our analysis if small transformations that give origin to a family of chemicals easily happen in two species that have independently acquired a new compound (Schulz et al. 1993, Symonds & Elgar 2004). Then, chemicals were also combined in biosynthetically related groups and the new data of presence and absence of such groups analyzed as explained above. Groups of known or assumed biosynthetic relationships are given in appendix 2.

Finally we reconstructed ancestral characters of specific volatile compounds with known behavioral function when they were present in both co-mimics using MP and maximum likelihood (ML) options implemented in MESQUITE (Pagel 1999, Maddison & Maddison 2008). For ML, a model of character evolution with asymmetric rates of gains and losses was tested against a symmetrical one-rate model (Mk1). Statistical significance of differences between two likelihoods was determined with a likelihood-

ratio test where the degree of freedom is equal to the difference in the number of free parameters in two nested models (Cunningham 1999, Pagel 1999, Whittall et al. 2006).

RESULTS The content of scent glands from Heliconius males varied considerably among species, both in composition and number of compounds (Figs 4.3, 4.4, appendix 2). Blends ranged from a few compounds, as in H. ismenius with only (E)-α-ionone and dihydro-β-ionone as major volatile components, to species with about 80 compounds like H. cydno and H. pachinus. The bouquets of the species studied included esters (41%), lactones (26%), and terpenes (15%), the remainder being alcohols, ketones and aromatic compounds. With exception of H. ismenius, male secretions have a similar general appearance across species, consisting of few major volatile compounds imbedded in a less volatile matrix made up mainly of esters of common fatty acids.

Both indexes used to estimate whether blends have evolved in congruence with the phylogeny indicate that the phylogenetic signal of blends were higher than expected by chance (all Z-tests, df = 1999, P < 0.001) (table 4.1A). The correspondence is however not very strong as calculated CI and RI fell around 0.5, half of the maximum possible value. Comparable results were obtained when chemical data was divided according to volatility which suggested that similar modes of evolution probably govern compounds that are part of signals and compounds that belong to the matrix (table 4.1A). Grouping compounds according to their biosynthetic relationships only slightly decreased the phylogenetic signal of abdominal blends indicating that the interdependence of chemical data did not bias our analyses with the entire data set toward any particular result (table 4.1A). Nearly half of the compounds that conform abdominal blends, and are present in more than one species, showed high degrees of congruence with the phylogeny (e.g. RI > 0.7, Figs 4.3, 4.4). Among those, half are lactones and esters found exclusively in the closely related pair H. cydno and H. pachinus. Values of CI and RI of blends not including those 25 chemicals are comparable with values obtained from the complete

data set and significantly different from random (CI = RI = 0.489, Z-tests, df = 1999, P < 0.001), suggesting that results of the congruence with the phylogeny for the complete data set have not been the product of the high chemical similarity between these two species alone.

In spite of the phylogenetic signal found with the consistency and retention indexes, no clear pattern of blend evolution emerged when Heliconius abdominal gland chemicals were mapped onto the molecular-based phylogeny of the group (Figs 4.3, 4.4). When chemicals were used as characters and analyzed with maximum parsimony (MP) species whose abdominal blends have evolved gradually clustered resembling their phylogenetic relationships (table 4.1B). Species associations obtained from this analysis show highest bootstrap support for the pair H. cydno-H. pachinus (clade a), which remain high regardless of the data set used. Although with lower bootstrap support, the clade that include H. sapho, H. hewitsoni, and H. sara (clade g) also emerged from trees done with all sets of chemical data. Support for other species associations was also found only with particular groups of chemicals. For example, H. sapho and H. hewitsoni (clade f) clustered in about half of the trees only when volatile compounds were taken into consideration. Likewise, the high support that appeared for the clade that comprise H. ismenius, H. hecale, and H. numata (clade d) was produced by the gradual change in chemical composition of the less volatile compounds but drops dramatically when only volatiles were included. Consensus trees for all data sets are shown in (Fig. 4.5).

Divergence in composition of abdominal blends between species were slightly correlated with their phylogenetic distances (Pearson’s correlation r = 0.335, Mantel test P = 0.02). Blends were in general very different across all levels of phylogenetic distance with an average close to the maximum value (0.981 ± 0.10). Some of the closest related species, however, exhibited chemical composition similarities higher than the average, although they rarely shared more than half of their compounds. Heliconius cydno and H. pachinus, and H. hewitsoni, H. sara, and H. sapho were the species with highest blend similarity among all species analyzed. When only pair of species within the

two main clades were analyzed (pupal and non-pupal mating groups, Fig. 4.1), correlations between genetic and phenotypic distance were stronger than with the complete set of comparisons (Pearson’s correlations r = 0.8, P = 0.005 and r = 0.82, P = 0.013, for clades of species with and without pupal mating behavior respectively). Interestingly at any level of genetic distance pair of species that belong to the clade with pupal mating behavior (squares, Fig. 4.5) were phenotypic more similar that equivalent pairs in the other clade (triangles, Fig. 4.5). Identical results were obtained using sets of data separated by chemicals volatility (not shown).

Contrary to expectations, no evidence was found for the suggestion that species with similar warning coloration have converged in abdominal chemical composition. Consensus trees after bootstrap analyses never clustered co-mimic species with confidence values higher than 5%. For example H. cydno and H. pachinus appeared together in almost all resulting trees and were never grouped with their co-mimics H. sapho, H. hewitsoni and H. sara. Similarly H. erato and H. melpomene which exhibit the red, yellow and black coloration were never clustered together. Heliconius ismenus, H. hecale and H. numata, which converge in wing color patterns in some location within their geographic ranges, were often grouped in consensus trees, however, they are also closely related, and thus we cannot separate both aspects in the evolution of their abdominal blend. Furthermore, the later species have only few compounds in their glands, with few commonalities between H. hecale and H. numata but none with H. ismenius. Their clustering in consensus trees of MP was then due to the likely loss of multiple compounds in their closest ancestor and not to chemical similarity of their blends.

While it is possible that the whole mixture of volatile compounds serve as pheromone signal in intraspecific communication or as allomone to advertise the toxicity of the butterflies, individual compounds or simpler mixtures might also be enough to signal conspecifics and predators. For example, 2-methoxy-3-isopropylpyrazine and 2- methoxy-3-isobutylpyrazine, well known warning odors present in Heliconius blends

(Moore et al. 1990), were found only in few of the species studied all of which exhibit different wing coloration (Fig. 4.3). Compounds known to trigger rejection of females by courting males (antiaphrodisiacs) are (E)-β-ocimene in H. melpomene (Schulz et al. 2008), hexyl isopentanoate in H. cydno (chapter 5), and benzyl salicylate in H. charithonia (Estrada C., Yildizhan S., Schulz S., Gilbert L.E., unpublished data). Analogous to pyrazines no evidence of convergence between co-mimic species in these compounds was found. Hexyl isopentanoate, the main volatile with antiaphrodisiac function found in H. cydno and H. pachinus, is not present in either of their co-mimics. The latter species also lack benzyl salicylate, the main compound in their co-mimics’ bouquet, which presumably also works as an antiaphrodisiac. In contrast, (E)-β-ocimene is present in the co-mimic species H. melpomene and H. erato, where it is the major volatile in both blends. This terpene, however, also occurs in other species, either as a minor compound (H. hewitsoni, H. sapho, H. charithonia and H. numata) or as the main volatile in the mixture (H. hecale) (Fig. 4.3). When tracing the ancestral states of (E)-β- ocimene, the hypothesis of this compound converging between H. melpomene and H. erato is not supported. The compound appeared as present in the closest ancestor of the two species (j in Fig. 4.4) when using MP, and with a likelihood proportion of being present of 0.53, when using the Mk1 probability model of maximum likelihood (ML) (Mk1 rate: 0.02573). Results of ML using symmetric and asymmetric probability models were not different (total likelihood Mk1= 7. 491, asymmetric = 6.947, likelihood-ratio = 1.087, P = 0.29).

DISCUSSION

Patterns of evolution of abdominal scents in Heliconius butterflies

Our results do not support the idea of a consistent mode of evolution in the chemical composition of abdominal blends across Heliconius species. Both, gradual and major shifts in composition were detected among closely related species. Changes

mirrored the phylogenetic relationships of species, and thus represented gradual evolution of blends in the pair H. cydno-H. pachinus, and in the group formed by H. sapho, H. sara, and H. hewitsoni (clade g). The opposite was true for H. melpomene, whose blend exhibit substantial changes in composition when compared to their closely related species H. cydno and H. pachinus, or for H. ismenius, which only has one compound in common with its sister species H. numata. Similar patterns of blend evolution were found using all groups of chemicals divided by molecular weight, suggesting that signals and matrix might be evolving under similar selective forces. Gradual and major shifts are also suggested when considering only those compounds with known behavioral function. For example, benzyl salicylate, identified as an antiaphrodisiac for H. charithonia males (Estrada C., Yildizhan S., Schulz S., Gilbert L.E., unpublished data) is also a major compound found in blends of all species within the clade i, presumably serving the same function. Heliconius cydno and H. pachinus have the same antiaphrodisiac too (hexyl isopentanoate), but differ from their closest relative H. melpomene that uses (E)-β- ocimene instead (Schulz et al. 2008, chapter 5).

What is driving the variation in scents among Heliconius?

The large variation in the chemical composition of blends from abdominal glands in Heliconius was unexpected, particularly when detected between closely related species. Levels of congruence with the phylogeny, although significantly different from random, were in general low. These values were the outcome of most compounds being scattered across species, suggesting that gains and losses throughout the evolutionary history of the group have been common. Contrary to our initial expectations, observed patterns of variation in blends from abdominal glands do not support the hypothesis that intrageneric mimicry had selected for convergence in signals between species with similar warning coloration. This conclusion holds regardless of whether we analyzed mixtures or focused only on chemicals with known defensive or intraspecific communication function. One possible explanation of the lack of blend similarity

between co-mimics is that selection pressure for convergence might not be as strong as suggested by tests of interspecific attraction or by expectations of predator learning abilities (Moore et al. 1990, Estrada & Jiggins 2008). When considering antiaphrodisiac pheromones this could be true if interspecific courtships are rare in nature, not costly or if species recognition signals alternative to wing coloration become more important at short distance and often work before female rejection displays. In Heliconius convergence in warning coloration normally happens between members of distant clades (Fig. 4.1) (Gilbert 1991) and thus additional species recognition signals could be expected. Tests that measure attraction between co-mimic species in captivity have shown that males equally approach virgin females of both species but court co-mimic females significantly less time than their own. These studies also suggested that at close range complementary recognition signals, likely chemical, are provided by females (Estrada & Jiggins 2008, chapter 5). However, in butterflies abundant evidence of species-specific sex pheromones exist for males, but evidence of pheromones in females is limited and suggested only by observation of male behavior and some chemical analysis (Boppré 1984, Omura & Honda 2005, Dapporto 2007).

Alternatively, the potential confusion of mimetic pattern could have selected not for convergence in signals but for males able to recognize antiaphrodisiacs specific to each co-mimic and thus able to quickly end courtship when coming across mated females of either species. Convergence in the receiver is feasible given that several chemicals found in the abdominal blends are also part of flower scent of common floral resources for Heliconius and thus males having already acquired ability to sense them can coopt this signal for another role (Andersson et al. 2002, Andersson & Dobson 2003a, Schulz et al. 2008, chapter 3). Cross-species communication could also happen if perception for ancestral compounds remains in spite of pheromonal change (Ryan & Rand 1995) or if males learn to associate female rejection with multiple odors (Dukas 2008a). Learning to recognize signals in a sexual context has been proved in Drosophila (Dukas 2008b) and could also happen in Heliconius given the seemingly sophisticated memory exhibited by these butterflies (Gilbert 1975). Behavioral experiments with H. cydno have shown that

males in captivity significantly reduce courtship time toward virgin females painted with some of the main volatile compounds found in its co-mimic species H. sapho and other Heliconius (chapter 5). This result clearly indicates that males can sense and respond to heterospecific odors and that cross-species communication could happen, although leave the question open whether or not there has been a convergence in the perception of signals by the receiver.

Highly diverse and complex mixtures, as this observed in our data, could be expected when signals are under strong selective pressure for divergence which happens, for example, when there is need of species-specificity or when interests from sender and receiver conflict (Arak & Enquist 1995, Symonds & Wertheim 2005, Smadja & Butlin 2008). The evolution of abdominal scents in Heliconius could be in part driven by intrasexual selection. Antiaphrodisiacs could change as a result of pressures for overcoming resistance from receiver males that challenge the signal with courtship persistance. Following copulation butterfly females are not receptive to mate again for short time intervals or permanently (Wiklund et al. 2001, Wiklund et al. 2003). Therefore, typically antiaphrodisiac pheromones represent an honest signal of female receptivity and thus both sexes benefit from releasing females from time and energetically consuming harassment (Forsberg & Wiklund 1989, Andersson et al. 2000, Bateman et al. 2006). In this sense antiaphrodisiac pheromone can be seen as part of the nuptial gift that together with nutrients and defensive substances, are transferred to female during mating (Boggs & Gilbert 1979, Thornhill & Alcock 1983, Dussourd et al. 1991, Cardoso & Gilbert 2007, Cardoso et al. 2009). Likewise, when females are unreceptive, males that respond to antiaphrodisiac pheromones and quickly end probably unproductive courtships will also benefit because forced copulation in butterflies is not an option (Forsberg & Wiklund 1989). However, in Lepidoptera last-male copulations have considerable sperm precedence in egg fertilization (Boggs 1979, Bissoondath & Wiklund 1997, Solensky & Oberhauser 2009). Thus the potential for intrasexual selection might arise under circumstances in which ignoring this signal and being persistent in courting previously mated females could offer a slight chance for copulation. The amount of scent

released by females during the refusal posture vary considerably, both in proportion to the quality of the donor male and the number of times she has adopted such posture toward courting males since the mating event (Fig. 4.6) (Andersson et al. 2004). Males are sensitive to such variation and the amount of the pheromone seems to be a key factor involved in the decision of courtship persistence (Andersson et al. 2004). Moreover, it is likely that male and female reproductive interest sometimes differ and thus the presence of male-donated pheromones creates a continuum between cooperation and conflict as occurs with other nuptial gifts transferred at mating (Wiklund et al. 2001). A conflict could arise because females gain from multiple matings but are unable to voluntarily control releasing the signal that make them unattractive to courting males even when they become receptive again (Boggs & Gilbert 1979, Boggs 1990, Wiklund et al. 2001, Andersson et al. 2004). Therefore, the fact that receptive females might occasionally release pheromones, whose quality and quantity provide information of their willingness to re-mate might create selective pressures in donor males to transfer greater amounts of antiaphrodisiac or produce more effective pheromones. Better signals will be those that, 1) reduce the threshold that other males will perceive as worth pursuing copulation with the female (Fig. 4.6a), 2) increase the time pheromones stay above such thresholds (e.g. controlling more effectively evaporation) (Fig. 4.6b), or, 3) use both strategies (Fig. 4.6c).

Neither this study, nor other research done before on antiaphrodisiac pheromones provide direct evidence that complex mixtures or novel elements make these signals more effective repelling males, a key assumption for the model of antiaphrodisiac evolution described above. However, there is compelling theoretical and empirical evidence that the intensity of conflict between sender and receiver influences the evolution of complex signals as those observed during courtship displays or disputes for territories (Arak & Enquist 1995). Data here and elsewhere suggest that it is worth considering the possibility of male-male competition being a major driving force in the evolution of antiaphrodisiac signals. First, behavioral tests with H. cydno mentioned above, show that males respond to odors that are not present in their own abdominal scents and thus are

novel when appearing together with their own species-specific signals (e.g. wing coloration or chemical signals) (chapter 5). However why males respond to such odors remains an open question (see above). Second, the complex matrix of less volatile esters that characterize blends from abdominal glands of Heliconius (and related genera) has apparently evolved in a similar mode as the more volatile portions of the blend, which is presumable the one detected by males and conveying information (Table 4.1) (Ross et al. 2001, Schulz et al. 2007, Schulz et al. 2008). If as proposed before (Schulz et al. 2008), the matrix regulates volatiles evaporation during female gland exposure, it could be that changes in composition occur as a consequence of selection for controlling evaporation more effectively and thus allowing females to retain pheromones above meaningful thresholds for longer time (Fig. 4.6b).

Finally, the rate of pheromone evolution in the two major clades of Heliconius differs in the direction expected under the influence of sexual selection given their contrasting mating systems (Fig. 4.1). Chemical composition has diverged faster among species that belong to the non-pupal mating clade than among those in the pupal mating clade (Fig. 4.6). Based on spermatophore counts, and field and greenhouse observations of courtship and mating (Gilbert, personal observations), females in the former group are considered to mate more frequently than females in the latter clade (Ehrlich & Ehrlich 1978, Boggs 1990). Males of the pupal mating clade, although search for and court adult females, also seek pupae that they then guard with the goal of mating with eclosing females (Gilbert 1976, Deinert et al. 1994). In some species of this clade, mating at the pupation site is the predominant mode of reproduction (L. Gilbert, personal observation) which would reduce further the pressure of courtship toward mated females. In butterflies the size of the spermatophore and amount of nuptial gifts alter female receptivity, and, because they correlate with the degree of polyandry, are considered male strategies that reduce sperm competition (Svärd & Wiklund 1989, Wedell 2005, Cardoso et al. 2009). It is then likely that the contrasting rates of pheromone evolution between Heliconius clades might also be the result of differences in the levels of competition for females in both mating systems. Variation in signals that indicate female receptivity is

not exclusive of Heliconius but has also been found among closely related polygamous butterfly species. For example, methyl salicylate reduce attractiveness of females in Pieris napi (Lepidoptera: Pieridae), methyl salicylate and indole in P. rapae, and benzyl cyanide is used by P. napi’s sister species, P. brassicae (Andersson et al. 2000, 2003, Chew & Watt 2006). However, antiaphrodisiacs of species in this or related genera with monogamous behavior have not been studied yet.

At least two additional factors could generate some of the chemical variation found in blends from the abdominal glands of Heliconius, and explain the observed differences in blend evolution between clades. First, compounds or precursors of compounds included in the mixture could be sequestered by butterflies from their larval host plants (Nishida 2002). For example, edulan I and II, major constituents of the flavor in fruits of Passiflora edulis, are biosynthetically related to dihydroedulans, ionones and ionols, some of which are also present in both plants and butterflies (Prestwich et al. 1976, Dhawan et al. 2004, and Figs 4.3, 4.4). If sequestration happens, variation could arise as species of Heliconius are often locally monophagous using only few of the species of Passiflora available and partitioning resources with other heliconians in the community (Benson 1978, Smiley 1978, Gilbert 1991). In fact butterflies in the sub-clade that include H. sapho, H. hewitsoni and H. sara, some of the species with the most similar abdominal blends, are the only Heliconius known to sequester cyanogens from their host plants (Engler et al. 2000, Engler-Chaouat & Gilbert 2007). Nevertheless, experiments feeding H. melpomene males with isotopically labeled compounds have shown they are able to synthesize terpenes and esters of the scent glands de novo (Schulz et al. 2008). Furthermore, no major differences were found among blends originated from butterflies of each species reared on different host plants suggesting that, if any, the role of host plants driving blend divergence seems to be limited.

Second, selection pressure for divergence in abdominal gland compounds that are different from sympatric species might arise if predators and parasitoids include those signals as part of their foraging strategies. Eavesdropping on sexual or aggregation

84 signals are widespread in nature and several changes in signals and signaling behaviors have been suggested to be adaptation to escape detection by predators and parasitoids (Stowe et al. 1995, Zuk & Kolluru 1998). Although evidence of the evolution of chemical signals resulting from such exploitation is limited, studies in bark beetles suggest that minor modifications in the pheromone help them to evade some of the natural enemies attracted to their signals (Cardé & Haynes 2004, Raffa et al. 2007). The antiaphrodisiac pheromone of the butterfly P. brassicae not only attracts the egg parasitoid Trichogramma brassicae, but also triggers phytochemical changes in this butterfly’s host plant that arrest females of this parasitic wasp (Fatouros et al. 2005, Fatouros et al. 2008). The extent to which antiaphrodisiac signals are exploited by parasitoids in Heliconius is unknown. However, if it occurs, high egg mortality by these natural enemies could be a strong selective force driving the divergence of pheromone. This selective factor could well exceed the cost of being a less effective signal at repelling unwanted males.

When considering chemicals that are produced to warn predators variation is more difficult to explain when happens between closely related species. Experiments with birds have demonstrated that these predators learn to discriminate palatable from unpalatable prey using odors as well as colors, and that odors alone can elicit biases against novel conspicuous colorations (Marples & Roper 1996, Roper & Marples 1997, Rowe & Guilford 1999, Lindström et al. 2001). Therefore, the spread of new patterns of aposematic wing colorations, which presumably drive speciation in Heliconius butterflies (Mallet et al. 1998, Gilbert 2003), could be facilitated if defensive odors remain unchanged providing protection against local experienced predators. Along with the pyrazines mentioned above, many others chemicals found in abdominal blends of Heliconius are also part of defense or repellents substances in several insects (e.g. benzyl cyanide, salicyladehyde, hexyl acetate, hexyl butanoate, (E,E)-α-farnesene) (Woolfson & Rothschild 1990, Seidelmann & Ferenz 2002, El-Sayed 2008). Such diversity of compounds that are ubiquitous across taxa strongly suggest that abdominal blends could

have evolved in large part to facilitate avoidance learning in diverse predators (Speed 2000).

Our results with Heliconius, although consistent across analyses and data sets, have been obtained from information of only one third of the species in the genus, include chemicals whose function has not been identified yet, ignore quantitative information of blend components, and inadequately sample intraspecific and intrapopulation variation. Each of these limitations suggests potential sources of error that could obscure real patterns of evolution among portions of chemical blends involved in communication. Nevertheless, we can be confident that blends as a whole have evolved at different rates in the species studied. Analyses of aggregation pheromones of well- studied groups have revealed similar models of evolution in chemical signals to those found here for Heliconius. For example, distribution of chemical components suggested that pheromones have predominantly evolved in a gradual way across species of Drosophila and with major changes in bark beetles (Curculionidae), but also revealed group of species, like those we studied in Heliconius, where the alternative mode of pheromonal evolution can be identified (Symonds & Elgar 2004, Symonds & Wertheim 2005). Clearly the complex structure of blends and variation of abdominal scents among Heliconius requires more research before the causes of variation and apparent differences in rates of evolution of those signals between major clades can be determined. However our results to date show the importance phylogenetic context and natural historical details in the developing of testable hypothesis capable of elucidating selective forces shaping abdominal scent evolution in butterflies.

FIGURES

Eueides H. melpomene rosina * H. cydno galantus * H. heurippa H. pachinus * H. timareta H. besckei H. ismenius clarescens H. numata H. pardalinus H. elevatus H. hecale zuleika H. atthis H. ethilla Non‐pupal H. nattereri mating N. aoede N. metharme group H. egeria H. burneyi ¾ H. wallacei H. hecuba L. doris H. xanthocles H. hierax H. hecalesia H. erato petiverana H. himera H. hermathena H. hortense H. clysonimus H. teleciphe H. charithonia vasquezae H. peruvianus Pupal mating H. ricini group H. demeter H. antiochius H. sara theudela H. leucadia H. hewitsoni H. sapho leuce H. eleuchia H. congener

Figure 4.1. Phylogeny of Heliconius inferred from combined mitochondrial (Co and 16S) and nuclear data (Ef1α, dpp, ap and wg) modified from Beltrán et al (2007). Names for the main two clades which differ in mating behavior are showed next to the tree. Species included in this study are indicated by boxes. Asterisk indicates species whose information about chemical composition of abdominal blends has been partly reported earlier (Schulz et al. 2007, 2008).

Figure 4.2. Female (A) and male (B) abdominal glands in Heliconius charithonia

(C8/C6-Z3-en, (C8/C6-Z3-en, . Information 5

Volatiles B .

Heliconius -olid), Compounds marked with asterisk Compounds -olid),

cases rows represent multiple chemicals as

(C12-4-olid, C13-4-olid, C14-4-olid), C14-4-olid), C13-4-olid, (C12-4-olid, 4

(Schulz et al 2007, 2008). Filled boxes indicate the presence of a (Schulz et al 2007, 2008).

(C5-en/C6, C5/C6), (C5-en/C6, 3

male abdominal glands of eleven species

(C16-dien-9-olid, C16-dien-11-olid, C16-en-9 C16-dien-11-olid, (C16-dien-9-olid, 7 than one species are shown and in some

has been partly reported earlier (C4/C6-Z3-en, C4/C6), (C4/C6-Z3-en, 2

(Geranoic/C6-Z3-en, Geranoic/C6), (C2/C6-Z3-en, C2/C6), (C2/C6-Z3-en, 6 1 have known function either in defense or intraspecific communication. in defense or intraspecific communication. either have known function compound. Only compounds present in more compounds present in compound. Only follow: from species followed by asterisk from species followed by asterisk Figure 4.3. Chemical composition of blends from Chemical compositionof blends Figure 4.3. C8/C6), melpomene* cydno* pachinus * ismenius numata hecale erato charithonia sara hewitsoni sapho

(C18- 11-olid), 12

(C18-dien-olid, (C18-dien-olid, 8

. Information from

No volatiles B No volatiles

. and we made reference to these letters in follow butterfly names indicate the total follow j to (Octadecyl acetate, Eicosenyl acetate ) a t multiple chemicals as follow: Heliconius

11 C18-9-en-11 olid, olid, C18-9-en 13-olid, C18- C18-9-en-11

the presence of a compound. boxes indicate Filled 2008). et al 2007, (Schulz

n and in some cases rows represen (C18-dien/C6, C18-en/C6). Numbers that 14

Nodes in the phylogeny are marked with letters Nodes in the phylogeny C18-Z9,E11-dien-18-olid, Z15-trien-18-olid,

C20-dien-15-olid), C20-dien-13-olid, (C20-trien-15-olid, 10

earlier has been partly reported (C24-dien-olid, C24-en-olid), C24-en-olid), (C24-dien-olid, 13 melpomene*(42) (77) cydno* * (89) pachinus (6) ismenius (19) numata hecale (15) erato (48) charithonia (20) (28) sara hewitsoni (33) (19) sapho

a c f b d g

h e i j (C18-9-en12-olid, C18-18-olid), C18-18-olid), (C18-9-en12-olid, en/iC3, C18-dien/iC3), en/iC3, C18-dien/iC3), C18-Z9, E11, Z15-trien-13-olid, C18-Z9,E11- Z15-trien-13-olid, C18-Z9, E11, number of chemicals detected in male glands. number of chemicals table 4.1 and in the text. in the text. table 4.1 and Only compounds present in more than one species are show species followed by asterisk Figure 4.4. Chemical composition of blends from male abdominal glands of eleven species Chemical compositionof blends Figure 4.4. 9

A) All chemicals B) Volatiles / no volatiles A

100 100 98

67 73 98 97 57 56 80 53 81 62 80

94 89 58 59 55

C) Volatiles / no volatiles B D) Biosynthetically related

99 100 99

74 84 97 58 50 95 84 72 99 56 58 53

Figure 4.5. Consensus trees obtained from bootstrap analysis with 2,000 replicates on trees constructed by maximum parsimony using heuristic search with TBR swapping algorithm. Different set of data were used for trees A to D: The entire data (A), chemicals divided by molecular weight in two groups at 300 g/mol (B) or at 270 g/mol (C), and chemicals combined in biosynthetically related groups (D). Numbers in branches represent bootstrap support.

0.9

0.8 distance 0.7

0.6

Chemical 0.5

0.4 0 100 200 300 400 500

Phylogenetic distance

Figure 4.6. Divergence in chemical composition of blend from abdominal glands and phenotypic distance between pair of Heliconius species. Phenotypic distances were calculated as D = 1- Jaccard similarity coefficient. Squares represent pairs of species that belong to the clade with pupal mating behavior, triangles pair of species within the clade of non-pupal maters, and circles pairs of species that belong to two different clades. Closed symbols represent pair of species with similar warning coloration.

Figure 4.7. A model for antiaphrodisiac pheromone evolution. After mating (*) the quantity of pheromone in a female decreases as a function the number of times it is used during adoption of the refusal posture. The initial quantity transferred during mating varies along this line according to the donor male quality (e.g. his mating history). Dotted lines represent mean thresholds quantities (± sd) below which males perceives the female as likely to accept mating. A decrease in the threshold (a), increase in the number of displays necessary to reach the threshold quantity (b) or both (c) represent ways to make an antiaphrodisiac more efficient and thus to increase the reproductive success of the donor male. The place along the pheromone quantity axis where females become sexually receptive again determine whether there is cooperation (if below male threshold) or conflict (if above male threshold) between females and donor males.

TABLE

Table 4.1. Measures of congruency with the phylogeny of blends from abdominal scent glands. Columns represent different set of data used for analysis with the number of characters included indicated in parenthesis. A. Values of consistence and retention indexes (CI, RI) obtained from chemical data (top) are compared with the mean (± SD) of 2,000 replicates obtained after shuffling the matrix of data (bottom) to create a null hypothesis. Congruency with the phylogeny was significantly higher than in a random distribution of chemicals across species with p < 0.0001 for all data sets (Z test with two tails). Only chemicals present in more than one species were used for calculations. B. Bootstrap support of species association from 2,000 trees obtained with maximum parsimony and using heuristic search algorithm. Letters represent group of species associated in the pruned molecular-based phylogeny of the group (clades) as shown in figure 4.4. A dash represents bootstrap values lower than 5%.

All Biosynthetic Volatile A Volatile B No volatile A No volatile B compounds groups (67) (55) (26) (38) (93) (36) A

0.553 0.623 0.486 CI 0.567 0.573 0.534 0.378 ± 0.013 0.396 ± 0.011 0.348 ± 0.011 0.397 ± 0.007 0.405 ± 0.008 0.399 ± 0.009 0.567 0.558 0.5 0.588 0.662 0.542 RI 0.139 ± 0.026 0.129 ± 0.03 0.138 ± 0.034 0.16 ± 0.047 0.144 ± 0.001 0.186 ± 0.041 B a 100 100 99 98.7 100 99.3 b ------c ------d 67.5 12.3 6.3 73.5 74.3 84.7 e ------f 45.9 55.7 53.5 - - 9.1 g 80.2 59.2 58.5 58 56.8 72.3 h ------i - 13.9 11.2 - - -

CHAPTER 5: Antiaphrodisiac Pheromones in Butterflies: Intra and Interspecific Communication in a Community of Mimetic Species

ABSTRACT Reproductive interference resulting from interspecific sexual interactions can have strong effects on intraspecific communication. It also can select for the ability of individuals of one species to detect pheromones of sympatric related species. Interspecific attraction is common among coexisting mimetic Heliconius (co-mimics) because these butterflies use wing coloration in mate recognition, a phenotypic trait that has converged between species. In this study we tested whether such interference might have resulted in the ability to recognize and respond to heterospecific courtship pheromones signaling female receptivity. In many butterflies courtship toward mated females is quickly terminated as odors previously received from males, and passively released by their abdominal glands, make them unattractive. We identified one such ‘antiaphrodisiac’ pheromone in H. cydno by comparing courtship behavior of males toward virgin females painted with male-transferred compounds, unmanipulated virgin and mated females. We also tested responses of H. cydno males toward virgin and mated females of its co-mimic species H. sapho, as well as toward conspecific females painted with male-transferred odors from H. sapho and other Heliconius. Results from these behavioral tests showed that males of H. cydno were equally attracted to females of H. sapho and conspecifics but courted the co-mimic species for shorter time. However there was no difference between the courtship duration with virgin and mated H. sapho, thus we found no evidence for a role of antiaphrodisiac pheromones in reducing courtship time between co-mimics. Nevertheless, adding H. sapho compounds to H. cydno virgin females reduced their attractiveness to courting males, often to levels comparable to females displaying their own antiaphrodisiac pheromone. Adding a pheromone from a congener with different color pattern also repelled courting males. Thus our results clearly indicate that males can sense and respond to heterospecific odors, and that cross- species communication can happen. But the question whether such ability is an

95 adaptation to mitigate interspecific sexual interactions, or if biases against ancestral compounds remains in spite of pheromonal change, is still open.

INTRODUCTION Interspecific sexual interactions, which can range from transitory attraction to hybridization, can affect the spatial and temporal distribution of species (Brower 1959, Hochkirch et al. 2007, Thum 2007), or can also be a strong selective agent for evolution of species recognition systems (Butlin 1987, Saetre et al. 1997, Jiggins et al. 2001b). Chemical communication, like other sensory modalities, can also evolve as the result of reproductive interference between species (McElfresh & Millar 2001, Cardé & Haynes 2004, Groot et al. 2006, Symonds & Elgar 2008). Selection pressures to avoid wastage of energy or gametes favor shifts in pheromones and recognition strategies that not only allow tracking of chemical changes but can also result in the ability to sense and respond to heterospecific signals. Interspecific communication is not rare in nature, and compounds that are part of sex pheromones often have antagonistic effects in sympatric closely related species (Cardé & Haynes 2004, Symonds & Elgar 2008). Recently, we demonstrated that interspecific attraction among butterfly species that have converged in wing coloration has not influenced the evolution of courtship pheromones that signal female receptivity (chapter 4). In this study we tested whether males have the ability to recognize and respond to heterospecific signals, which could suggest that communication interferences have instead affected receiver recognition strategies.

Following copulation, females of many species are no receptive to mate again for a limited time interval or permanently. This condition is then communicated to males in several ways. For example, changes in odors after mating are common as females either stop producing sex pheromones, or modify their odor profiles by the synthesis of new compounds, or acquisition of antiaphrodisiac pheromones from males (e.g. Gilbert 1976, Scott 1986, Ayasse et al. 2001, Johansson & Jones 2007). In butterflies, with a few exceptions, mating is preceded by courtship (Wiklund 2003). Females typically respond

to courting males by adopting a refusal posture that consists of raising the abdomen and releasing scents toward hovering males (Wiklund 2003). Courtships toward virgin females is sustained for long time or until mating, but displays toward mated females are often quickly terminated as odors previously received from males, and passively released by their abdominal glands, make them unattractive (Gilbert 1976, Forsberg & Wiklund 1989, Andersson et al. 2000, Schulz et al. 2008). The existence of such antiaphrodisiac pheromones in butterflies was first suggested for Heliconius erato (Nymphalidae) and have since been demonstrated and identified in a few Heliconius and three Pieris (Pieridae) (Gilbert 1976, Andersson et al. 2000, 2003, Schulz et al. 2008). In both genera, large variation in the chemical composition of odors released by females has been found. In Heliconius for example, blends appear to have evolved gradually in some species, showing changes congruent with the phylogeny, while in others, blends have diverged dramatically in composition, away from those of closely related species (chapter 4).

Heliconius is a speciose genus of unpalatable and aposematic butterflies whose wing pattern coloration, habitat use and flight characteristics have converged between species to form extraordinary examples of mimicry (e.g. Brown 1981, Turner 1981, Mallet & Gilbert 1995, Srygley & Ellington 1999). Like most butterflies, males use wing colour patterns for mate searching, species recognition and mate choice (Crane 1955, Jiggins et al. 2001b, Kronforst et al. 2006). Males commonly approach similar looking butterflies and insistently chase conspecific males and females alike probably due to the lack of sexual dimorphism. As expected by this behavior, there is also substantial heterospecific attraction toward congeneric species that have converged in warning coloration (co-mimics) (Brower et al. 1963, Estrada & Jiggins 2008). Mimicry in Heliconius generally has evolved between members of distant clades (Gilbert 1991). Thus initial attraction by males to co-mimics, even if followed by courtship, do not lead to mating, probably due to additional signals exchanged after the initial approach. Nevertheless, reproductive character displacement can happen even in the absence of hybridization (Butlin 1987). Thus, given the ample opportunity for interspecific

97 attraction, and data of courtship behaviors between distant co-mimics (Estrada & Jiggins 2008), we had predicted that antiaphrodisiac pheromones in Heliconius should converge between species that share warning coloration. However, despite similarities in blends between some species pairs, analysis of chemical composition of four mimicry rings showed no evidence of signal convergence (chapter 4).

Time and energy spent courting co-mimics might also be reduced if males are able to sense and respond to their antiaphrodisiac pheromones, and if such signals, which are greatly variable across species, also confer species identity. In this study we tested responses of males of H. cydno to compounds normally transferred to females during mating, both present in their abdominal scent glands and in those of other species with whom this species usually coexist. More than 70 compounds have been identified in abdominal scent glands of H. cydno (Miyakado et al. 1989, Schulz et al. 2007, chapter 4). In this species, as in other Heliconiinae studied so far, blends from abdominal scent glands are characterized by the presence of a few major volatile compounds and a complex matrix of less volatile esters (Ockenfels et al. 1998, Ross et al. 2001, Schulz et al. 2007, Schulz et al. 2008, chapter 4) (Fig. 5.1). At least for H. melpomene, only volatiles repel males while the matrix presumably regulates evaporation of volatiles during female gland exposure (Schulz et al. 2008).

The aims of the present study were, first, to test whether two major compounds in the scent from H. cydno reduce mated female attractiveness to courting males and hence are antiaphrodisiacs. Second, we tested whether H. cydno males also approach and court females of their co-mimic species, H. sapho, and whether the presence of antiaphrodisiac pheromones in mated females of this species would reduce time males spent on heterospecific courtships. Previous work with co-mimics H. erato and H. melpomene, has shown that after attraction to heterospecific unmated females, males display courtship behavior that, although significantly shorter than those directed to conspecific females, could last for relatively long time (Estrada & Jiggins 2008). Finally, we investigated the possibility of interspecific recognition by testing responses of H. cydno males to

conspecific females where odors from other Heliconius species were added. Compounds tested included two major components of the signal in its co-mimic species H. sapho, and the antiaphrodisiac pheromone of H. melpomene. The latter is a closely related species with contrasting wing color pattern, with whom H. cydno occasionally hybridizes (Gilbert 2003, Mavarez et al. 2006, Mallet et al. 2007, Jiggins et al. 2008).

METHODS Butterfly populations

We maintained populations of Heliconius cydno galanthus and H. sapho leuce in greenhouses at Brackenridge Field Laboratory (BFL) and Patterson building at the University of Texas, Austin. Stocks started with wild-caught butterflies and were supplemented periodically with individuals purchased from the pupae supplier ‘El Bosque Nuevo’ from Costa Rica. Greenhouses, kept at about 30°C and high humidity, contained host plants on which butterflies breed freely year-round: several species of Passiflora for H. cydno and P. pittieri for H. sapho. Sugar and honey water solution (10%), and pollen and nectar from Psiguria spp., Psychotria poeppigiana and Lantana camara flowers where also available for adult butterfly feeding. Pupae were collected periodically from these populations and isolated in rearing boxes for a constant supply of unmated individuals to use in behavioral tests.

Behavioral tests

The effect of male-transferred compounds on female attractiveness was assessed by comparing responses of H. cydno males toward virgin females painted with test chemicals, conspecific unmanipulated males, mated females, and virgin females. Six different chemicals were tested, two from scents of abdominal glands of H. cydno, three from scents of other Heliconius, and one control. The esters Hexyl isopentanoate and (9Z, 11E)-octadeca-9,11-dien-13 olide (coriolide) are the main volatile and semivolatile

components present in scent glands of H. cydno (Fig. 5.1a) (Schulz et al. 2007, chapter 4), while, aromatic compound benzyl salicylate, and terpens (E)-β-ocimene and dihydro- β-ionone are major components of scents from several species that coexist with H. cydno. (E)-β-Ocimene is known to reduce attractiveness in mated females of H. melpomene and is also present, although in lower quantities, in most other Heliconius studied so far including H. sapho (Schulz et al. 2008, chapter 4). Benzyl salicylate and dihydro-β- ionone dominate scents of H. sapho and close relative H. hewitsoni, and the former is also known to reduce attractiveness of H. charithonia mated females to courting males (Chapter 4, unpublished data) (Fig. 5.1b). Finally, virgin females were painted with analytical graded hexane to test for the effect of this solvent used to prepare chemical solutions in other treatments. In addition to observing their responses to conspecifics of both sexes, we observed the behavior of caged H. cydno males towards mated and virgin females of their co-mimic species, H. sapho. Both species posses near identical similar white and black wing color pattern and have converged in habitat use across their geographic range (Brown 1979, Estrada & Jiggins 2002). Races of these two species H. c. galanthus and H. s. leuce, used here, are part of forest butterfly communities from Costa Rica to southern Mexico

Tests were performed inside a 1.75 x 1.75 x 3.5 m cage, a sub-compartment of the 22 x 22 x 10 m greenhouse at BFL. Temperature fluctuated daily from about 20 to 32 oC and Light/dark periods followed natural cycles as walls and roof are made with a clear polycarbonate panels partially covered with 50% shade cloth. Five to ten males, unmated and more than three days old, were maintained in this cage with a constant supply of sugar and pollen, and their forewings were color-coded using Sharpie® permanent markers to facilitate individual recognition. Males in the group were replaced when tested with at least three females for a given treatment, or when they died. Trials consisted of introducing test butterflies into the cage and recording of the behavior of individual males toward this butterfly during a 30 minute observation period. Such test females (virgin, mated or virgin painted with chemicals) and males were between one and three days old, and were used only once. We registered the number and duration of

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courtship events, characterized by a clear diversion in a cage male flight path and flutter of his wings in a sustained manner behind a test butterfly. Copulation attempts, identified as a male bending his abdomen in search of the female abdomen, where also recorded. If copulation happened the couple was immediately separated to minimize chances of transference of spermatophore or scents, and females were then isolated for two minutes before continuing observations. We tested groups of males once or twice a day until responses of at least 10 males towards three different test butterflies each were obtained (five males for tests with mated H. sapho) (table 1). Mean courtship time and mean copulation attempts were calculated for each male and only those that courted more than three different test butterflies for a given treatment where included in the analysis. Similarly, trials where less than one third of males were actively approaching butterflies were excluded as that could indicate general low activity due to unfavorable light and temperature conditions. Data was then square-root transformed and means compared among treatments using one-way ANOVA and a Tukey’s HDS test for post hoc pair-wise comparisons. Analyses were done with SPSS (16.0.1 for Windows).

Chemicals

The abdomens of virgin H. cydno females were painted with 1 µl of test compounds dissolved in hexane. For trials with highly volatile compounds (hexane, (E)- β-ocimene, hexyl isopentanoate and dihydro-β-ionone), additional 0.5 µl was applied after 15 minutes of observation. The quantity of compound applied matched average amounts present in extracts of mated females glands: 10 µg /individual of coriolide and 1µg of hexyl isopentanoate as contained in abdominal glands of H. cydno, 1 µg/individual of 80% 96:4 (E/Z)-β-ocimene as found in scents of H. melpomene (Schulz et al. 2008), and 100 ng/individual of benzyl salicilate (98% Acros Organics), and dihydro-β-ionone (90% SAFC Supply Solutions) as in glands of H. sapho (unpublished data). Coriolide was synthesized using the enzymatic oxidation of linoleic acid with soy

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bean lipoxygenase, reduction and macrolactonisation (Matsushita et al. 1997, Schulz et al. 2007), hexyl isopentanoate with the esterification of hexanol and isopentylchloride, and (E)-β-ocimene with a new synthesis pathway (S. Yildizhan, unpublished data).

RESULTS Responses of cage males toward test butterflies across treatments were significantly different (Fig. 5.2). Both, the amount of time males courted and sought copulation, differed among test butterflies (one-way ANOVAs, F = 21.291 and F = 8.515 respectively, P < 0.001) depending on their sex, mating history, species and added chemicals. As expected, males quickly terminated encounters with conspecific males or mated females. Such females were in general very active and often responded to males by first flying away and then effectively discouraged further courtship by perching and adopting rejection postures. In contrast, most virgin females, regardless of treatment, flew little and while perching adopted rejection or acceptance postures. Similarly, test males were not disturbed by approaching of males, but when courtship hovering was sustained they opened their wings and often lifted their abdomen opening and closing their claspers. Applying hexane to the abdomen of virgin females while not affecting male courtship behavior (Fig. 5.2), slightly reduced the probability of mating. The proportion of attempts that ended up in copulation was lower, but not statistically significant, in females with hexane than in those without the solvent (0.28 and 0.19 for unmanipulated virgin females and those painted with hexane respectively, χ2, df = 1, P = 0.244). However, ratios of copulation to attempts were similar for virgins painted with hexane and those painted with other test chemicals (χ2, df = 1, P > 0.05 for all pairwise comparisons), suggesting that the patterns of courtship duration observed were the result of attraction of cage males to test butterflies and not to female behaviors.

Antiaphrodisiac pheromones in Heliconius cydno

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Only one of the two tested compounds that are part of scents of H. cydno decreased female attractiveness to courting males. Coriolide had little effect on males as they courted and attempted to mate with coriolide-added females as much as with unmanipulated virgins and those with just the solvent (Fig 5.2, Table 5.1). In contrast, highly volatile, hexyl isopentanoate, both reduced the duration of courtships and amount of copulation attempts (Fig 5.2, table 5.1). Cage males courted virgin females painted with this compound for as long as they courted males or mated females and copulation never happened. Courtship time toward mated females was slightly higher than that with females with hexyl isopentanoate. The tendency to escape from harassment before displaying the rejection posture in mated females, which often results in long chases by males, accounts for this difference.

Attraction to co-mimics females and responses to pheromones of other Heliconius species

As expected by the wing color similarity, H. cydno males constantly approached females of their co-mimic species H. sapho. In the 30-minute trials individual males approached a similar number of times virgin females of either species (2.61 ± 2.19 and 2.46 ± 1.59, average ± sd for H. cydno and H. sapho virgin females respectively). Nevertheless, the average duration of such encounters with co-mimic virgin and mated females was very short and in both cases not significantly different that time spent fluttering behind conspecific males (Fig. 5.2). Although some individuals initiated courtships that occasionally lasted up to 14 s, copulation attempts were never observed (table 5.1). Moreover, when fluttering was sustained, females often flew away or quickly opened their wings while perching but displaying of the typical rejection posture was rare.

Antiaphrodisiac compounds from scents of other Heliconius species when added to conspecific virgin females also had an effect on courtship behavior of H. cydno males. However the effect was less pronounced than that observed toward their H. cydno’s

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pheromone. While the duration of courtship toward females painted with benzyl salicylate, dihydro-β-ionone and (E)-β-ocimene were reduced compared to unmanipulated virgin females, the mean amount of copulation attempts were similar among all these test butterflies (Fig. 5.2, table 5.1). Responses of individual males toward virgin females painted with heterospecific odors were diverse and ranged from males consistently ending courtships promptly, resembling the behavior toward their own mated females, to males that courted extensively and attempted to mate with these females as if unscented. Such a range of behaviors contrast with the similar and predictable responses of males toward females painted with conspecific antiaphrodisiac hexyl isopentanoate (Fig. 5.3).

DISCUSSION

Antiaphrodisiac pheromones in Heliconius cydno

Results from behavioral tests with H. cydno males suggests that despite the complex mixture of compounds present in their abdominal scents glands, the main volatile compound, hexyl isopentanoate, is enough to reduce attractiveness of females to levels comparable to attractiveness of males. Similar results have been obtained for other Heliconius species (Schulz et al. 2008, unpublished data). Nevertheless, in H. cydno the lack of effect of less volatile coriolide on male courtship behavior is intriguing, since this is a major component of the blend that gives males the characteristic strong odor that human olfaction detects. Except for one rare Brazilian species Heliconius are sexually monomorphic for wing, and males are visually attracted to both sexes. Scents from abdominal glands are believed to allow rapid sex recognition that helps terminate male- male encounters (Schulz et al. 2008). Therefore, mated females are not longer attractive because they mimic males, apparently an effective strategy for avoiding harassment in other insects (Gilbert 1976, Scott 1986, Cook et al. 1994, Svensson et al. 2009). If, as suggested by our perception, the coriolide is part of H. cydno’s male odor and hence

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potentially an antiaphrodisiac signal, then its presence must be effective only when released along with the more volatile component, because as we show, it does not function alone. Adopting the refusal posture decreases the amount of scent stored in mated females (Andersson et al. 2004), and might also change the ratio of individual components in the blend that courting males perceive. It is thus possible that coriolide enhances the repellency of the scent when the amount of the more volatile hexyl isopentanoate has decreased in the mixture, and both the quantity and proportion of compounds of the blend give males information for assessing the optimum time to invest in courtship persistence (Andersson et al. 2004). Whether males are able to sense this chemical, or if coriolide’s function, if any, is other than intraspecific communication is still an open question.

It may be that coriolide’s role has more to do with signals to predators. Scents from abdominal glands in Heliconiinae are believed to also advertise the toxicity of these butterflies reinforcing warning colorations. Their protective function has been suggested because females, and occasionally males, release these odors while being handled (Eltringham 1925, Collenette & Talbot 1928), and because some of the components in the mixture are known to work as defensive signals in other insects (Ross et al. 2001, Schulz et al. 2008, chapter 4). We did not find any effect of coriolide on food acceptance by predatory spiders (unpublished data), but the roles of this chemical in defense cannot be ruled out in other potential predators and in fact that the compound is detectable by we vertebrates is suggestive.

Attraction to co-mimics females and responses to pheromones of other Heliconius species

As has been observed in other co-mimic pairs (Estrada & Jiggins 2008), H. cydno males approached H. sapho females as frequently as conspecifics. However, courtship duration toward such females was short, and in most cases ended without a display of female refusal posture. Furthermore, males never attempted to copulate with H. sapho.

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Therefore, under these experimental conditions we found little evidence for a role of antiaphrodisiac pheromones in reducing courtship time toward similar looking congeneric butterflies. This result contrasts with those found for other pair of co-mimic species studied previously where males occasionally attempted copulation with unmated co-mimic females and those often responded to sustained courtship with rejection postures (Estrada & Jiggins 2008). Minor differences in wing color pattern could also aid in identifying species at short distance perhaps explaining the differences in behavior between both pairs of species. In fact such differences exist between H cydno and H. sapho and are particularly conspicuous on the ventral surface of the hindwing (red vs. brown ommachromes on ventral hindwing’s base). However, this possibility seems unlikely since behavior of males toward H. sapho females was similar regardless of wing position (open or closed) and thus display of the hindwing color pattern. Moreover, differences in the shape of the white forewing band, which allows species identification by human eye even at flight, are probably unimportant for H. cydno males, as color rather than pattern that is used in mate recognition in this species (Kronforst et al. 2006).

This and past studies suggest that after attraction to potential mates by wing coloration, males recognize conspecifics by close range signals, likely chemical, provided by females (Boppré 1984, Takanashi et al. 2001, Estrada & Jiggins 2008). However our results for Heliconius indicate that such species recognition signals are not released by abdominal scent glands. For one thing, abdominal glands of unmated females of all Heliconius studied so far (N = 11) have almost identical chemical composition and thus are unlikely to provide species-specific information to courting males (unpublished data). Moreover, heterospecific courtship behavior suggests that species recognition of mated females apparently happens without release of antiaphrodisiac pheromone. In Lepidoptera, males produce chemical signals in androconial organs located in their wings or in abdominal scent glands. Those signals are often a necessary stimulus for acceptance by females, since they function in species recognition and in female assessment of male quality (e.g. Boppré 1984, Schulz et al. 1993, Andersson et al. 2007, Costanzo & Monteiro 2007). While, females by contrast lack androconial organs and the existence of

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chemical signals has been suggested in some species by observations of male behavior during courtship, and in others by sexual dimorphism in volatile composition of wing and body extracts (Boppré 1984, Takanashi et al. 2001, Omura & Honda 2005, Dapporto 2007). However, to date , studies that either combine chemical analysis and behavioral tests, or that identify where such signals originate, have not been done. Removing cuticular odors from wing models decreases the power of species discrimination between co-mimics H. erato and H. melpomene by courting males, suggesting that wings are the source of additional species-specific chemical signals of females in this genus (Estrada & Jiggins 2008).

The question that arises from our results is why males are repelled by chemicals found in scents of other species if apparently courtship towards heterospecifics is seldom long enough for females to release their antiaphrodisiacs, and different species-specific chemical signals are used in species recognition. One possible explanation is that our experiments between co-mimics females in enclosures do not reflect accurately what happens in the wild. Attraction to females might happen when they are flying and thus a rapid chase will precede courtship and species identification by the putative chemical signals. Highly volatile antiaphrodisiacs could then be released by the females and detected by males before more close range species-specific pheromones come into play. Although such chases are common in our insectaries, it is unknown if females can release antiaphrodisiac pheromones in flight, or if the quantity or chemical properties of antiaphrodisiacs make them easier to detect. High motivation to mate in males, on the other hand, is known to drive failures in discrimination, and courtship or mating attempts toward males, heterospecifics or inappropriate objects (Thornhill & Alcock 1983). Perhaps, additional signals that contribute to species discrimination (e.g. antiaphrodisiac pheromones in mated females) might occasionally help in discouraging courtship by those very motivated males. Thus, responses to signals of congeners could indicate that heterospecific courtship, particularly between co-mimics, is more common than our experiments suggest and that males have been selected to sense and respond to these signals.

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Males were repelled by conspecific females when major components of abdominal scent glands from the co-mimic species were added (dihydro–β-ionone and benzyl salicylate), but also responded, though slightly less, to (E)-β-ocimene, the antiaphrodisiac of the closely related H. melpomene. Despite major differences in color patterns between these closely related species, and the display of assortative mating driven by this phenotype (Jiggins et al. 2001b), hybridization between them is common in captivity and in the wild (Gilbert 2003, Mallet et al. 2007, Jiggins et al. 2008). This implies not only that under some ecological conditions color differences are not adequate barriers for attraction, but also that in these butterflies additional reproductive isolating mechanisms (e.g. chemical signals) are not always effective or might have evolved slower than wing coloration and ecological differentiation (Mallet et al. 1998, Gilbert 2003). Hybrids are strongly selected against since their intermediate wing color pattern makes them more vulnerable to predators and less likely to mate (Benson 1972, Mallet & Barton 1989, Kapan 2001, Naisbit et al. 2001), and in most cases of intra clade interspecific mating Haldane’s rule applies (Jiggins et al. 2001a, Gilbert 2003) . Thus, reproductive interference between closely related species with different mimetic coloration might have not only reinforce mate preference using such coloration (Jiggins et al. 2001b, Kronforst et al. 2007) but also select for recognition of heterospecific chemical signals. Finding added heterospecific antiaphrodisiacs along with conspecific recognition signals, both visual and chemical, could create signal confusion in courting male explaining the diversity of responses and lack of difference in copulation attempts toward test butterflies with heterospecific odors compared to unmanipulated virgin females.

Alternatively, male ability to sense chemicals from pheromone mixtures of other species could have evolved in a different context. Several chemicals found in the abdominal blends of Heliconius, including ‘heterospecific’ odors tested here, are ubiquitous floral scent components and occur in common sources of pollen for these species (Andersson et al. 2002, El-Sayed 2008). This could explain why males are able to sense them (Andersson & Dobson 2003a), but if they are indeed floral attractants, their

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repellence when present in female butterflies is not expected unless such compounds are also associated with female receptivity to mate or heterospecific odors. Contrasting responses to common attractants when sensed in a sexual context could be the result of selection to reduce wastage of reproductive effort if interspecific attraction is common. However, biases against certain chemicals could also happen if responses to chemicals that occurred in ancestral blends remain in spite of pheromonal change (Ryan & Rand 1995, Endler & Basolo 1998, Ryan et al. 2001). Given the rapid change in antiaphrodisiac composition and the irregular distribution of most components across species, this possibility also seems likely (chapter 4).

Biases against heterospecific compounds, whether adaptive or not, suggests a mechanism for the rapid evolution of this chemical signal (Arak & Enquist 1995, Endler & Basolo 1998). Modification to pheromones via addition or exchange of compounds are difficult since traits involved in production, reception and behavioral responses of a chemical are often not genetically linked (Roelofs et al. 2002, Cardé & Haynes 2004). Theoretical and empirical evidence in evolution of sex pheromones in moths suggest that shifts in blend composition start with the synthesis of new compounds, usually by simple genetic switches that modify enzymatic pathways, and then such changes are tracked by receiver responses (Löfstedt 1993, Roelofs et al. 2002, Roelofs & Rooney 2003, Symonds & Elgar 2008). Shifts in antiaphrodisiac pheromones in Heliconius could happen by a similar mechanism as suggested by the apparent facility for gaining and losing particular chemicals throughout the evolutionary history of the genus (chapter 4). Enzymatic pathways that lead to the production of these or related compounds used by ancestral species might remain in the genome and be switched on and off in evolutionary time. Shifts could be further accelerated if males have retained an ancestral ability to sense and respond to such chemicals in a similar behavioral context.

Our evidence for interspecific communication in Heliconius, although suggestive, is still limited. Tests with different species in the group might help establish whether the behavior of H. cydno males found here has resulted from selection for the ability respond

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to congeneric antiaphrodisiac pheromones or from biases against compounds from ancestral blends. If the former is true, males from different Heliconius should only respond to those chemicals present in co-mimics or in sympatric closely related species with potential of interspecific attraction and hybridization. For example, similar tests using H. melpomene males would show repellence only toward females painted with (E)- β-ocimene (own and co-mimic pheromone), and hexyl isopentanoate (antiaphrodisiac from H. cydno), but not to major compounds in H. sapho like benzyl salicylate and dihydro-β-ionone tested here.

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FIGURES

H. cydno

H. sapho

Figure 5.1. Total ion chromatogram of pentane extracts of male abdominal scent glands from Heliconius cydno and H. sapho. Extracts were analyzed with GC-MS using a BPX5 fused silica capillary column (chapter 4). Hexyl isopentanoate (1), (9Z, 11E)-octadeca- 9,11-dien-13 olide (coriolide) (2), (E)-β-ocimene (3), dihydro-β-ionone (4), benzyl salicylate (5).

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a) 30 d

25 d

20 dc

15 bc

ab ab 10 ab Courtship time (s)

5 a a a a

1.4 b)

bc bc 1.2 c c c 1

bc 0.8

0.6

0.4 ab Copulation attemps Copulation 0.2 a a a a 0 VF MF Male Male VF+ hexane VF+ VF H. sapho VF +iC5/C6 MF H. H. MF sapho VF + ocimene VF + coriolide VF + D-ionone + VF VF + benzyl Sa. benzyl + VF

Figure 5.2. Average courtship time (a) and average copulation attempts (b) (± standard error) of H. cydno males toward conspecific virgin (VF), mated females (MF), males and females of the co-mimic species H. sapho. Virgin females were painted with one of several chemicals. Coriolide and hexyl isopentanoate (iC5/C6) are esters found in the scents of abdominal glands of H. cydno , while dihydro-β-ionone (D-ionone), benzyl salicylate (Sa.), and (E)-β-ocimene are part of scents of other Heliconius species including H. sapho . The latter two compounds are known antiaphrodisiac pheromones for H. charithonia and H. melpomene respectively (schulz et al 2008, unpublished data). Bars labeled with different letter are significantly different (P < 0.05 Tukey’s HDS post hoc analysis).

112

1 VF VF + hexane MF a) 0.75 0.5 0.25 0 1 VF + coriolide VF + iC5/C6 VF H. sapho 0.75 0.5 0.25 0

Relative frequency 1 VF + benzyl Sa. VF + ocimene VF + D-ionone 0.75 0.5 0.25 0 51525 35 51525 35 5 15 25 35 Courtship time (s)

1 VF VF + hexane MF b) 0.75 0.5 0.25 0 1 VF + coriolide VF + iC5/C6 VF H. sapho 0.75 0.5 0.25 0

Relative frequency 1 VF + benzyl Sa. VF + ocimene VF + D-ionone 0.75 0.5 0.25 0 0 1 2 3 0 1 2 3 0 1 2 3 Copulation attempts

Figure 5.3. Relative frequency histograms showing the distribution of average courtship time (a) and average copulation attempts (b) that individual H. cydno males exhibited with virgin (VF) or mated females (MF). Virgin females were painted with hexane, coriolide or hexyl isopentanoate (iC5/C6) from abdominal glands of H. cydno, or dihydro-β-ionone (D-ionone), benzyl salicylate (benzyl Sa.), or (E)-β-ocimene found in other Heliconius species . Histograms of distribution for both behaviors toward males and mated females of H. sapho have a frequency of one for the first category and were not included here.

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TABLE

Table 5.1. Summary of number of trials and male responses toward butterflies across treatments.

Trials Males Attempted Initiated Treatments tested copulation* copulation* Virgin female (VF) 20 16 15 12 VF + hexane 9 10 10 7 Mated female (MF) 13 11 2 0 Male 13 10 1 0 VF+ coriolide 10 13 10 6 VF + hexyl isopentanoate 13 12 1 0 VF + benzyl salicylate 11 10 6 1 VF + (E)-β-ocimene 11 14 9 4 VF + dihydro-β-ionone 8 10 4 2 MF H. sapho 6 5 0 0 VF H. sapho 7 11 0 0 * Number of males that attempted or succeeded engaging in copulation with at least one of the three butterflies they courted for each treatment.

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APPENDIXES

Appendix 1. Butterfly species with mate-locating behavior involving host plants. Sources includes Google Scholar, ISI web of science, Journal of lepidopterist’s society, Journal of the Research on Lepidoptera, Scientific Electronic Literature Online (SciELO) and regional entomology journals available online.

Butterfly species1,2 Host plant3 Strategy Host plant role4 Reference Lycaenidae

Apodemia mormo Eriogonum jamesii perch Host plant habitat (Scott 1975a) Brephidium exilis Atriplex spp. patrol Around and close to (Scott 1975a) branches of host plant Callophrys dumetorum perch Host plant habitat (Scott 1975a) C. muiri (Mitoura muiri) Cupressus macnabiana perch Leks close to host (Nice & plant Shapiro 2001) C. nelson (Mioura Calocedrus decurrens perch Leks close to host (Nice & nelson) plant Shapiro 2001) C. sheridanii perch Host plant habitat (Scott 1975a) Cupido comyntas Trifolium repens patrol Host plant habitat (Scott 1975a) (Everes comyntas) Euphilotes battoides patrol On and between the (Scott 1975a) host plant E. enoptes (Philotes patrol On and between the (Scott 1975a) enoptes) host plant E. rita patrol On and between the (Scott 1975a) host plant Glaucopsyche lygdamus patrol Host plant habitat (Scott 1975a) G. piasus Lupinus argenteus patrol Host plant habitat (Scott 1975a) Habrodais crysalus Quercus chrysolepsis patrol Around and close to (Scott 1975a) branches of host plant H. grunus Quercus chrysolepsis patrol Around and close to (Scott 1975a) branches of host plant Hypaurotis crysalus Quercus gambellii patrol On and around host (Scott 1975a) plant 1Jalmenus evagoras Acacia Cluster Attracted to plants (Elgar & in pupa particularly those with Pierce 1988) larva, pupa and mutualist ants Lycaena cupreus perch Host plant habitat (in (Scott 1975a) and one locality only) patrol L. gorgon Patrol Host plant habitat (Scott 1975a) and perch Phaeostrymon alcestis Sapindus drummondi patrol On and around host (Scott 1975a) plant Plebejus acmon patrol Host plant habitat (Scott 1975a) P. argyrognomon patrol Host plant habitat (Scott 1975a) P. glandon (Agriades Androsace patrol Host plant habitat (Scott 1975a)

115 glandon) P. icarioides Lupinus patrol Host plant habitat (Scott 1975a) P. idas (Lycaeides idas) Fabaceae patrol Around host plant (Nice et al. 2002) P. melissa (Lycaeides Fabaceae patrol Around host plant (Scott 1975a, melissa) Nice et al. 2002) P. saepiolus Trifolium patrol Host plant habitat (Scott 1975a) P. shasta patrol Host plant habitat (Scott 1975a) Satyrium fulginosa Lupines perch On and around host (Scott 1975a) plant Sibataniozephyrus kuafui Fagus hayatae Perch an Host plant habitat (Yen & Jan patrol 1995) 1Tarucus theophrastus Zizyphus vulgaris perch Host plant habitat (Courtney & and Parker 1985) patrol Nymphalidae

1Aglais urticae nettles perch Host plant habitat (Baker 1972) Anartia jatrophae Bacopa monnieri perch Defend territories (Lederhouse always containing host et al. 1992) plant 1Asterocampa leilia Celtis pallida perch On or around host (Rutowski & plant Gilchrist 1988) Boloria freija patrol Host plant habitat (Scott 1975a) Chlosyne damoetus patrol Host plant habitat (Scott 1975a) (only in one locality) C. leanira fulvia Perch Host plant habitat (Scott 1975a) and patrol C. palla Erigeron speciosus Perch Host plant habitat (Scott 1982) and patrol Eueides aliphera Passiflora perch Defend territories (Benson et al. always containing host 1989) plant 1Euphydryas chalcedona Penstemon perch Host plant habitat (Rutowski et michrophyllum and al. 1988) patrol Junonia coenia (Precis Plantago lanceolata Adults Host plant habitat (Scott 1975b) coenia) 1Heliconius 18 spp. Passiflora Cluster Pupa on or close to (Gilbert in pupa host plant 1976) and patrol Hypolimnas bolina Sida rhombifolia perch Host plant habitat (Rutowski (occasionally) 1992) Melanitis leda perch Host plant habitat (Kemp 2002) Phyciodes phaon patrol Host plant habitat (Scott 1975a)

Pieridae

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1Anthocharis cardamines Several crucifers patrol In edges of forest and (Wiklund & around plant Ahrberg 1978) Neophasia menapia patrol Around and close to (Scott 1975a) branches of host plant Perrhybris pyrrha Capparis isthmensis perch Host plant habitat (Devries 1978) Pieris rapae crucivora Brassica oleracea patrol Around and close to (Hirota & (planted) branches of host plant Obara 2000) Papilionidae

Ornithoptera priamus Aristolochia tagala patrol males attracted to pupa (Borch & caelestis on host plant Schmid 1973) Papilio glaucus Prunus, Fraxinus, patrol Host plant (Lederhouse Liriodendron 1995) P. polyxenes Patrol Host plant habitat (Scott 1975a) and (occasionally) perch P. canadensis Prunus, Populus Patrol Host plant (Lederhouse 1995) P. gracialis Corydalis incisa Patrol Host plant habitat (Yamashita 1995) P. troilus Sassafras, Lindera Patrol Host plant (Lederhouse 1995) P. xuthus Zanthoxylum, Citrus, Patrol Host plant (Lederhouse Poncirus 1995, Yamashita 1995) P. zelicaon Patrol Host plant habitat (Scott 1975a) and (occasionally) perch Parnassius clodius patrol On and around host (Scott 1975a) plant P. phoebus patrol Host plant habitat (Scott 1975a) Hesperiidae

Atrytone arogos Andropogon perch Host plant habitat (Scott 1975a) Erynnis brizo Quercus gambellii perch On and around host (Scott 1982) and plant patrol E. martialis Ceanothus fendleri perch On and around host (Scott 1982) and plant patrol E. pacuvius Ceanothus fendleri perch On and around host (Scott 1982) and plant patrol Ochlodes venata Morinia caerulea Perch Host plant habitat (Dennis & and Williams patrol 1987) O. yuma Phragmites communis perch On and around host (Scott 1975a, plant Scott et al.

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1977) Pholisora graciellae Atriplex spp. patrol Host plant habitat (Scott 1975a) Riodinidae

Apodemia nais Ceanothus fendleri Perch On and around host (Scott 1975a, and plant 1982) patrol 1 Species included in Rutowski (1991) 2 When butterfly scientific name changed since reported in references, the more recent name appears first and old name is given in parenthesis. We used Brower (2008) taxonomic nomenclature. 3 Host plant species described in references. 4 ‘Host plant habitat’ means that host plants were reported as abundant in locations where males perch or patrol.

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Appendix 2. Chemical composition of blends from male abdominal glands of eleven species of Heliconius. Information from three first species (M, C, P) was reported earlier (Schulz et al. 2007, Schulz et al. 2008).

Group #1 Molecular MW M C P I N H E Ch S He Sa Formula 1-Hexen-3-one on 13 C6H10O 98 x (Z)-3-Hexenol ol 13 C6H12O 100 x x x x x 3-Hexanone on 13 C6H12O 100 x Hexanol ol 36 C6H14O 102 x x x x 3-Hexanol ol 13 C6H14O 102 x Butyl acetate est 24 C6H12O2 116 x 1-Hydroxy-3- hexanone on 13 C6H12O2 116 x Benzylcyanid ar 17 C8H7N 117 x x x x x Salicylaldehyd ar 16 C7H602 122 x x x 1-Octen-3-one on 13 C8H14O 126 x 3-Octanone on 13 C8H16O 128 x Isobutyl acetate est 24 C7H14O2 130 x Pentyl acetate est 24 C7H14O2 130 x (Z)-Ocimene ter 38 C10H16 136 x x (E)-Ocimene ter 38 C10H16 136 x x x x x x x Myrcene ter 38 C10H16 136 x x Alloocimene ter 38 C10H16 136 x x Alpha-pinene ter 38 C10H16 136 x 4-Nonen-3-one on 12 C9H16O3 140 x (Z)-3-Hexenyl acetate est 24 C8H14O2 142 x x 3-Nonanone on 12 C9H18O 142 x Hexyl acetate est 24 C8H1602 144 x x 3-Nonanol ol 12 C9H20O 144 x 2-Methoxy-3-isopropylpyrazine ar 18 C8H12N20 152 x x x β-Cyclocitral ter 38 C10H16O 152 x x x Isopentyl pentanoate est 32 C10H20O2 152 x Linalool ter 38 C10H18O 154 x x 2-Methoxy-3-isobutylpyrazin ar 18 C9H14N20 166 x x x Decadienolide lac 1 C10H14O2 166 x Undecadien-3-one on 12 C11H18O 166 x Dec-2-en-4-olide lac 1 C10H16O2 168 x 4-Undecen-3-one on 12 C11H20O 168 x (Z)-3-Hexenyl butanoate est 27 C10H18O2 170 x x 5-Decanolide lac 1 C10H18O2 170 x x Undecen-3-ol ol 12 C11H22O 170 x 4-Decanolide lac 1 C10H18O2 170 x 3-Undecanone on 12 C11H22O 170 x (Z)-3-Hexenyl lactate est 33 C9H16O3 172 x x Hexyl butanoate est 27 C10H20O2 172 x x 3-Undecanol ol 12 C11H24O 172 x (Z)-3-Hexenyl pentenoate est 28 C11H28O2 182 x (Z)-3-Hexenyl isopentanoate est 32 C11H20O2 184 x x x x x x Hexenyl pentenoate est 28 C11H20O2 184 x x x 4-Undecanolide lac 2 C11H20O2 184 x x Hexyl isopentanoate est 32 C11H2202 186 x x x Hexyl pentanoate est 28 C11H2202 186 x x x Nonyl acetate est 24 C11H22O2 186 x Helional ar 19 C11H12O3 192 x x x x (E)-α-Ionon ter 39 C13H20O 192 x x x β-Ionon ter 39 C13H20O 192 x x x 4-(2,6,6-Trimethyl-1,3- ter 39 C13H20O 192 x cyclohexadienyl)-2-butanon Dihydro-α-ionon ter 39 C13H22O 194 x x Dihydro-β-ionon ter 39 C13H22O 194 x x x x x x Dihdyroedulan II ter 39 C13H22O 194 x x x x x x x x x x Dihydroedulan I ter 39 C13H22O 194 x x x (E)-α-Ionol ter 39 C13H22O 194 x (Z)-3-Hexenyl hexenoate est 29 C12H20O2 196 x x x Dyhydro-β-ionol ter 39 C13H24O 196 x x (Z)-3-Hexenyl hexanoate est 29 C12H22O2 198 x x Hexyl (Z)-3-Hexenoate est 29 C12H22O2 198 x x 4-Dodecanolide lac 3 C12H22O2 198 x x 12-Dodecanolide lac 3 C12H22O2 198 x 11-Dodecanolide lac 3 C12H22O2 198 x Hexyl hexanoate est 29 C12H24O2 200 x (E,E)-α-Farnesen ter 37 C15H24 204 x x x

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Hexyl benzoate ar 16 C13H18O2 206 x (E)-α-3-Oxoionone ter 39 C13H18O2 206 x 4-Tridecanolide lac 4 C13H24O2 212 x x Heptenyl hexanoate est 29 C13H24O2 212 x 2-Tetradecanone on 14 C14H28O 212 x Hexyl heptanoate est 30 C13H26O2 214 x Heptyl hexanoate est 29 C13H26O2 214 x 9,10-Epoxyfarnesene ter 37 C15H24O 220 x (Z)-3-hexenyl octanoate est 31 C14H26O2 226 x x x 4-Tetradecanolide lac 5 C14H26O2 226 x x 5-Tetradecanolide lac 5 C14H26O2 226 x x x 2-Pentadecanone on 15 C15H30O 226 x x Octenyl hexanoate est 29 C14H26O2 226 x 14-Tetradecanolide lac 5 C14H26O2 226 x Benzyl salicylate ar 16 C14H12O3 228 x x x x x Hexyl octanoate est 31 C14H28O2 228 x x x Octyl hexanoate est 29 C14H28O2 228 x 2,3-Dihydrofarnesenic acid ter 37 C15H22O2 234 x Unknown 1 un 40 238 x x Unknown 2 un 40 238 x x x 2-Hexadecanone on 14 C16H32O 240 x x (Z)-3-Hexenyl decatrienoate est 20 C16H24O2 248 x x (Z)-3-Hexenyl decadienoate est 20 C16H26O2 250 x x x Hexadecadien-9-olide lac 6 C16H26O2 250 x x Hexadecadien-11-olide lac 6 C16H26O2 250 x x (Z)-3-Hexenyl geranoate ter 41 C16H26O2 250 x x Hexadecadien-16-olide lac 6 C16H26O2 250 x Hexadecadien-13-olide lac 6 C16H26O2 250 x Hexadecen-9-olide lac 6 C16H28O2 252 x x Hexyl geranoate ter 41 C16H28O2 252 x x (Z)-3-hexenyl decenoate est 20 C16H28O2 252 x Hexadecenolide lac 6 C16H28O2 252 x Hexadecen-11-olide lac 6 C16H28O2 252 x 16-Hexadecanolide lac 6 C16H30O2 254 x x x x x 15-Hexadecanolide lac 6 C16H30O2 254 x Hexyl tetrahydrogeranoate ter 41 C16H32O2 256 x Hexyl decanoate est 20 C16H32O2 256 x x (Z)-3-hexenyl decanoate est 20 C16H30O2 256 x 3Brassicalacton ter 262 x (Z9, E11, Z15)-Octadeca-9,11,15- lac 7 C18H28O2 276 x x trien-13-olide (Z9, E11, Z15)-Octadeca-9,11,15- lac 7 C18H28O2 276 x x trien-18-olide Octadecatrienolide lac 7 C18H28O2 276 x Octadecadienolide lac 7 C18H30O2 278 x x (Z9, E11)-Octadeca-9,11dien-18- lac 7 C18H30O2 278 x x olide Neophytadien isomer ter 40 C20H38 278 x x Octadec-9-en-12-olide lac 7 C18H32O2 280 x x x Octadec-9-en-11-olide lac 7 C18H32O2 280 x x Octadec-9-en-13-olide lac 7 C18H32O2 280 x x Octadecenolide lac 7 C18H32O2 280 x x (E)-Octadec-9-en-13-olide lac 7 C18H32O2 280 x Octadecadecen-17-olide lac 7 C18H32O2 280 x Octadecadecen-10-olide lac 7 C18H32O2 280 x 11-Octadecanolide lac 7 C18H34O2 282 x x 18-Octadecanolide lac 7 C18H34O2 282 x x x 12-Octadecanolide lac 7 C18H34O2 282 x 17-Octadecanolide lac 7 C18H34O2 282 x Hexyl dodecanoate est 21 C18H36O2 284 x x x x Hexadecyl acetate est 24 C18H36O2 284 x 2Isopropyl hexadecanoate est 22 C19H38O2 298 x x x Eicosatrien-15-olide lac 8 C20H32O2 304 x x (E)-2-Pentenyl (E)-2,3- ter 37 C20H32O2 304 x dihydrofarnesenoate Eicosadien-13-olide lac 8 C20H34O2 306 x x Eicosadien-15-olide lac 8 C20H34O2 306 x x Octadecatrienyl acetate est 24 C20H34O2 306 x Pentyl (E)-2,3-dihydrofarnesenoate ter 37 C20H34O2 306 x Octadecadienyl acetate est 24 C20H36O2 308 x Eicosen-13-olide lac 8 C20H36O2 308 x 2-Heinecosanone on 15 C21H42O 310 x x x Octadecenyl acetate est 24 C20H38O2 310 x 13-Eicosanolide lac 8 C20H38O2 310 x Ethyl ocadecanoate est 23 C20H40O2 312 x x

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Butyl hexadecanoate est 22 C20H40O2 312 x Octadecyl acetate est 24 C20H40O2 312 x x Isopropyl hexadecanoate est 22 C20H40O2 312 x (Z)-3-hexenyl (E)-2,3- ter 37 C21H32O2 316 x dihydrofarnesenoate (Z)-3-hexenyl (Z)-2,3- ter 37 C21H32O2 316 x dihydrofarnesenoate Hexyl dihydrofarnesenoate ter 37 C21H34O2 318 x Propyl octadecadienoate est 23 C21H38O2 322 x Isopropyl octadecadienoate est 23 C21H38O2 322 x x x Docosenol ol 34 C22H44O 324 x x Propyl octadecenoate est 23 C21H40O2 324 x Isopropyl octadecenoate est 23 C21H40O2 324 x x x Isopropyl octadecanoate est 23 C21H42O2 326 x Docosanol ol 34 C22H46O 326 x x Butyl octadecatrienoate est 23 C22H38O2 334 x Isobutyl octadecatrienoate est 23 C22H38O2 334 x Eicosatrienyl acetate est 24 C22H38O2 334 x Docosadien-15-olide lac 9 C22H38O2 334 x Docosen-15-olide lac 9 C22H40O2 336 x x Butyl octadecadienoate est 23 C22H40O2 336 x Isobutyl octadecadienoate est 23 C22H40O2 336 x (Z)-3-Hexenyl hexadecenoate est 22 C22H40O2 336 x 22-Docosanolide lac 9 C22H42O2 338 x (Z)-3-Hexenyl hexadecanoate est 22 C22H42O2 338 x x x x x x x Ethyl eicosenoate est 25 C22H42O2 338 x Butyl octadecenoate est 23 C22H42O2 338 x Isobutyl octadecenoate est 23 C22H42O2 338 x Eicosenyl acetate est 24 C22H42O2 338 x x 15-Docosanolide lac 9 C22H42O2 338 x Hexyl hexadecanoate est 22 C22H44O2 340 x x x x Butyl octadecanoate est 23 C22H44O2 340 x Isobutyl octadecanoate est 23 C22H44O2 340 x Eicosyl acetate est 24 C22H44O2 340 x x x 1,22-Docosanediol ol 34 C22H4402 340 x 2-Phenylethyl (E)-2,3- ter 37 C23H32O2 340 x dihydrofarnesenoate Phenylmethyl hexadecenoate ar 22 C23H36O2 344 x Pentyl octadecatrienoate est 23 C23H40O2 348 x Pentyl octadecadienoate est 23 C23H42O2 350 x Pentyl octadecenoate est 23 C23H44O2 352 x Tetracosenol ol 35 C24H48O 352 x Pentyl octadecanoate est 23 C23H46O2 354 x (Z)-3-Hexenyl octadecatrienoate est 23 C24H40O2 360 x x x x Hexyl octadecatrienoate est 23 C24H42O2 362 x x (Z)-3-Hexenyl octadecadienoate est 23 C24H42O2 362 x x x x x x x Tetracosadienolide (a) lac 10 C24H42O2 362 x x Tetracosadienolide (c) lac 10 C24H42O2 362 x Octyl-3-oxo (E)-2,3- ter 37 C23H38O3 362 x dihydrofarnesenoate (Z)-3-hexenyl octadecenoate est 23 C24H44O2 364 x x x x x x Hexyl octadecadienoate est 23 C24H44O2 364 x x x x x Tetracosenolide (a) lac 10 C24H44O2 364 x x Tetracosenolide (b) lac 10 C24H44O2 364 x Tetracosenolide (c) lac 10 C24H44O2 364 x Tetracosenolide (d) lac 10 C24H44O2 364 x Hexyl octadecenoate est 23 C24H46O2 366 x x x x x (Z)-3-Hexenyl octadecanoate est 23 C24H46O2 366 x x x x x Docosenyl acetate (a) est 24 C24H46O2 366 x Docosenyl acetate (b) est 24 C24H46O2 366 x Docosenyl acetate (c) est 24 C24H46O2 366 x x Tetracosanolide lac 10 C24H46O2 366 x Hexyl octadecanoate est 23 C24H48O2 368 x x x Docosyl acetate est 24 C24H48O2 368 x 1,2-Tetracosanediol ol 35 C24H48O2 368 x 6,10,14,18-C18-tetraenoate- est 34 ~C25H4102 373 x 3,7,11,15,19-pentamethyl-, methyl ester Benzyl octadecenoate ar 23 C25H42O2 374 x Pentacosadienolide lac 11 C25H44O2 376 x Pentacosenolide lac 11 C25H46O2 378 x Hexacosenol ol 35 C26H52O 380 x Pentacosanolide lac 11 C25H48O2 380 x Tetracosenyl acetate (a) est 24 C26H50O2 394 x Tetracosenyl acetate (b) est 24 C26H50O2 394 x

121

Tetracosyl acetate (a) est 24 C26H52O2 396 x (Z)-3-Hexenyl docosenoate est 26 C28H52O2 420 x x Hexyl docosadienoate est 26 C28H52O2 420 x Hexacosenyl acetate est 24 C28H54O2 422 x Hexyl docosenoate est 26 C28H54O2 422 x Hexyl docosanoate est 26 C28H56O2 424 x Hexacosyl acetate est 24 C28H56O2 424 x 1 Compounds with similar number were grouped by known or assumed biosynthetic relationships. 2 Division of compounds at 300 g/mol (volatile/no-volatile A) 3 Division of compounds at 270 g/mol (volatile/non-volatile B) Compound groups: ar = aromatics, est = esters, lac = lactones, ol = alcohol, on = ketone, ter = terpenes, MF= molecular formula, MW= molecular weight, M = H. melpomene, C= H. cydno, P = H. pachinus, I = H. ismenius, N = H. numata, H = H. hecale, E = H. erato, Ch = H. charithonia, Sa = H. sara, He = H. hewitsoni, Sa = H. sapho.

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VITA

Catalina Estrada was born in Ibagué (Tolima) Colombia, on May 28, 1972. She is the daughter of Fernando Estrada Robledo and Olga Montes de Estrada. After graduation from Colegio Santa Teresa de Jesús in Ibagué in 1989, she entered the program of biology in Universidad de los Andes at Bogotá, where she received her bacherlor degree in 1996, and then Master of Science degree in biology in 1998. In fall 2002 she entered graduate school at the University of Texas at Austin.

Permanent Address: Conjunto Residencial Balcones del Vergel Apt. 501, Torre 2. Ibagué, Colombia.

This dissertation was typed by the author.

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