SPATIAL DYNAMICS OF NIGHT ROOSTING IN erato petiverana (: )

By

CHRISTIAN SALCEDO

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2006

Copyright 2006

by

Christian Salcedo

“Perfect as the wing of a may be, it will never enable the butterfly to fly if unsupported by the air. Facts are the air of science. Without them a man of science can never rise.” Adaptation from phrase by Ivan Pavlov (1849-1936)

ACKNOWLEDGMENTS

This work could not have been possible without the great help and advice of Dr.

Thomas C. Emmel, Dr. Miriam Medina Hay-Roe, Dr. Jacqueline Y. Miller, Dr. Andrei

Sourakov, and Dr. Peter Teal. I want to thank also Ernesto Rodriguez, from El Bosque

Nuevo Preserve, , for providing some of the used in this study. Student assistants Vanessa Walthall and Kari Ellison contributed with help in breeding and colony maintenance.

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TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... iv

LIST OF FIGURES ...... vii

ABSTRACT...... viii

CHAPTER

1 INTRODUCTION AND RESEARCH GOALS ...... 1

2 HELICONIUS ...... 3

3 ROOSTING PATTERNS IN HELICONIUS...... 5

Introduction...... 5 Roosting in Heliconius ...... 5 Objectives ...... 8 Materials and Methods ...... 8 Study Organisms ...... 8 Butterfly Rearing...... 9 Butterfly Marking and Wing Length Measurement ...... 9 Roost Structure ...... 9 Butterfly Roost Recruitment...... 10 Statistical Analysis for Roost Structure...... 11 Determining Level of Clustering...... 11 Analyzing Spatial Distribution related to Individual Traits ...... 11 Results...... 12 Butterfly Roost Recruitment and General Observations...... 12 Level of Clustering...... 12 Spatial Distribution related to Sex, Age, and Size ...... 13 Discussion...... 13 Within Roost Interactions and Trends...... 13 Level of Gregariousness...... 16 Conclusions...... 17

LITERATURE CITED ...... 21

v BIOGRAPHICAL SKETCH ...... 26

vi

LIST OF FIGURES

Figure page

3-1. Species used in this study ...... 18

3-2. Measurement of butterfly wing length...... 18

3-3. Example of grid used to locate each individual position in a roost...... 19

3-4. sub-aggregations in captivity...... 20

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

SPATIAL DYNAMICS OF NIGHT ROOSTING IN Heliconius erato petiverana (LEPIDOPTERA: NYMPHALIDAE)

By

Christian Salcedo

August 2006

Chair: Thomas C. Emmel Major Department: Entomology and Nematology

Communal roosting occurs when multiple of one or more species assemble

in close proximity to one another for a certain period of time. Roosts may be

synchronized with circadian rhythms (day-night cycles), or with seasons, or they can be

permanent. Some species within the Heliconius display night roosting behavior.

This particular behavior has been addressed several times over more than a century, but there is still no clear explanation for it. In order to better understand this behavior, I analyzed clustering and roost structure related to individual’s sex, age, and size using

Heliconius erato petiverana individuals. The results show that the roost is frequently formed by sub aggregations of individuals but there is no spatial pattern related to sex, age, or size. This suggests that the roost spatial distribution is not affected by selective pressure on these particular traits. Other factors that could be involved in the formation and structure of the roost are discussed.

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CHAPTER 1 INTRODUCTION AND RESEARCH GOALS

Communal roosting occurs when multiple insects of one or more species assemble

in close proximity to one another for a certain period of time (Yackel 1999). Roosts may be synchronized with circadian rhythms (day-night cycles), or seasons, or they can be

permanent (Waller and Gilbert 1982). This behavior has been documented in several

groups such as butterflies, moths, dragonflies, bees, and wasps for over a century, and in each case authors have proposed different hypotheses to explain its relevance

(Evans and Linsley 1960; Benson and Emmel 1973; Joseph 1982; Greig and DeVries

1986; Rehfeldt 1993). Nevertheless, the functional and adaptive significance of gregarious roosting in insects is not fully understood. It is a complex behavior indeed and several factors might have been responsible for its . An interesting group where communal roosting occurs and that can help to further understand this behavior is

Heliconius butterflies.

Heliconius butterflies belong to the Nymphalidae within the order

Lepidoptera (Penz 1999). They comprise a widespread genus over the tropical and subtropical regions of the New World (Brown 1981; Emsley 1965; Turner 1981) and have been subject to a wide range of studies due to their abundance and relative ease in breeding under laboratory conditions. Even though considerable information has been published on their genetics, ecology, and behavior (Benson 1971, 1972; Cook et al. 1976;

Crane 1955, 1957; Jiggins et al. 2001; Jones 1930; Mallet 1980, 1986; Mallet and Gilbert

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1995; Mavárez et al. 2006; Murawski and Gilbert 1986; Poulton 1931; Salcedo 2003;

Turner 1971, 1975, 1981; Waller and Gilbert 1982; Young 1978), little is known about

their night roosting habits (Beebe 1949; Cook et al. 1976; Crane 1955, 1957; Edwards

1881; Jones 1930; Mallet 1980, 1986; Mallet and Gilbert 1995; Murawski and Gilbert

1986; Poulton 1931; Turner 1971, 1975, 1981; Waller and Gilbert 1982; Young 1978).

In the typical situation, several individuals (males and females) begin to land usually on twigs, tendrils, and dry leaves under the shade of a tree just before sunset.

After sunset, usually a group has been formed and most of them remain together until sunrise (Crane 1955, 1957; Jones 1930; pers. observations). In addition to the mentioned studies on Heliconius roosting, several authors have speculated about the possible function of this type of aggregation, and within these speculations, the evolution of this

behavior as a social trait has been frequently addressed (Benson 1971; Gilbert 1975,

1977; Mallet 1986; Turner 1981). However, to date, only one author has gathered

evidence that suggests that avoidance of disturbance and are likely reasons to

explain this behavior (Mallet 1986; Mallet and Gilbert 1995).

Why do Heliconius species roost gregariously? The answer remains in obscurity.

Consequently, further observations and experiments are required. The present work

analyzes roost structure patterns to further understand this remarkably complex behavior.

CHAPTER 2 HELICONIUS BUTTERFLIES

History, Distribution and Biology: Relevant Facts and Traits

Heliconius butterflies do not represent a rare isolated genus for the scientific community. They have been studied and described since the times of Darwin, and have became a widespread model in evolutionary studies (Turner 1981). In Wallace’s contributions to the Theory of Natural Selection, he wrote:

There is in an extensive family of these insects, the Heliconidae, which are in many respects remarkable. They are so abundant and characteristic in all the woody portions of the American tropics, that in almost every locality they will be seen more frequently than other butterflies. They are exceedingly beautiful and varied in their colors, spots and patches of yellow, red, or pure white, upon a black, blue or brown ground…. Yet, although they are so conspicuous and could certainly be caught by insectivorous birds more easily than any other insect, their great abundance…shows that they are not persecuted. (Wallace 1870, p.77).

Wallace was certainly right. Heliconius butterflies include approximately 46 species distributed in tropical and subtropical America, and their higher diversity is concentrated in the tropical region of South America (Brown 1981; Emsley 1965; Turner

1981). They can be found between from 0 to 2000 m elevation, usually in habitats with mild temperatures that range between 25 to 30ºC. Their life cycle averages 28 days and passion vines () are their host plants. Heliconius butterflies sequester cyanogenic glycosides from their host plants (Engler et al. 2000; Hay-Roe 2004) and synthesize de novo cyanogenic compounds (Nahrsdet and Davis 1983, 1985) which are known to be unpalatable to predators (Chai 1986, 1988; Srygley and Chai 1990). This

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particular trait plays a key role in large, complex rings involving phenotypic

resemblance in which they are mimicked, by relatively distantly related species of the

same genus. This is called Müllerian mimicry. Evolutionarily speaking, its occurrence

appears to involve natural selection driving the color patterns of diverse species, living in

the same community, towards a convergence upon one color pattern that may lessen the

learning trace for naïve (e.g. newly fledged birds) predators and enhance predation recognition and hence effectiveness of a color pattern for a distasteful insect (Benson

1972; Brown et al. 1974; Brown 1981; Mallet 1993; Mallet and Gilbert 1995; Brower

1996; Linares 1996, 1997a, b; Mallet et al. 1998; Mallet and Joron 1999; Mallet 2001;

Turner 1981).

CHAPTER 3 ROOSTING PATTERNS IN HELICONIUS

Introduction

Behavior is perhaps one of the most challenging traits to study in biology. Its study is usually constrained to a complex experimental design, which usually involves non- traditional approaches of data analysis and long periods of observation in order to detect possible patterns. As with any other characters, behavioral traits have a genetic basis and hence are under selective pressure (Mallet and Gilbert 1995). Night roosting is perhaps one of the most interesting behaviors presented by Heliconius, and this behavior has dazzled Heliconius experts and enthusiasts for a long time (Beebe 1949; Cook et al.

1976; Crane 1955, 1957; Darwin 1871; Edwards 1881; Jones 1930; Mallet 1980, 1986;

Mallet and Gilbert 1995; Murawski and Gilbert 1986; Poulton 1931; Turner 1971, 1975,

1981; Waller and Gilbert 1982; Young 1978). The famous Amazon explorer Alfred

Russel Wallace set the stage nicely for future workers when he recorded in his observations on Heliconius: “they may be observed after sunset suspended at the ends of twigs and leaves, where they have taken up their station for the night, fully exposed to the attacks of enemies, if they have any.” (Wallace 1870, p. 77).

Roosting in Heliconius

Jones (1930) was the first who performed experiments on Heliconius roosting behavior. Jones tested how butterflies in Florida identified the roosting place, in particular whether if it was by place-memory or by scent. After

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removing their usual roosting twigs, he found that the butterflies came again to the same place and roosted on new twigs. Thus he suggested that recognition of the roost is by place-memory, not by scent. Based on his data, he also suggested these butterflies showed non-long-term roost fidelity, and he observed that they usually occupied new positions nightly or conserved the same position for only a short succession of nights.

After Jones, Jocelyn Crane (1955, 1957) recorded controlled observations on

Heliconius erato, H. melpomene, and H. ricini in Trinidad at the Simla Research Station in the Arima Valley. She observed in screened outdoor insectaries that males and old females tend more to occupy crowded roosts together, while younger females, except when at their most receptive state to males (on the second through the fourth days post- emergence), showed less tendency to be gregarious. However, these receptive females usually roosted on the more crowded perches, or sometimes next to a somewhat isolated male. She also reported the tendency to maintain separate roosts from other species of the same genus, but noted instead that they often roosted with congeners. In addition, she

noted that in both sexes and all species, the habit of assortative roosting (according to

species) begins or is first expressed on the second or third night after an adult emerges.

Two decades later, Young and Thomason (1975) found that sex ratio was 1:1 for

roost membership in H. charithonia in Costa Rica. They also did not find any particular

pattern with respect to age as indicated by relative condition of the wings. Fidelity of

individuals to a roost was often high and was not correlated with sex or age.

After more than a century of observations and experiments, a plausible

explanation for roosting behavior in heliconiines is still obscure. Nonetheless, some

interesting approaches to the question of its purpose have been proposed. Gilbert (1972)

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suggested that communal roosting in these butterflies evolved as the result of young,

inexperienced individuals needing to improve their ability to find scarce or inconspicuous

sources by following older adults from a central point, as pollen feeding is of

major importance to long life and reproductive success in many Heliconius species.

Subsequently, Turner (1975) refined this hypothesis and proposed that communal roosts give added protection by aggregating individuals within the home ranges of a small number of educable predators, compared with the number that would require education if the butterflies were dispersed. The small size of the breeding populations, restricted home ranges, and broadly overlapping generations, allowing a butterfly to fly with several generations of its descendants, could be expected to lead to moderately high levels of kinship within populations and hence to the evolution of cooperative altruistic behavior

through kin selection.

One of the most complete studies on Heliconius roosting combined data on

gregarious roosting with accurate mapping of the daily movements of individual

butterflies (Mallet 1986). The results added evidence that make the anti predatory approach a likely reason to explain the nature of gregarious roosting and, contrary to the early proposal of Jones (1930), Mallet suggested that the roosts could be formed with the help of . In addition, Mallet and Gilbert (1995) recently gave more support to his hypothesis by discovering roosting stratification and inter and intraspecific roosting habits. They found that Heliconius species demonstrate a particular correlation between roosting height and the mimicry ring to which they belong. For example, black and red

Heliconius erato and H. melpomene roost low (0.5-3.5 m), while three species of the yellow-orange mimicry ring (H. ismenius, H. hecale, and H. doris) roost higher in trees,

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(up to 10 m and more). Mallet and Gilbert also concluded that on average, individual

Heliconius roost next to a co-mimic with probability of 0.17 and to a non-mimic with probability of 0.06. Individuals are thus 2.8-fold more likely to roost with mimics than with non-mimics.

Even though there is evidence supporting an anti-predatory function for the gregarious roosting in Heliconius, the ultimate and proximate reasons why they roost remain elusive. Further evidence needs to be gathered in order to continue finding the pieces of this dazzling behavior.

Objectives

1. Identify roosting patterns in Heliconius:

9 Do the butterflies tend to present a clumped spatial distribution?

If they do show this spatial distribution:

9 How long do the butterflies take to begin to express this behavior after they

emerge from the ?

9 Do the butterflies roost always in the same site?

9 Does the roosting group possess any particular distribution of individuals

related by sex, age, or size?

2. Address the evolutionary significance of roosting using the obtained roosting

spatial data and the information obtained in previous studies:

9 Does the spatial distribution imply or suggest an anti-predatory strategy?

Materials and Methods

Study Organisms

Heliconius erato petiverana, rosina, and Heliconius charithonia were used in this study (Fig. 3-1)

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Butterfly Rearing

Heliconius erato petiverana and Heliconius melpomene rosina were initially purchased as pupae from a butterfly farm located in Costa Rica (El Bosque Nuevo preserve). Heliconius charithonia was bought as pupae from a local butterfly farm in

Naples, Florida, and also wild adult individuals were collected in Gainesville, FL, USA.

The experimental colonies were started with 15 individuals of each species. Adults were kept in 1.8 x 2 x 5 m indoor insectaries at 27ºC, 80-95% ºH, that were inside a temperature controlled Lord & Burnham glass house (5 x 8 m) at the University of

Florida, Department of Entomology and Nematology, together with appropriate larval host plants ( biflora, P. punctata, and P. auriculata), where they mated and laid eggs. Once eggs were laid, they were collected and transferred to environmental chambers for hatching and larval growth until pupation and eclosion; chambers were maintained under constant laboratory conditions (27ºC, 75% ºH and a 14L: 10D photoperiod).

Butterfly marking and Wing Length Measurement

After eclosion, each individual was sexed and marked on the underside of both hind wings using a permanent ink Sharpie® marker. Wing length was measured using a

Vernier caliper. The wing was measured from the wing base to the apex (Fig. 3-2).

Roost Structure

Heliconius butterflies roost naturally with their legs’ tarsal claws clinging to twigs, tendrils, and dead leaves, and wings hanging downward. Based on preliminary studies and personal observations, these butterflies also will roost on the screen of the walls of the insectary. However, they were given the “choice” of using twigs and passion vines

(Passifloraceae), which have tendrils, within the cage enclosures.

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Observations were made after sunset during the months of July, August,

September, October, and November 2005. White light was found to awaken individuals in some cases; therefore, a low-intensity, red-beam flashlight, which apparently did not disturb the butterflies, was used to identify each of them. Initially, the wall that most individuals frequented during the first week was identified. After this, a grid was created using natural fiber thread. The grid began at the outside of the insectary wall. Each cell of the grid was 5 x 5 cm and was identified by a letter-number code (Fig. 3-3), so that the position of each individual could be easily and precisely recorded. Apparently, the placing of the natural fiber grid did not affect the roosting site, since adults continued to use the same wall as roosting site.

For “solitary” individuals or new roosting patches in other walls of the insectary, another procedure was developed to record each individual position. Taking into account the dimensions of each wall, a grid was designed and printed for each of them, so the relative position of each individual could be located and recorded in the printed grid. This permitted me to keep the same data format for the subsequent analysis.

Additionally, dry and slender passion vine stems were attached to the top screen of the cage. In this case, each individual position was recorded by making a two- dimensional sketch of the stem with 5 cm cells.

Butterfly Roost Recruitment

Once the roosting behavior was observed consistently, the arrival order was recorded by observing adults and the time that they landed on the dry passion vine stem, over a period of 30 days. I also recorded the number of days from emergence a butterfly took to become a member of the roost.

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Statistical Analysis for Roost Structure

Determining Level of Clustering

In order to determine the level of clustering, I used the Monte Carlo test of the

Index of Cluster Size (ICS) (Cressie 1993). The ICS is a very useful index that can be used to determine clumping: ICS = (Observed variance/ Observed mean) – 1.

The rationale for using this ICS statistical index is as follows. If the butterflies are distributed among quadrats, there will be a certain number in each quadrat each day. The mean number and the variance can be calculated each day, and after adding all ICSs and dividing them by the number of days, one can obtain an average ICS. Then, using statistical software (Insightful S+ 7.0 for Windows 2005, Insightful Corp. Seattle,

Washington), a Monte Carlo test (Manly 1997) can be made to compare this ICS with the hundreds of possible random arrangements which the butterflies could present in the quadrats. The ICS obtained in the results will determine if the butterflies have a clustering pattern. ICS would be zero if all the butterflies are randomly distributed among the quadrats.

Analyzing Spatial Distribution related to Individual Traits

I used a multinomial cumulative logit design (Manly 1997) where the whole plot factor was sex and the experimental whole-plot unit was individual. The subplot "effect" was age which is continuous. I also used randomized block design to test whether sex or wingspan affected the decision as to where to settle.

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Results

Butterfly Roost Recruitment and General Observations

After a period of 60 days, colonies of each species were relatively stable. Each colony was composed by at least 15 individuals. The only species that presented the night roosting behavior in a consistent way was Heliconius erato petiverana. Once the individuals were placed inside the cage, they took an average of three days to start joining the roost. Newly emerged individuals perched solitarily and randomly on the walls or roof of the cage. They always used the same area of the cage to roost and the order in which the individuals landed in the roosting area (passion vine stems or wall) was completely random. A separate isolated colony of 10 individuals (5 males and 5 females) was bred from a new pupae shipment from Costa Rica, and then placed in the cage of the bigger colony; the individuals were able to join the roost of the original colony.

The other two species, Heliconius melpomene rosina and Heliconius charithonia, were not able to form a roost within the cages. They basically perched randomly on the roof of the cage during the night. This could be possibly due to the fact that in the wild, the different Heliconius species exhibit height stratification (Mallet 1986; Mallet and

Gilbert 1995) and the experimental cages were not high enough to allow these species to express roosting behavior.

Level of Clustering

The wall grid data provided useful information to test whether there was presence or absence of clumping. The statistical method used (see Methods) showed the strong clustering tendency of this species when there were no passion vine stems present in the cage; they perched on one of the cage walls (ICS = 6.433).

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A more natural scenario occurs when the butterflies roost on a twig or a dry passion

vine stem. Several nights after a long dry passion vine stem (1.2 m) was placed next to

the wall where the butterflies liked to roost, they moved to the stem and used it exactly as

they do in the wild. This new set-up was almost perfect to identify patterns in the spatial

distribution within the roost related to sex, age, and size (wingspan). After observing the

butterflies’ distribution along the stem, I frequently identified subgroups formed by 3-5

individuals. Even though ICS was used to test clustering at a bigger scale (i.e. the entire

cage), the model could also be set to test clustering at a different scale. To test this, the

stem set-up was divided into quadrats. Two quadrat sizes were used. In one, the quadrat

size was barely the size of a butterfly, while in the other one the quadrat size matched the

observed group size. The test showed strong clustering at the subgroup quadrat size

(ICS=1.530).

Spatial Distribution Related to Sex, Age, and Size

Neither the multinomial cumulative logit model nor the randomized block design

showed any trend related to sex, age or size. The sex ratio was 1:1, and the roost was

composed on average by 10.1 females and 10.6 males.

Discussion

Within Roost Interactions and Trends

The most detailed and recent study on Heliconius roosting dynamics addressed

different interactions observed during the roost formation and between-roost interactions

(Mallet 1986). During the roost formation, the behavior ranged from hovering closely

with probable antennal and wing contact (“fanning”), to grasping at the perch or the

wings of the roosted butterfly with their tarsal claws (“clutching”). These interactions are remarkably similar to courtship; Mallet concluded that gregarious roosting evolved

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through a modification of courtship behavior, confirming Crane’s (1957) proposal. Mallet

also found movements of teneral adults among the roosts, overlapping in the daily home

ranges of individuals from different roosts, and ability of foreign individuals to join

gregarious roosts. I also found that adults from a separate colony are able to join the roost

successfully, which gives more support to Mallet’s (1986) conclusions against a role for

kin selection. He suggested that roosting aggregations and home ranges in Heliconius

erato are not defended or exclusive territories of family groups, and that exchange of resources information, such as pollen sources, among individuals is very unlikely.

Within-roost trends have never been addressed by investigators. Even though intra-

and inter-species communication among individuals of a roost seems unlikely, individual

distribution in the roost might give more clues towards understanding the true function of

these aggregations. Roost structure involves trends found to be adaptively significant in other insect groups. For example, dragonflies have roosts formed by immature individuals (Miller 1989) and the roost structure can change during a season. Damselflies can form adult or immature aggregations, possibly due to the proximity of the roost to resources used by adults (Neubauer and Rehfeldt 1995). In Heliconius, Waller and

Gilbert (1982) documented the occurrence of worn and intermediate wear butterflies in the roosts early in the season. They evaluated age based on wing wear, an estimate that is not as precisely accurate as following the fate of captive adults with known emergence dates.

The observations in the present study were done during summer and fall 2005

(from July to late November) with a daylight period from approximately 14 h in July decreasing to 11 h in November. Daylight in the natural habitat (Costa Rica) of these

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Heliconius species (H. erato and H. melpomene) has less variation; daylight is approximately 12 h all year round. These conditions apparently did not affect the roosting habits.

Multi-age composition and 1:1 sex ratio were found in the experimental roost.

Because of the constant input of laboratory-reared individuals to the experimental colony, sex ratio and age composition might differ from the natural states; in fact, a previous study shows that in the wild, there is a 2:1 male bias at roost sites (Mallet 1986). The butterflies’ distribution among the experimental roost was not affected at all by sex; in other words, there are no preferential or assortative male or female aggregations in the roost, and there is no special positioning according to sex. These findings eliminate the possibility of a probable enhanced protection from predators that might occur from the higher toxicity of gravid females (Hay-Roe 2004) which could be more exposed if on the periphery of a roost, or on the other hand, could be more protected in less exposed areas of the roost, in order to secure the survival of the next generation.

Age was not also a condition that influenced butterfly distribution, which shows that younger “less experienced” individuals behave in the same way as two-month-old or older conspecifics, at least regarding the gregarious roosting. However, recently-emerged butterflies took an average of 3 days to start perching on the roosting stem. This finding supports Mallet (1986) assertion as to the possible role of learning in generating this behavior. However, this 3-day period might be indicating also that the adults take 3 days to mature and start producing aggregation pheromones (Borden 1986). Previous authors have suggested that gregarious roosting evolved through modification of courtship behavior (Crane 1957; Mallet 1986), so it would not be surprising if it were found that

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aggregation pheromones are modified sexual pheromones and that the 3-day maturation

period to start producing and releasing the compounds is linked to the consumption of a certain resource, such as pollen.

Wingspan was also analyzed with spatial statistics. Individual size might be

important in fanning interactions, where there are brief approaches involving aerial wing

contact and also where (once settled) the wings are flapped vigorously to defend a perch

during the roost formation (Mallet 1986; Young and Carolan 1976). Larger wings could

also enhance the ability to exchange chemical cues during the roost formation. However,

wingspan is not involved in this process; my results show that the butterflies’ location in

the roost is not affected by wingspan, which suggest that smaller wingspans might be as

successful as larger ones when defending or looking for a perch.

Level of Gregariousness

The Heliconius erato roosts in the present experiments were characterized by linear

non-touching arrays of as many as five individuals per stem (Fig. 3-4). Statistical analysis

of the data confirmed this trend; H. erato shows a high tendency to cluster at this scale.

The same behavior has been documented in the wild with H. erato subspecies from

Mexico and , and has been related partially to population density (Mallet 1986).

The experiments at the University of Florida were done under artificial conditions, yet the butterflies exhibited the same level of gregariousness shown in wild established populations. The constant input of laboratory-bred butterflies and general conditions correctly mimicked the natural conditions, or the level of gregariousness might not depend strongly on population density. Other agents might affect the level of clustering.

H. erato roosting height can be very low in the wild at only 0.5 m (Mallet and Gilbert

1995), and even lower in captivity 0.3 m (pers. observations). At this height, it might be

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advantageous to have small units of few individuals; in case of a predator attack or a breakage in the stem or twig, fewer individuals will be at risk. However, further observations are needed to establish whether predation on roosts is common enough to constitute a significant selective force for small clusters at low roost heights.

Conclusions

The results of this work show that rather than being a complex structure, gregarious

roosting involves a simple randomly formed group with some degree of complexity in

spatial distribution. Heliconius erato internal roost structure is not defined by individual traits such as sex, age, or wingspan, and kin selection is unlikely to be playing any role in these aggregations. Nevertheless, the process by which Heliconius butterflies present this behavior remains obscure, but chemical cues are likely to be involved.

The observations in this present research neither support nor weaken an anti- predatory strategy. However, the level of gregariousness tested with spatial models implies that perching in smaller sub units might give some adaptive advantage to

Heliconius erato.

Finally, further research is needed to find the significance of the different levels of gregariousness in Heliconius, to look for strong evidence regarding the possible role of predation in the roost sites, and to conduct analysis of the probable role of chemicals in the ecology and evolution of Heliconius gregarious roosting behavior.

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Figure 3-1. Species used in this study: Heliconius erato petiverana (top left), Heliconius melpomene rosina (top right), and Heliconius charithonia (center).

Figure 3-2. Measurement of butterfly wing length.

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A B C D E F G H I J K L M ... 1 2 3 4 5 6 7 8 9 10 11 12 13 14 …

Figure 3-3. Example of grid used to locate each individual position in a roost. Each cell is 5 x 5 cm.

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Figure 3-4. Heliconius erato sub-aggregations in captivity. Note the three subgroups in this picture.

LITERATURE CITED

Beebe, W. 1949. High Jungle. Duell, Sloan, and Pearce. New York, U. S. A.

Benson, W. W. 1971. Evidence of unpalatability through kin selection in the Heliconinae (Lepidoptera). Amer. Nat. 105 (943): 213-226.

Benson, W. W. 1972. Natural selection for Müllerian Mimicry in Heliconis erato in Costa Rica. Science 176 (4037): 936-939.

Benson, W. W., and T. C. Emmel. 1973. Demography of gregariously roosting populations of the nymphaline butterfly berania in Costa Rica. Ecology 54: 326-335.

Borden, J. H. 1986. Aggregation pheromones. In Comprehensive Physiology Biochemistry and Pharmacology. Kerkut, G. A. and Gilbert, L. I. eds. Pergamon Press. Oxford, U. K.

Brower, A. V. Z. 1996. A new mimetic species Heliconius (Lepidoptera: Nymphalidae), from southeastern Colombia, revealed by cladistic analysis of mitochondrial DNA sequences. Zool. Journ. Linn. Soc. 116: 317-332.

Brown Jr., K. S. 1981. The biology of Heliconius and related genera. Ann. Rev. Entomol. 26: 427-456.

Brown, K. S., P. M. Sheppard and J. R. G. Turner. 1974. Quaternary refugia in tropical America: evidence from race formation in Heliconius butterflies. Proc. R. Soc. Lond. B. 187: 369-378.

Chai, P. 1988. Wing coloration of free-flying Neotropical butterflies as a signal learned by specialized avian predator. Biotropica 20: 20-30.

Chai, P. 1986. Field observations and feeding experiments on the responses of rufous- tailed jacamars (Galbula ruficauda) to free-flying butterflies in a . Biol. J. Linn. Soc. 29: 161-189.

Cook, L. M. Thomason, E.W. and A. M. Young. 1976. Population structure, dynamics and dispersal of the tropical butterfly Heliconius charitonius. J. Anim. Ecol. 45: 851-863.

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BIOGRAPHICAL SKETCH

Christian Salcedo was born on November 6, 1979, in Bogotá, a city in the Andean

Mountains of South America located at 2600m.o.s.l. (8900ft), and capital of Colombia.

His interest in Biology started in his last years of high school (1997-1998) stimulated by

Biology professor Leopoldo Arrieta. In search of a benign and intellectually spirited climate, Salcedo went to the Universidad de los Andes in Bogotá. Coursework in invertebrate life, evolution, and ecology, together with fieldtrips to local natural parks, gave him confidence and enthusiasm towards his future in biological sciences. Two years before graduation, Salcedo volunteered at the Population Genetics Institute at

Universidad de los Andes. There he performed his first formal research project under the supervision of Dr. Mauricio Linares, which later became his undergraduate thesis. After graduation (2003), he was accepted in the master’s program at the Entomology and

Nematology Department at the University of Florida, under the advisorship of Dr.

Thomas C. Emmel. He currently works as a graduate research assistant at the McGuire

Center for Lepidoptera and Biodiversity at the Florida Museum of Natural History in

Gainesville, Florida.

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