Sexual Selection and Female Ornamentation in a Role- Reversed Dance Fly

SEXUAL SELECTION AND FEMALE ORNAMENTATION IN A ROLE- REVERSED DANCE FLY

Jill Wheeler

A thesis submitted in conformity with the requirements for the degree of MSc Graduate Department of Ecology and Evolutionary Biology University of Toronto

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While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. Canada Sexual selection and female ornamentation in a role-reversed dance fly

Jill Wheeler (MSc, 2008)

Department of Ecology and Evolutionary Biology

University of Toronto

THESIS ABSTRACT

Elaborate morphological traits (ornaments) can evolve if they increase the reproductive success of the bearer during competition for mates. However, such ornaments are very rare in females, potentially due to trade-offs between fecundity and ornaments. I examined sexual selection on female ornamentation in the dance fly

Rhamphomyia longicauda. Female R. longicauda have two female-specific ornaments— inflatable abdominal sacs and pinnate leg scales. Using models of real females, I found that males prefer females with large ornaments, irrespective of the swarm sex-ratio. A selection analysis revealed stabilizing selection for the scale ornament only. However, only inflated abdomen area had a significant positive relationship with fecundity.

Future studies should investigate the influence of viability selection and intrasexual competition on female ornamentation of this species.

ii ACKNOWLEDGEMENTS

The entire process of completing my Masters has been one of an exceptionally challenging but rewarding experience for me. I may have never started it—let alone completed it—without the support of many people. First, I would like to thank my advisor Darryl Gwynne for his guidance and support throughout this project. Darryl struck the ideal balance of offering help when I needed it, but also letting me figure things out for myself. His enthusiasm for the study system was a constant source of inspiration. I am amazed by his ability to see things from various perspectives and brainstorm alternate hypotheses, and it has pushed me to see the bigger picture. I am also very grateful to him and his family for putting me up. Not many graduate students live in their advisor's basement during their field work, but Darryl and his family — especially Sarah—made me feel very comfortable. I am also tremendously appreciative of my lab members: Laura, Edy, Murray and especially Kevin. Kevin always knew the answer to every question I asked him, and is one of the most selfless and helpful people I have ever met. The grueling hours at the microscope were made much easier by being surrounded by such caring and fun people. I'd also like to thank the community of graduate students at UTM and EEB, who were always willing to share their expertise. I probably would have never pursued graduate work if I hadn't had such a positive experience in my early academic career. I thank my first advisor Locke Rowe for hiring me to work in his lab in my second year of undergrad, and for his continued support. I was very fortunate to work with Russell Bonduriansky while in Locke's lab, and I thank him for all that he taught me. I also want to bestow a huge thank you to my 'academic mom' Helen Rodd who has always welcomed me into her office to talk about school, life, etc.

in My thesis was made possible by the expertise contributed by the following individuals. Luc Bussiere has been remarkably generous in sharing his dance fly and statistics knowledge. I am grateful to David Funk for sending me the templates he used for his dance fly models. Janice Ting offered valuable assistance in constructing the models. I thank John Hunt for his help with the selection analyses. Thank you to Vicki Simkovic for her assistance in the field. Finally, I want to acknowledge the tremendous support of my family and friends. I thank my family— Mum, Jenn, Fam, Dad and Erick~for their love, patience and understanding. I am incredibly fortunate to have the friends I do- especially Michelle, Matthew, Heath, Christine and Lorissa—who never fail to check in on me, listen to my rants or take me out when I need stress relief. I also want to thank my 'bosses'—Petra and Myriam—for their patience and support.

IV CONTENTS

CHAPTER 1

GENERAL INTRODUCTION

1.1 AN OVERVIEW OF SEXUAL SELECTION

1.1.1 THE THEORY OF SEXUAL SELECTION

1.1.2 THE DIRECTION OF SEXUAL SELECTION

1.1.3 FEMALE ORNAMENTATION

1.2 THE STUDY SPECIES

1.2.1 FEMALE ORNAMENTATION IN THE EMPIDIDAE

1.2.2 THE LONG-TAILED DANCE FLY RHAMPHOMYIA

LONGICAUDA

1.3 OUTLINE OF THESIS

CHAPTER 2

MALE MATE CHOICE FOR FEMALE ORNAMENTS OF THE DANCE FLY

RHAMPHOMYIA LONGICAUDA

2.1 ABSTRACT

2.2 INTRODUCTION

2.3 METHODS

2.4 RESULTS 2.5 DISCUSSION

2.6 FIGURES

2.7 TABLES

CHAPTER 3

SEXUAL SELECTION FOR FEMALE ORNAMENTS AND THEIR

RELATIONSHIP TO FECUNDITY IN THE DANCE FLY RHAMPHOMYIA

LONGICAUDA

3.1 ABSTRACT

3.2 INTRODUCTION

3.3 METHODS

3.4 RESULTS

3.5 DISCUSSION

3.6 FIGURES

3.7 TABLES

CHAPTER 4

CONCLUSIONS

LITERATURE CITED

VI 1

Chapter 1: General Introduction

1. GENERAL INTRODUCTION

1.1 An overview of sexual selection

1.1.1. Theory of sexual selection

Darwin (1871) was inspired by showy morphological traits to formulate his theory of sexual selection; he posited that traits such as ornaments and weapons arise within a species through differential reproductive success. This difference in reproductive success can occur if one sex invests more resources in reproduction, and thus will be the limiting sex (Trivers, 1972). The limiting sex consequently will require more time between matings and will therefore have a lower potential reproductive rate (PRR: Clutton-Brock & Vincent, 1991; Clutton-Brock & Parker,

1992). These conditions lead to the limiting sex being less abundant, and the operational sex ratio (OSR) will be biased towards the non-limiting sex (Emlen &

Oring, 1977). Sexual selection will act more strongly upon the non-limiting sex.

Sexual selection can act through two mechanisms: mate choice and intra- sexual competition. Mate choice occurs when the limiting sex bases its mating decisions on the expression of certain traits of the non-limiting sex. Why these traits 2 are the target of mate choice—and how mating preferences are maintained—has been the focus of extensive research in the field of sexual selection (Bateson, 1983;

Andersson, 1994). Exercising mate choice is thought to offer the choosy sex either direct benefits (e.g., nutrition in a nuptial gift (Gwynne, 1984) or a high quality territory (Bensch & Hasselquist, 1992)) or indirect benefits (e.g. enhanced offspring viability and/or attractiveness). Intra-sexual competition occurs when there is competition within the non-limiting sex over access to mates, or access to resources that may increase reproductive success. Within this context, the sexually-selected traits of the non-limiting sex enhance its competitive ability.

1.1.2. The direction of sexual selection

Typically, sexual selection acts more strongly on males than females, but sexual selection can act on females (e.g. Gwynne & Simmons, 1990). Since eggs are more costly to produce than sperm, females are typically the limiting sex. However, females can become the target of sexual selection if males incur high breeding costs

(Kokko & Monaghan, 2001; Kokko & Johnstone, 2002) and/or if there is high variation in female quality (Parker, 1983; Owens & Thompson, 1994; Johnstone et ah,

1996; Kvarnemo & Simmons, 1999). Males can incur breeding costs by producing or obtaining nuptial gifts (Gwynne, 1981,1984; Davies & Dadour, 1989), performing long courtship or copulatory behaviours (Saeki et al., 2005), or by the increased mortality risk of engaging in these activities (Jiggins et ah, 2000). Female quality can vary with respect to fecundity (Kvarnemo & Simmons, 1999) or stage of egg 3 development (Funk & Tallamy, 2000). If there is high sperm competition, females may vary with respect to the likelihood of using males' sperm for egg fertilization: males may reject recently mated females if there is first-male sperm precedence (e.g.

Simmons et al., 1994) or females with immature eggs if there is last-male sperm precedence (e.g. Parker, 1970). When there are high breeding costs to males and/or high variance in female quality, the typical mating roles can become reversed: male mate choice and female-female competition (Gwynne, 1991).

Male mate choice has been documented in a variety of taxa (see review in

Bonduriansky, 2001). Males have demonstrated mating preferences for fecund females (Pitafi et al., 1995), or for correlates of fecundity, such as body weight

(Gwynne, 1981; Byrne & Rice, 2006). Sperm competition has been shown to also influence male mating decisions (e.g. Schwagmeyer & Parker, 1990). In species where males have ample mating opportunities, female phenotypic quality (e.g. fecundity) is presumed to be more important in mate choice than female genetic quality, since, compared to the fitness effects of offspring quality, male reproductive success is more likely to vary with the number of offspring he sires (Bonduriansky,

2001). However, in the relatively few species where the number of females a male can inseminate is low (e.g. monogamy), female genetic quality may be important in male mating decisions (Roulin et al., 2000). Thus, for most species, male mating preferences typically select for female phenotypic quality, which is mainly manifested as male mate choice for fecund females. 4

Sexual selection can also act upon females through intra-sexual competition.

In order to gain access to mates, and, in particular, the resources that mates may supply such as nuptial gifts (Gwynne, 1984), females may fight amongst themselves

(Petrie, 1983) or interrupt coitus (Bro-Jorgonson, 2007). A species may display both female-female competition and male mate choice (e.g. Gwynne & Simmons, 1990), and these two processes may interact to influence mating behaviour (Santangelo &

Itzkowitz, 2006). However, mating roles may not completely reverse, and both female-female competition and female mate choice (Owens et ah, 1994), or mutual mate choice (Jones & Hunter, 1993; Johnstone et al, 1996) may occur within a species.

1.1.3. Female ornamentation

Although sexual selection on females is well-documented (Clutton-Brock,

2007), reports of female ornamentation are relatively scarce; female sexually-selected weaponry is unknown (Bro-Jorgensen, 2007). Females may display the same ornament as males of the species, although they may differ in the expression of the ornament (mutual ornamentation). Several hypotheses have been proposed to explain mutual ornamentation (see review Kraaijeveled et al, 2007). Female ornaments may be a by-product of selection on male ornaments, due to a strong genetic correlation between male and female ornaments (Lande, 1980,1987; Rice

1984; Price, 1996). In some species, females adopt the male phenotype by mimicking the ornaments of males to avoid male harassment (Burley, 1983; Robertson, 1985). 5

Mutual sexual selection can give rise to mutual ornamentation, either through mutual mate choice (Jones & Hunter, 1993; 1999) and/or mutual intra-sexual competition (Moore et ah, 1997). Moreover, mutual social competition can also select for mutual ornamentation (West-Eberhard, 1979,1983); both sexes may compete for non-sexual resources such as high quality territories (Rohwer, 1975).

However, not all ornamentation is mutual; there are reports of ornamentation specific to females in a variety of taxa such as birds (Heinsohn et ah, 2005), fish

(Amundsen & Forsgren, 2001; Berglund & Rosenqvist, 2001), lizards (Weiss, 2002), and insects (Funk & Tallamy, 2000). Female-specific ornamentation can not be explained by most hypotheses concerning mutual ornamentation; only sexual selection or social competition can explain female-specific ornaments. Female- female competition can select for ornaments in females only (e.g. the female-specific red colouration of Eclectus parrots is important in intra-sexual competition for scarce nesting hollows: Heinsohn et ah, 2005). Sexual selection for female-specific ornaments may arise through male mating preferences. For example male two- spotted gobies prefer to mate females with colourful belly ornaments than drab females (Amundsen & Forsgren, 2001). The benefits males receive from mating ornamented females remains unclear but may involve fecundity and mate quality.

Female ornament expression has been positively associated with fecundity

(Berglund et ah, 1997), egg carotenoid concentration (Svensson et al, 2006), mite load

(Weiss, 2006) and female condition (Weiss, 2006). However, there have been reports 6 of negative associations between degree of female ornamentation and both egg quality (Nordeide et al, 2006; 2008), and female condition (Nordeide et al, 2008).

Fitzpatrick et al. (1995) suggested that females might face a trade-off between fecundity and ornaments; since resources are finite, investment in ornaments will divert resources from fecundity. However in species where males cannot assess females directly, female ornaments may function as fecundity indicators despite ornament expression being limited by potential fecundity costs (Chenoweth et al,

2006). The Chenoweth et al. (2006) model predicts stabilizing selection for female ornamentation; females with intermediate levels of ornament expression having the greatest mating success. Stabilizing sexual selection for female ornaments has been shown in laboratory studies of Drosophila serrata (Chenoweth et al, 2005), but these ornaments are not specific to females.

1.2 The study species

1.2.1. Female ornamentation in the Empididae

The empid dance flies (Diptera: Empididiae) display a number of interesting mating behaviours. Males form courtship swarms, bearing nuptial gifts that they present to their mates. A number of species of empid dance flies show female- specific ornaments - modifications of the wings, legs and abdomens (Cumming,

1994; Svensson, 1997; Funk & Tallamy, 2000; LeBas et al, 2003). Male mate choice for wing size (a sexually dimorphic trait) has been demonstrated in the empid Empis borealis (Svensson & Petersson, 1988; Svensson et al, 1989), and this female trait is an 7 indicator of fecundity (Svensson & Petersson, 1987). In the empid Rhamphomyia tarsata, only females possess pinnate scales on their tibiae; pinnate length serves as a fecundity indicator (LeBas et al, 2003). Pinnate area is under escalating quadratic sexual selection; there is strong sexual selection for females with relatively large scale areas only. The female-specific ornament of R. marginata—wing coloration—is significantly correlated with fecundity (Svensson, 1997). Thus, evidence has accumulated that the female-specific ornaments of the empids arise through sexual selection. However, formal selection analyses are rare for this family; only Lebas et al. (2003) conducted a selection analysis for R. tarsata.

1.2.2. The long-tailed dance fly, Rhamphomyia longicauda

Female long-tailed dance flies Rhamphomyia longicauda possess two ornaments. Only females have eversible abdominal sacs, which they inflate by swallowing air; an inflated abdomen is three to four times wider than an uninflated abdomen (Funk &Tallamy, 2000). Females also have pinnate tibial scales, found on other congeners such as R. tarsata, which render their legs thicker in appearance than males' legs. They display their scaled legs alongside their inflated abdomen prior to entering the courtship swarm, presumably to exaggerate the size of their abdomen as perceived by males. Females form swarms at dusk and dawn under gaps in the tree canopy. The courtship swarms are typically female-biased during the swarming period, but male-biases have been reported (Steyskal, 1950; Downes, 1958;

Chapter 2). Males enter the female swarms from below bearing a nuptial gift 8

(winged insect prey). Females require the nutrition from nuptial gifts to develop their eggs to maturity (Dowries, 1970). Females vary with respect to this aspect of quality; all stages of egg development can be observed even late in the mating season (Funk & Tallamy, 2000; Wheeler, unpublished data). This variation in female phenotypic quality, in addition to the presumed costs to males associated with providing a gift, may provide the necessary conditions for male mate choice and female-female competition to occur; thus females are predicted to be the target of sexual selection.

Previous studies of R. longicauda females support the hypothesis that female ornaments are maintained by sexual selection. Funk & Tallamy (2000) constructed models from enlarged photographs of natural inflated females to show a male preference for the largest models. They interpreted their result as a male mating bias for large abdomen size; however, they enlarged the entire female image in addition to abdomen size and thus their result could be evidence of male mating preference for overall large body size. Furthermore, their largest models exceeded the natural range of female R. longicauda and their observed male response may be influenced by this super-normal stimulus (Tinbergen, 1951). Funk & Tallamy (2000) also found a significant positive relationship between inflated abdomen area and egg size, and this result has been duplicated (Bussiere et ah, in press). However,

Funk & Tallamy (2000) claimed that inflated abdomen area was a dishonest signal of fecundity since egg size only explained 23% of the variation in inflated abdomen area compared to 72% explained by the egg size of a congener R. sociabilis, a species 9 without abdominal inflation. A recent selection analysis comparing wild-caught single and mated females showed that mated females had longer wings and shorter tibiae than single females, but they were unable to include ornament measures in their analyses (Bussiere et al, in press). Interestingly, Bussiere et al. (in press) found that females in the bottom of courtship swarms have larger tibiae than females at the top. Since females at the bottom of swarms may have greater mating opportunities, females may compete for optimal positions in the swarm. Although Bussiere et al.

(in press) did not investigate the relationship between female ornaments and swarm position, it is possible that ornaments may play a role in female-female competition.

Thus, the evidence remains equivocal in support of the hypothesis that female ornaments in R. longicauda are the result of sexual selection for ornaments, either via male mate choice or female-female competition.

1.3 Outline of Thesis

This thesis aims to investigate the hypothesis that the female ornaments of

Rhamphotnyia longicauda are under sexual selection by male mate choice.

Chapter 2: Investigates male mating preferences for female ornament size. Models were constructed using the same template as Funk & Tallamy (2000). However, I only manipulated ornament size and I produced two sizes of models (small and large) that fall within the natural range of female sizes that I have observed in the population I studied. I asked whether male approaches to models were influenced by model size. Male response to the two sizes of models was recorded and 10 analyzed. There was variation between and sometimes within the sex bias of each swarming event; the effect of sex ratio on male response was thus included in these analyses.

Chapter 3: Examines the influence of female ornaments on female mating success. I asked if female ornaments are under sexual selection. A selection analysis was conducted comparing the phenotype of wild-caught single and mated females. The relationship between female ornamentation and fecundity was also investigated, in addition to the allometric relationship between ornaments and body size.

Chapter 4: Conclusions 11

Chapter 2: Male Mate Choice for Female Ornaments of the

Dance Fly Rhamphomyia longicauda

2.1. Abstract

Elaborate ornaments can arise through sexual selection, either by mate choice or intra-sexual competition. Female long-tailed dance flies Rhamphomyia longicauda have two female-specific ornaments—inflatable abdominal sacs and pinnate tibial scales—that they display to males bearing gifts within courtship swarms. A previous study used models of natural females to show a male preference for large ornaments. However, they manipulated the entire model size as opposed to ornament size alone, and their models exceeded the natural size range of females.

This experiment investigates male mate choice for natural models of these female ornaments. I constructed models in which ornament size was manipulated independently of body size. All models were within the natural size range of females. Males approached the large model more frequently than the small model, 12 suggesting male mating preference for large ornaments. This preference persisted despite large fluctuations in the swarm sex ratio. These results are discussed in light of a recent sexual selection analysis of R. longicauda females.

2.2. Introduction

Given that, in most species offspring are more costly to produce for females than males (Trivers, 1972), males tend to compete for access to females that are usually choosy about their mates (Emlen & Oring, 1977; Clutton-Brock & Vincent,

1991; Clutton-Brock & Parker, 1992). And as a result, typically, it is the males of a species that display showy behaviours or ornaments in order to compete for access to mates and/or attract mates. However, females can become the target of sexual selection if there is high variation in female quality (Owens & Thompson, 1994;

Johnstone et ah, 1996; Kvarnemo & Simmons, 1999), and/or males incur high breeding costs (Kokko & Monaghan, 2001; Kokko & Johnstone, 2002), leading to a reversal in mating roles: male mate choice and female-female competition (Gwynne,

1991). In these situations, females may display showy behaviours or ornaments to enhance their mating success. Female ornamentation has now been reported in a variety of taxa, including birds, insects and fishes (Amundsen, 2000; Funk &

Tallamy, 2000; Amundsen & Forsgren, 2001; Heinsohn et ah, 2005). Since females investing in costly ornaments are likely to suffer decreased fecundity (Fitzpatrick et ah, 1995), males may be selected to avoid highly ornamented females as mates.

However, in species where males cannot directly assess female fecundity, ornaments 13 may act as fecundity signals to males, but extreme ornament expression may still be limited by fecundity costs (Chenoweth et ah, 2006). Thus, in order to understand the evolution of female ornamentation, studies should assess the influence of female ornaments on male mate choice.

A number of species of empid dance flies (Diptera: Empididae) exhibit female-specific ornaments—modifications of the wings, legs or the abdomen

(Cumming, 1994). Female long-tailed dance flies Rhamphomyia longicauda possess two ornaments not found in males: eversible abdominal sacs and pinnate tibial scales (Figure 1). Females inflate their abdomen and wrap their thick legs alongside it prior to entering swarms where courtship occurs. It is hypothesized that the two ornaments act in tandem to exaggerate the size of the female silhouette, and flying males may assess silhouette size or shape. For several reasons, it is thought that this species shows a reversal of the mating roles, with males being the choosy sex. First, males offer a nuptial gift (winged insect prey) during mating swarms. Females require the nutrition from nuptial gifts to develop their eggs to maturity (Downes,

1970). Second, females vary in mate quality. Females displaying in the courtship swarms vary in egg maturity and thus quality; even late in the mating season, all stages of egg development are observed (Funk & Tallamy, 2000; Wheeler, unpublished data). Lastly, males have been observed to fly between females prior to mating (Svensson, 1997; Gwynne & Wheeler, personal observation), suggesting they may be choosing their mates. This study asks if male R. longicauda express mating 14 preferences, and if they base their mating decisions on the female-specific ornaments.

Tests of mate choice involve the manipulation of ornament size to evaluate its effect on mating success. Unfortunately, given the small size of R. longicauda, direct manipulation of live females is difficult. Instead, I constructed natural models of females and varied ornament size independent of body size to ask whether male mate choice is based on female ornament size. I presented these models to males during the courtship period, and scored male responses to them.

In a previous study, Funk & Tallamy (2000) used models to test the effects of female size on male mating preferences in R. longicauda by using photographs of real females enlarged 0.75X, 1.5X and 2X the mean size of females to create two- dimensional models, which were then presented in female swarms. Males approached the 2X model significantly more often than the other models, which the authors interpreted as evidence for male preference for large abdomen size in females. However, the largest model was double the size of an average female, and fell beyond the size range of natural females. Their result may thus be explained as a species-typical response to a species-atypical stimulus (super-normal stimulus:

Tinbergen, 1951). Moreover, Funk and Tallamy (2000) interpreted their result as male mate choice for large abdomens. However, the abdomen size of models perceived by males may be a composite of a female's inflated abdomen and her scaled legs alongside it. Males may thus have responded to the entire silhouette as opposed to the exaggerated abdomen size alone. 15

The goal of my experiment was to investigate male mating preferences in R. longicauda by offering them models with a natural range of female ornament sizes.

My models were constructed to allow the effect of ornament size to be studied independently of body size. I predicted that large models would be accepted more frequently than small models, as per Funk and Tallamy's study. During my trials, there was variation from female- to male-bias between and sometimes within certain swarming events. I took advantage of this natural variation in swarm sex-ratio to ask whether male choosiness declined with a drop in the number of available males

(Gwynne, 1985; Shelly & Bailey, 1992). Mating roles can be sensitive to the ratio of available males to females; the less abundant sex is expected to be more choosy than the more abundant sex since they have potentially greater opportunities to mate

(Emlen & Oring, 1977; Clutton-Brock & Vincent, 1991; Clutton-Brock & Parker,

1992). If sex-bias does not influence male choosiness, the large model preference will persist even in male-biased swarms.

2.3. Methods

Mate Choice Trials

Models were constructed from a template used in a previous mate choice study (and kindly sent to me by D. Funk; Funk & Tallamy, 2000). I reduced the template to the proportions of natural females using Adobe Photoshop (version 5.0).

The mean (3.5mm) and standard deviation (0.45mm) of the width of inflated female abdomen was calculated from the previous year's swarm samples. The abdomen 16

width was then enlarged by two standard deviations from the mean to create the

large model (4.4mm), and reduced by two standard deviations from the mean to

create the small model (2.6mm). Since abdomen width co-varied with abdomen

length in the template, a change in one measure resulted in a proportional change in

the other measure. Thus total abdomen size was manipulated. A reduction in

abdomen size can create a gap between the abdomen and legs in the template;

similarly, an increase in abdomen size can cause an overlap between the abdomen

and legs. Thus, I rotated the legs in the model to minimize the gaps or overlap

between abdomen and legs.

Models were printed on to transparencies with a photocopier. A silhouette

comprised of abdomen, thorax and legs was cut out; the head and wings of the

model were excluded. The transparency models were then traced and cut out from black foamcore (a sheet of fine VA" thick Styrofoam). Each model was glued to a 25- cm long piece of transparent fishing line. Two fishing weights were attached to the line, above the model, to ensure that the model remained horizontal.

An array of models was suspended between two stakes inserted into the

ground on either side of the swarm site, approximately 165 cm apart. A single strand of fishing line was run between the two stakes, 100 cm from the ground.

Alternating large and small models were attached 15 cm apart along the line, for a total of eight models (four large and four small models). The order of presentation was haphazardly determined for each trial. A trial consisted of a single swarming event, either at dusk or dawn. 17

I ran trials from June 16th to 30th, 2008 for a total of 17 trials. Trials were run at both dawn (500-600 hours) and dusk (2030-2130 hours). The study site was located on the banks of the Credit River, near Glen Williams (Halton Co., Ontario,

Canada: 43°41'11"N, 79°55'34"W), and is the same site that has been used in a number of previous studies on this system (Bussiere et ah, in press; Gwynne &

Bussiere, 2002; Gwynne et ah, 2007). I set up the models before the swarms formed.

Each trial commenced when males first approached the models. I recorded the time of male approach to a model and the size of the model (large or small). I scored

'male approach' as a male flying less than 5cm beneath a model for 3 or more seconds, before rapidly flying back and forth several times and ascending to the model. If a male approached more than one model, only his first approach was included; males were recognized by the nuptial gift they carried. Also, the swarms were comprised of dozens of individuals, which lessens the chances that a male was represented more than once. I tallied the number of male approaches for each size of model for each trial. Since the height of swarms ascends throughout the swarming period, I moved the models 10cm higher once or twice on the stakes in order to keep the models roughly in the centre of the swarm. Each trial concluded when more than 5 minutes had elapsed since a male had approached a model.

The swarms are typically female-biased but male-biased swarms have been observed (Steyskal, 1950; Downes, 1958; Wheeler, personal observation). Since the sex bias fluctuated both between and sometimes within a trial during my experiment, I recorded the sex bias for each trial. This was based on qualitative 18 observations of the relative abundance of each sex during a trial. For this project, I classified swarms as either female-biased throughout the duration of the trial, male biased throughout the duration, or as a trial where the sex ratio changed from male- biased to female-biased or vice versa. Although I did not measure the operational sex ratio (males:females) at each trial, my estimate of trial sex bias was confirmed with swarm sex ratio when they were measured at the same times by another observer (Gwynne, unpublished data): sex ratios of female-biased trials ranged from 0 to 0.12, male-biased trials ranged from 1.25 to 8.5, and the sex ratio fluctuated within a trial from 2.5 (male-biased) to 0.29 (female-biased).

Statistical Analyses

Male approach scores were log-transformed to remove the right-skew of the distribution. A two-way analysis of co-variance (ANCOVA) included 'model' and

'sex bias' on log male approaches, and 'trial' as a co-variate. Tukey's Honest

Significant Difference was used to test for significant differences between the means of male approaches for the three sex biases.

2.4. Results

Model size, sex bias and trial all had significant effects on male approaches

(Table 1). There was not a significant interaction between model and bias (P = 0.23).

As predicted, males approached large models significantly more frequently than small models (Figure 2). The mean number of male approaches to the models in trials when the swarm sex ratio was female-biased was significantly lower than in 19 male-biased trials (Tukey's 1150=1.03, p<0.001) and trials when the sex bias fluctuated Tukey's HSD=0.98, p<0.001), but there was no significant difference between male-biased trials and trials when the sex bias fluctuated (Tukey's HSD=-

0.05, p=0.98).

2.5. Discussion

As predicted, large models were approached significantly more often than small models, irrespective of the sex bias. I interpret approaches to models as a preference by males since Rhamphomyia males have been observed to assess and leave a real female before approaching and mating another female (Svensson, 1997;

Wheeler, personal observation). Therefore my results indicate a male preference for large females in R. longicauda. Furthermore, because I manipulated only the sizes of two ornaments (inflated abdomen size and scaled legs), my results suggest that males prefer females with large ornaments. It is thought that both ornaments act together to exaggerate abdomen size as perceived by males. Since females swarm in low-light crepuscular conditions and males approach females from below, males are probably assessing the composite silhouette of females as opposed to responding to inflated abdomen size and/or scaled legs directly. In a separate study, comparing the traits of mated and unmated females, I investigated sexual selection on female ornaments in R. longicauda and found evidence for stabilizing selection on scale ornaments but no significant selection on inflated abdomen area (Chapter 3).

Furthermore, I found that inflated abdomen area was a reliable indicator of egg size. Despite male mating preferences for large ornament size~and abdomen area being a

reliable indicator of fecundity—there is weak selection on inflated abdomen area.

However, my selection analyses only examined ornament size and did not consider

how ornament shape—or the composite shape of the two ornaments—may affect

female mating success. Furthermore, selection for abdomen area may be

constrained by intense sperm competition in fecund females with large abdomens,

or by fecund females exceeding male load-lifting capacities.

My results with models are consistent with those of an earlier study; males

responded to large models more frequently than small models (Funk & Tallamy,

2000). However, my results give more reliable information about mate choice

preferences since I offered males models in the range of natural phenotypes; Funk &

Tallamy's models exceeded the natural size distribution of females. Furthermore,

Funk & Tallamy enlarged the entire female image, so that males could be

demonstrating a preference for overall body size as opposed to large ornament size.

I manipulated ornament size but held body size constant, thus my result of a large model preference can be interpreted as a male mating preference for large

ornaments.

The male preference for large models persisted even in male-biased trials.

Two hypotheses they may explain this result. First, variation in mate quality and/or

costs incurred from mating can be more important than the operational sex ratio in

determining the choosy sex in a mating system (Owens & Thompson, 1994;

Johnstone et at, 1996; Kvarnemo & Simmons, 1999; Kokko & Monaghan, 2001). 21

Based on egg size, female R. longicauda present in a swarm vary considerably in their

stage of egg development (Funk & Tallamy, 2000; Wheeler, personal observation). If

there is last-male sperm precedence in this species, males would benefit from

selecting females with mature eggs since they increase their paternity assurance and

also increase the probability that their mate will oviposit before perishing.

Furthermore, male R. longicauda make a considerable mating investment by

providing a nuptial gift, as they probably face associated predation and energetic

costs with this behaviour. Thus, male mate choice may persist in male-biased trials

due to high variation in female quality and/or high breeding costs to males.

Alternatively, males may be choosy despite an abundance of males because of the

brief nature of the male biases during this experiment. The sex ratio varied between

and sometimes within trials; a male-biased ratio rarely persisted for consecutive

trials. Mating roles can reverse over a short time period if the relative abundance of

each sex changes (e.g. two-spotted gobies Gobiusculus fiavencens switch from female

to male mate choice over the breeding season as male abundance declines: Forsgren et ah, 2004), but the change in the sex ratio must remain consistent and not fluctuate

back and forth between male- and female-bias. If the conditions favouring female

mate choice are not consistent for a sufficient period of time, male mate choice may

persist.

Why might males prefer females with large ornaments? Ornament size may

act as a phenotypic indicator of fecundity and/or stage of egg development. Since

male R. longicauda cannot directly assess female fecundity in flight, they may rely on 22 fecundity indictors such as ornament size (Chenoweth et al, 2006). In a related species Empis borealis, males prefer females with long wings (Svensson & Petersson,

1988; Svensson et al, 1989), and female wing length is positively correlated with fecundity (Svensson & Petersson, 1987). LeBas et al. (2003) found that mated R. tarsata females had a greater area in their pinnate scale ornaments than unmated females, and scale length had a significant positive relationship with egg size. Thus, some female ornaments appear to be honest signals of fecundity and egg maturity in empids. However, Funk & Tallamy (2000) claimed that the inflated abdomen area of

R. longicauda is a dishonest signal of egg maturity. They found a significant positive relationship between inflated abdomen size and egg size for R. longicauda females, but argued abdomen size was a 'dishonest' since egg size explained only 23% of the variation in abdomen size of R. longicauda compared to 72% in a congener R. sociabilis that lacks inflatable abdomens. Recently, the positive phenotypic co- variance between egg size and inflated abdomen area in R. longicauda females has gained further support (Bussiere et al, in press; Chapter 3). Moreover, females who over-invest in abdomen ornaments (i.e., have a greater abdomen area than predicted by body size) have relatively high fecundity and mature eggs for their body size

(Chapter 3), suggesting that abdomen area is an honest indicator of fecundity.

Furthermore, males may prefer females with large ornaments if ornament size signals female genetic quality. Females of high genetic quality can overcome the costs associated with ornament expression and have larger ornaments than females of low genetic quality, and thus ornament size may act as an indicator of 23 genetic quality (Pomiankowski, 1987; Grafen, 1990; Iwasa & Pomiankowski, 1994).

Ornaments increase predation risk: females with inflated abdomens tend to become entangled in spider webs more frequently than uninflated females (Gwynne et ah,

2006). Additionally, female JR. longicauda may face potential fecundity costs by investing in ornaments, since investment in ornaments may divert resources from fecundity (Fitzpatrick et ah, 1995), but this trade-off can only arise if there is low variation in females' ability to acquire resources. Females can vary with respect to both acquisition and allocation of resources (Noordwijk & de Jong, 1986), and high variation in resource acquisition may override any trade-offs in allocation between fecundity and ornaments. Since fecundity and ornaments have a positive relationship for R. longicauda females, it is possible that ornaments signal females' ability to acquire resources, and thus their condition. However, in polyandrous species, males are more likely to vary in the quantity of offspring they sire as opposed to the quality of offspring, and thus males are more likely to base their mating decision on female traits associated with fecundity than those associated with genetic quality (Bonduriansky, 2001). But inflated abdomen area may signal both phenotypic and genetic quality in female R. longicauda; females that 'over- invest' in ornaments (i.e., have larger ornaments than predicted by body size) have high fecundity (Chapter 3). This result suggests that high quality females can invest more in both ornament expression and fecundity than low quality females.

Finally, both my result of males approaching large models more frequently than small models, and Funk & Tallamy's (2000) result of male preferences for 24 models of supernormal ornament size, may be attributable to males having a pre existing bias for large silhouettes. Male R. longicauda have been observed to court such objects as small burrs and seedpods (Newkirk, 1970). This bias may in fact be a pre-existing bias for large females, since body size generally signals fecundity in many taxa (Bonduriansky, 2000). Females may be exploiting this male bias by exaggerating their size with their inflatable abdominal sacs and scaled legs. Thus, the evolution of female ornaments of R. longicauda may have involved sensory exploitation (Ryan, 1990; Endler & Basolo, 1998). Unfortunately, a phylogeny of neither the family nor the genus is available, so whether the male bias for large silhouettes pre-dates the origin of female ornaments cannot be investigated.

Furthermore, although female ornaments may have originated from sensory exploitation, different selective pressures may be involved in their maintenance

(Fisher, 1930; Arnqvist, 2008). Additionally, the male preference for a supernormal stimulus may arise from asymmetrical selection pressures on male mating preferences for ornament size (Staddon, 1975); the negative selection for mating females with small ornaments is weaker than the positive selection for mating females of increasing ornament size. Since egg maturity varies with female abdomen size, males would not benefit from mating females with small ornaments and immature eggs. However, there appears to be no limit to the benefit to males of mating females with large ornaments. There is no evidence that females trade off fecundity with ornament investment (Chapter 3), and thus the benefits to males escalate with increasing female ornament size. 25

In summary, my experiment showed male R. longicauda prefer females with large ornaments. This male preference persisted regardless of sex ratio. Future studies should focus on the benefits to males of mating females with large ornaments. 26

Figure 1. Female long-tailed dance flies Rhamphomyia longicauda prior to joining the courtship swarm. Photo courtesy of Darryl Gwynne. 27

2.5 l/> i V JC U ? (0 i 1 o1. a a 1.5 f < * u (0 1 Z en o ^™ 0.5 *

0 Smal Large Model

Figure 2. The mean and standard error of log male approaches to small and large models. The circles represent trials with a female-biased sex ratio, the triangles represent male-biased trials and the squares represents trials where the sex bias fluctuated. 2.7. Tables

Table 1. Two-way analysis of co-variance of model and sex bias on log male approaches, with trial as the co-variate. Models were either large or small. Bias indicated the sex-ratio of each trial: female-, male- or mixed-bias. N = 17 DF SumSq Mean Sq F-value P-value Model 1 3.44 3.44 20.4 <0.01 Sex Bias 2 8.31 4.16 24.7 <0.01 Model* Sex Bias 2 0.55 0.28 1.64 0.21 Trial 1 2.15 2.15 12.8 <0.01 Residuals 27 4.54 0.17 29

Chapter 3: Sexual selection for female ornaments and their

relationship to fecundity in the dance fly Rhamphomyia

longicauda

3.1. Abstract

Although sexual selection on females is widespread, female ornaments

(secondary sexual characters) are rare. Given the greater investment of females in offspring than males, female-specific ornaments can in theory signal fecundity yet be constrained by fecundity costs. A recent theoretical model suggests that female ornaments can arise through stabilizing sexual selection. Female long-tailed dance flies Rhamphomyia longicauda display two female-specific ornaments in mating swarms — inflatable abdominal sacs and pinnate tibial scales. The ornaments enlarge the appearance of the abdomen to choosy males that provide prey gifts to their mates. This study investigates sexual selection for female ornaments and the relationship between ornaments and fecundity in females. As predicted by the model, there was stabilizing selection on scale ornaments, but only weak selection for the abdominal ornament. Fecundity had a significant positive relationship with inflated abdomen area. The two female specific ornaments of this species appear to have differing relationships with mating success.

3.2. Introduction

Elaborate ornaments, such as showy morphological or behavioural traits, can arise via sexual selection if they increase the reproductive success of the bearer during competition for mates or to make the bearer more attractive to mates

(Darwin, 1871). Since producing eggs is costly and mates are usually readily available, the number of gametes a female can produce primarily determines her reproductive success (Trivers, 1972). However, sexual selection on females can arise if there is high variance in female quality and/ or certain kinds of mating costs to males (Parker, 1983; Gwynne, 1991; Owens & Thompson, 1994; Kokko & Monaghan,

2001). Females may display variation in quality through variation in fecundity

(Kvarnemo & Simmons, 1999) or stage of egg development (Funk & Tallamy, 2000).

Costs to males can come with the effort in producing or obtaining nuptial gifts

(Simmons, 1990) or performing long courtship or copulatory behaviours (Saeki et ah,

2005), as well as increased male mortality from these activities or from other sources

(Jiggins et ah, 2000). If either or both of these conditions arise, females will come under sexual selection and males may exercise mate choice. Males of various taxa have demonstrated mating preferences for an indicator of fecundity, or for body size, which usually correlates with fecundity (see review in Bonduriansky, 2001). 31

Sexual selection on females is quite common (Clutton-Brock, 2007), but examples of female ornamentation are relatively scarce (Amundsen, 2000; Funk &

Tallamy, 2000; Amundsen & Forsgren, 2001). Amundsen (2000) outlined three hypotheses to explain female ornamentation. In the first hypothesis, sexual selection is not operating directly on females, but female ornamentation is an artifact of selection for male ornament genes expressed in females. However, this hypothesis does not hold in species where the ornament is limited to females. In such cases, ornament expression is the result of direct selection on females for ornament expression either to attract mates (e.g. female-specific bright-yellow bellies in two- spotted gobies Gobiusculus flavescens: Amundsen & Forsgren, 2001) or to compete with other females. An example of the latter is the female-specific bright blue and red coloration in a parrot, Ecelctus roratus, which is important in female-female competition for scarce nesting hollows (Heinsohn et ah, 2005, Heinsohn, 2008).

The majority of models of ornament evolution predict positive directional selection for ornament expression (see reviews: Andersson, 1994; Kokko et ah, 2003); only Fisher (1930) proposed there may be stabilizing selection for ornaments. Fisher

(1930) predicted that a genetic correlation between ornament expression and mating preference could lead to positive selection for ornaments. However, ornamentation would be constrained by natural selection if there were viability costs associated with ornaments, leading to stabilizing selection on ornaments. But Fisher's theory fails to explain why ornaments are initially preferred. Subsequent theories have proposed that ornaments act as indicators of genetic quality or condition, and mating preferences may offer direct benefits (e.g. high fecundity) or indirect benefits

(enhanced offspring fitness) to the choosy sex (see review Kokko et ah, 2003).

Ornament expression would still be constrained by viability costs, but individuals with large ornaments would be of a higher quality than individuals with small ornaments, leading to directional selection for ornaments.

For females, investment in the expression of ornaments could come at a cost to gamete production, and therefore her reproductive success (Fitzpatrick et ah,

1995). Given the potential fecundity cost of ornaments, perhaps males should not base mating decisions on ornament expression. If this is the case, the origin and maintenance of female ornamentation is puzzling: males should maximize reproductive success by preferring females with smaller ornaments if these females are more fecund (Fitzpatrick et ah, 1995). While recognizing a potential fecundity cost of female ornaments, Chenoweth et ah (2006) stated that male mate choice for female ornaments might be adaptive if males cannot directly assess female fecundity and must rely on a fecundity indicator. In such systems, females with a low expression of the indicator (ornament) may be less detectable by males, whereas females with a high expression and potentially low fecundity would be avoided.

This model predicts stabilizing selection on ornament expression. This prediction was generated with mathematical models, but it has yet to be supported in the wild

(Chenoweth et ah, 2005).

A group with a diversity of female ornamentation—including modified wings, abdomens and legs—are empidine flies (Diptera: Empididae) (Cumming, 33

1994; Svensson, 1997). Rhamphomyia longicauda is one of the most ornamented species in this group with two female-specific ornaments that are displayed in mating swarms at dusk and dawn. First, eversible pleural sacs are inflated just prior to swarming by swallowing air. The result is an inflated abdomen three to four times wider than that of an uninflated female (Funk & Tallamy, 2000). Females also have pinnate leg scales, which render their legs much thicker in appearance than males. While displaying in courtship swarms, females position their legs alongside their inflated abdomens, greatly exaggerating the size of their body distal to the thorax. Males enter the swarms from below and assess the females' silhouettes against the twilight above. Although males may differentiate between the two ornaments, it is possible that, in such low-light conditions, these ornaments may be perceived as a single trait and have a combined effect of exaggerating the female silhouette.

Several factors may have led to reversed mating roles in this species, i.e. male mate choice and female-female competition. Female R. longicauda vary greatly in the maturity of their eggs at any given time in the mating season; even late in the season, all stages of egg development are present within swarming females (Funk &

Tallamy, 2000; Wheeler, unpublished data). Male R. longicauda and other empidines present females with a 'nuptial gift' (insect prey) prior to copulating. Females have lost the ability to hunt and must rely upon gift nutrients to develop their eggs to maturity (Downes, 1970). The restricted (swarming) time interval when nuptial gifts are available, the variance in female mate quality (egg maturity) and the presumed 34 male effort in obtaining 'nuptial gifts' may explain the apparent reversal of the mating roles of this species.

It is thought the scaly legs and inflated abdomens of R. longicauda females are sexually-selected ornaments. Since swarming occurs in crepuscular low-light conditions, these ornaments may function as fecundity indicators. In a related species Rhamphomyia tarsata that has female-only pinnate leg scales, LeBas et al.

(2003) found that the length of pinnate leg scales significantly predicted egg number, and there was positive sexual selection for pinnate scale length. For R. longicauda,

Bussiere et al. (in press) conducted selection analyses comparing wild-caught mated and unmated female R. longicauda, and found significant linear selection for larger wings and smaller tibiae. However, they were unable to include ornament measures in their selection analyses. Although larger wings may be a part of female ornamentation in this species (wings appear to be under sexual selection in an Empis dance fly: Svensson & Petersson, 1987; 1988), it is imperative to perform analyses incorporating the abdominal and leg ornaments in order to fully evaluate sexual selection on females in R. longicauda.

This study aims to test two hypotheses in a natural population of R. longicauda. First, I ask if female ornaments are subject to sexual selection. I first examine the allometric relationship between ornament size and body size. A trait is considered to be sexually-selected if it has a positive allometric relationship with body size (Green, 2000); however, it is possible for a trait to be sexually selected but exhibit isometry or negative allometry (Bonduriansky & Day, 2003). I then conduct 35 a selection analysis to investigate the effect of ornamentation on female mating success. Most models of ornament evolution predict positive directional selection for ornaments. The models of Fisher (1930) and Chenoweth et al. (2006) predict stabilizing selection on ornament expression: individuals with intermediate levels of expression achieve the greatest mating success. Secondly, I ask if ornaments act as phenotypic indicators of fecundity. I scored the fecundity of wild-caught females and examined its relationship with female ornamentation. If ornaments are fecundity indicators, I predict females with large ornaments will be more fecund and/or have more mature eggs than females with smaller ornaments. Alternatively, if females pay a fecundity cost by investing in ornaments (Fitzpatrick et al., 1995), females with large ornaments will be less fecund and/or have less mature eggs than females with smaller ornaments. Finally, Chenoweth et al. (2006) predict that the relationship between ornament expression and fecundity will be hump-shaped, since higher quality females will have the greatest ornament expression, but females who display too strongly for their quality will pay a fecundity cost.

3.3. Methods

Biology ofR. longicauda

R. longicauda gather in mating swarms from early June to early July. Swarms form under gaps in the tree canopy, usually next to rivers or other running water.

Swarms occur at dusk and dawn only, and are comprised mainly of females; on average, the swarm is composed of 88% females (Gwynne et al, 2007), but swarm 36 sex ratios can vary temporally and spatially (Chapter 2). Once a male has ascended through the swarm and copulation occurs, the nuptial gift is transferred between the mating pair and they leave the main swarm. Unlike many other empidines (e.g.

Svensson & Petersson, 1987; Preston-Mafham, 1999; LeBas et al., 2003,2004;

Daugeron & Grooterat, 2005), copulation occurs 'on the wing' - inflated mating females remain in flight while consuming the nuptial gift.

Sample Collection

Samples were collected from 11th of June to 3rd of July in 2007. The collection site is located on the banks of the Credit River, near Glen Williams (Halton Co.,

Ontario, Canada: 43°41'11"N, 79°55'34"W), and is the same site that has been used in a number of previous studies on this system (Bussiere et ah, in press; Gwynne &

Bussiere, 2002; Gwynne et al., 2007).

At each swarming event, mating pairs were caught in a 15 x 12cm dipping net and immediately transferred to a plastic test tube. The pair was then doused with

100% ethanol super-cooled in dry ice and immediately placed in a cooler filled with dry ice. A single female from the swarm was simultaneously caught and treated in the same manner. The paired female was recorded as a 'mated' female (N = 115) and the singleton female netted from the swarm as an 'unmated' female (N = 149). As some samples were damaged during collection or processing, there are unequal numbers of mated and unmated females. Since it is possible that the sampled unmated females may have either previously mated or might have been about to mate, this protocol provides a conservative test of potential differences between the two categories of females.

All samples were transferred to a -10°C freezer upon return from the field. A few days later, samples were placed in alcoholic Bouin's solution for a minimum of

24 hours, to permanently harden the pleural regions of the abdomen in the inflated position (Funk & Tallamy, 2000). Samples were then preserved in 70% ethanol until measurements could be taken.

Morphological Measurements

Morphological and fecundity traits were measured using a dissecting microscope fitted with a digital camera (LeicaDFC290) connected to an iMac, and using Image J (version 1.38x), a digital imaging program. I included the following morphological measurements: wing length, tibia length abdomen area, thorax length, pinnate scale length (from base to tip of longest scale) and pinnate scale area

(the area of scales on the inferior surface of the tibia). I dissected a subset of females

(228/264) and counted the number of developing eggs (egg number). I measured the length of 5 randomly selected eggs from each female and calculated the mean egg length (egg size). Virgin females may either behave differently than mated females, or males may prefer virgin females, and thus may be a confounding factor.

To investigate whether any females had mated previously, I dissected the spermatheca and recorded the presence or absence of sperm. Only 6% of the females did not have sperm in their spermatheca, and one-third of these females were in the 'mated' category. 38

Statistical Analyses

Both area measurements (scale and abdomen area) were square root transformed to ensure that all traits were measured in the same units (mm). All continuous traits were normally distributed, with the exception of egg size, as per the Shapiro-Wilks' test of normality (P > 0.1). The non-normal distribution of egg size was caused by four females who had very long eggs. No transformations could make this distribution normal. All subsequent analyses were run including and excluding these four individuals; the results reported below were obtained from the analyses excluding these 4 individuals.

To examine the allomerric relationship between ornament size and body size,

I performed separate linear regressions of each ornament trait (scale length, scale area, inflated abdomen area) on thorax length (body size). Each trait was log transformed. I tested the slope from each regression model to see if it significantly differed from isometry (Ho: (3 = 1).

To investigate the relationship between female mating success and ornamentation, I conducted selection analyses. I used standard multiple regression techniques (Lande & Arnold, 1983), with standardized traits and relative fitness

(mating status). Relative fitness was obtained by dividing each individual's fitness score by the sample mean. The coefficients (linear, correlational and quadratic gradients) were obtained from these multiple regressions, and I employed logistic regression for their significance testing. I used a canonical analysis to further investigate the quadratic selection acting upon traits in multivariate space (Box & 39

Draper, 1987; Blows & Brooks, 2003; Phillips & Arnold, 1989). The quadratic terms in the gamma matrix were doubled for the analysis (Stinchcombe etal, 2008). The canonical analysis was performed using PopTools for Microsoft Excel (version 3.0.3,

CSIRO, 2008). This analysis involves rotating the coordinate space to find the major axes (m) of the fitness surface. The canonical rotation yielded six major axes as eigenvalues (since there are six traits in the analysis) and the loadings of the traits on each axis as the eigenvectors (Blows & Brooks, 2003). Because the off diagonal terms in the m-matrix are zero, we have more power to detect nonlinear selection using regression. I calculated the m-score for each individual on each major axis based on the trait loadings, and these m-scores and their squares were used as the independent variables in a linear regression model, with relative fitness as the dependent variable. Logistic regression was employed to test the significance of the coefficients obtained from the model. The three significant major axes (ml, m2 and m6) were plotted in pair-wise thin-plate splines (Schluter & Nychka, 1994), in order to visualize their relationship to mating success, using the 'Fields' package in R

(version 2.6.1). The same smoothing parameter (lambda = 0.3) was used for all three splines to allow for comparison between splines. Contour maps of each pair of major axes were also plotted to find any local fitness maximum or minimum for each pair.

To analyze the relationship between ornamentation and fecundity, I performed separate multiple linear and non-linear regressions of morphological traits on egg size and egg number. I also performed additional analyses to examine the investment in ornaments relative to body size, and how this residual ornament investment varied with fecundity. First, I performed separate linear regressions of each ornament trait (scale length, scale area and inflated abdomen area) on thorax length (body size). I used the residuals from these regressions in additional linear regressions of residual ornament investment on fecundity (egg size and egg number), and included a quadratic term to test for any curvature in their relationship.

Post Hoc Analyses

As the Results below reveal, there was disruptive selection indicating that females had the greatest mating success if they had long wings and short tibiae and thorax, or short wings and long tibiae and thorax. From this I hypothesized that the former may allow females to display longer in the swarms and thus increase the probability of attracting a mate. To examine this I used MANOVA with period

(start or end of swarming) and status (mated or single) as factors to test the prediction that females in the swarm at the end of the swarming period would have longer wings and shorter tibiae and thoraxes than females at the beginning of the swarm.

3.4. Results

Allometry

Each ornament trait had a significant, positive relationship with thorax length

(body size) (Table 3; Fig. 4-6). None of the slopes were greater than one, nor did 41

they significantly differ from one (rScaieiength=-0.088/ p=0.15; rScaiearea=-0.071, p=0.25; fabdomen area=" 0.039, p=0.52). Thus, all ornament traits are isometric with body size.

Selection Analysis

As predicted, the two ornaments—scale size and inflated abdomen area- were under sexual selection. However, the form of this selection was non-linear; scale area was under significant negative, quadratic selection (P = 0.04), suggesting stabilizing selection for scale area. The area of the inflated abdomen area showed a trend toward the same form of selection as scale area but it was not significant (P =

0.089). There was significant, positive correlational selection between scale area and scale length. There was no significant linear selection on any of the female morphological traits (Table 1).

Canonical rotation of the y matrix of nonlinear selection gradients resulted in three major axes with positive eigenvalues and three with negative eigenvalues

(Table 2). There was significant disruptive selection along both ml and m2, and significant stabilizing selection along m6, although the eigenvalues of ml and m6 are much greater than m2.

The ml axis contrasted wing length with tibia and thorax lengths. The m2 axis was mainly influenced by tibia length and scale length; tibia length was contrasted with scale length along this axis. The only axis experiencing significant stabilizing selection, m6, was heavily influenced by scale area and scale length.

Contrary to the positive correlational selection on these two traits, scale area loads positively on this axis but scale length loads negatively. Abdomen area loaded 42 strongly on m4, but its negative eigenvalue did not achieve significance; so this analysis provides no evidence for stabilizing selection on inflated abdomen area.

I used thin-plate splines to visualize the fitness surface of the three major axes

(ml, m2 and m6) (Figures 1-3). Since there was significant stabilizing selection along m6, the fitness surface of both ml-m6 and m2-m6 is saddle-shaped (Fig. la, 2a), with no single maximum nor minimum (Fig. lb, 2b). Females had the greatest mating success at the centre of the distribution of m6, indicating stabilizing selection along this major axis. Thus, females with either large scale areas and short scales, or small scale areas and long scales had the lowest mating success, with intermediate expression of both traits resulting in increased mating success. Intermediate ornament expression had the greatest mating success; there is stabilizing selection for the scale ornament. In relation to ml, the major axis with the largest eigenvalue, females had the greatest mating success if they had intermediate expression of scale length and area, but had either long wings and short tibiae and thorax, or short wings and long tibiae and thorax (Fig la, lb).

Predictors of fecundity

All morphological measures were significantly and positively correlated.

There was a significant positive linear relationship between inflated abdomen area and fecundity. Abdomen area was the only morphological trait that significantly predicted both egg size (Table 4) and egg number (Table 5). Abdomen area explained 49% of the variation in egg size (Figure 7), and 6% of the variation in egg number (Figure 8). Neither scale length nor scale area had a significant relationship with fecundity. None of the non-linear coefficients were significant; there was no evidence for the hump-shaped relationship between fecundity and ornament expression predicted by Chenoweth et al. (2006).

Residual inflated abdomen area also had a significant positive relationship with both egg size (Figure 9) and egg number (Figure 10). All three ornament traits

(scale length, scale area and inflated abdomen area) had a significant positive relationship with body size (thorax length). Both residual scale area and residual scale length had a significant relationship with egg number (Table 6), but not egg size (Table 7). None of the quadratic terms were significant when they were included in the regression of residual ornament investment on egg size or egg number.

3.5. Discussion

My study found stabilizing selection for a female ornament. Female R. longicauda possess two female-specific ornaments—inflatable abdominal sacs and pinnate leg scales. Although initial selection analyses revealed negative quadratic

(concave) selection on both scale area (P = 0.04) and inflated abdomen area (P =

0.089), the canonical analysis showed significant stabilizing selection only on scale area; females achieve the greatest mating success at intermediate levels of scale expression. Although these analyses suggest that selection is mainly acting upon scale expression alone, an effect of abdomen size on mating success cannot be ruled out. Since males assess females from below and in low-light conditions, they are likely assessing females based on a two-dimensional silhouette. This silhouette is comprised not only of the scaled legs, but also the abdomen as well. My selection analysis only included ornament size; it is possible that the shape of ornaments — and the shape of the silhouette—may also influence female mating success. Male R. longicauda prefer models of females with larger abdomens and scaled legs to smaller versions (Chapter 2). Future studies need to examine how sexual selection may be operating on the shape of the combined silhouette of inflated abdomen and scaled legs.

As far as I am aware, this study is the only example to date of stabilizing sexual selection on a female-specific ornament in a wild population. In another study looking at female ornaments, in this case cuticular hydrocarbons (CHCs) in the fruit fly Drosophila serrata, Chenoweth & Blows (2005) found that male preferences exerted stabilizing selection on female CHC expression. Although males and females differ in which CHCs influence mating success, both males and females produce the same CHCs, and it is therefore not a female-specific ornament.

For a related dance fly Rhamphomyia tarsata, LeBas et al. (2003) found escalating, quadratic selection on female pinnate scale area. In contrast to the stabilizing form of the quadratic selection on ornaments in our study, the selection on pinnate scales in R. tarsata took the form of weak selection over most of the distribution, but rapidly increasing selection in the positive tail; scale area enhanced the mating success of females with relatively large scales only. The differing patterns of selection on pinnate scales between these related species may be due to the fact that 45

R. longicauda has another ornament in addition to the pinnate scales (inflatable

abdominal sacs), and R. longicauda mates on the wing whereas R. tarsata mates on

vegetation, which may give R. tarsata males more opportunity to directly assess

female fecundity. Furthermore, the low-light conditions and thus the importance of

ornaments to signal fecundity in R. longicauda may not apply to the diurnal

swarming R. tarsata.

My study also revealed significant disruptive selection on wing length and tibia and thorax length; females with either long wings and short tibiae and thorax, or short wings and long tibiae and thorax had the greatest mating success.

Interestingly, a previous selection analysis, that was unable to include ornaments, found significant linear selection on females for longer wings and shorter tibiae

(Bussiere et ah, in press). The inclusion of ornaments in our analyses revealed non linear selection on wing and tibiae lengths, but selection was still operating in opposing directions. This conflicting selection on wing length and tibia and thorax lengths may represent alternative mating strategies, which has never been reported in a sex-role reversed system. One trait combination with high reproductive success—long wings and short tibiae and thorax—may be successful because it enhances female flight performance, i.e. allowing females to display in swarms for longer periods of time, thereby increasing mating opportunities. Evidence in another gift-giving empid with conventional mating roles indicated sexual selection for males with greater flight muscle mass relative to their body size (Marden, 1989).

To examine this possibility, I predicted that, in contrast to females early in the swarming period, females at the end would have longer wings and shorter tibiae and thorax if this trait combination allowed females to display longer. I found no significant differences between structure size of females at the start of the swarming period and females from the end (F6,i58 = 1.40, P = 0.22); however, this analysis did not control for females entering the swarm after swarming had begun. An alternative trait combination with high mating success—small wings and long tibiae and thorax—may be advantageous because tibia length may be important in female- female competition for the best swarm position. Females at the bottom of swarms have larger tibiae than females at the top of swarms (Bussiere et at, in press). Since males enter the female swarm from below, females at the bottom of the swarm have presumably greater mating opportunities than females at the top.

I found all female ornaments are isometric with body size. Although a trait is considered to be sexually-selected if it has a positive allometry (Green, 2000), a model by Bonduriansky & Day (2003) suggested that selective conditions necessary to produce positive allometry are narrower than previously assumed. A trait can still be the subject of sexual selection and exhibit isometry or even negative allometry. Isometry can arise if there is directional sexual selection on the body size to ornament size ratio, if there is stronger sexual selection on ornament size than body size, or if there is directional sexual selection on ornament size but stabilizing viability selection on the ornament size to body size ratio. The latter two situations may apply to R. longicauda. If males prefer females with large silhouettes (Chapter

2), females of an equal body size would increase their mating success by investing more in ornaments, thus sexual selection would be stronger on ornaments than body size. Additionally, ornament expression may be limited by predation risk, since there was a tendency for inflated females to become entangled more often than uninflated females (Gwynne et ah, 2007). The role of predation risk in shaping female phenotype requires further investigation.

I next asked whether ornaments in R. longicauda indicate fecundity. I found that the area of the inflated abdomen explained a significant amount of the variation in both egg size and number. The inflated abdomen size in particular predicted about half of the variation in egg number so that this ornament may be an important fecundity indicator for males (but see Funk and Tallamy, 2000). Furthermore, residuals of both ornaments (after controlling for body size) had a significant, positive relationship with egg number. Positive phenotypic co-variance of residual ornaments with fecundity indicates a positive relationship between overinvestment in ornaments and high fecundity. This positive co-variance may occur because females with large ornaments have a great ability to acquire resources, and can therefore invest in both ornaments and eggs. This suggests that ornaments may signal female genetic quality; the condition-dependence of female ornaments requires future study for this species. Moreover, the positive covariance between over-investment in the inflated abdomen ornament and high fecundity may arise because a larger abdomen is necessary to accommodate more eggs (inflated abdomen area as an honest indicator of fecundity). Funk & Tallamy (2000) argued that inflated abdomen area was a dishonest signal of fecundity since egg size 48 explained very little (23%) of the variation in inflated abdomen area. In my study, inflated abdomen area explained about half of the variation in egg size (possibly due to the larger sample size and thus greater power). This, taken together with the result that females that over-invest in abdomen ornaments have high fecundity, provides strong evidence that the abdomen ornament of R. longicauda is an honest signal of fecundity.

My results did not support the prediction of Fitzpatrick et al. (1995) that females would trade-off ornament investment with fecundity, nor did they support the prediction of Chenoweth et al. (2006) that the relationship between ornaments and fecundity would be hump-shaped; high quality females would have the greatest ornament expression, but females who signal above their quality by over-investing in ornaments would pay fecundity costs. However, both Fitzpatrick's and

Chenoweth's predictions rely upon low variation in females' abilities to acquire resources. Females can vary with respect to both the acquisition and the allocation of resources (van Noordwijk & de Jong, 1986). High variation among females in acquisition levels may offset any trade-offs in allocation between ornaments and fecundity, and there will be a positive phenotypic covariance between ornamentation and fecundity. However, if there is low variation in acquisition levels, then variation in allocation strategies may result in negative phenotypic covariance between ornamentation and fecundity. My finding of a positive phenotypic co-variance between inflated abdomen area and fecundity does not support the hypothesis that males who prefer ornamented females will incur a fecundity cost.

Although abdomen area appeared to be an honest indicator of fecundity, surprisingly it was pinnate scale area and length that had the significant effect on females' mating success. There are several hypotheses to explain the weak selection on inflated abdomen area. First, males may not be responding to inflated abdomen size or scale size alone, but may in fact be responding to the composite of the shapes of these traits in forming the female silhouette (recall that females wrap their scaled legs alongside their inflated abdomen). Male R. longicauda chose natural models with large silhouettes more frequently than small silhouettes (Chapter 2). The potential of the shape of the entire silhouette being a fecundity indicator itself needs to be investigated. A second hypothesis to explain the lack of sexual selection on the abdominal ornament is the risk of sperm competition. Females rely upon the nutrition gained from nuptial gifts to develop their eggs (Downes, 1970); they must mate in order to develop their eggs to maturity. Inflated abdomen area significantly co-varies with egg size, and thus stage of egg development. It is possible that females with large inflated abdomens and thus a greater number of mature eggs have gained more matings (and thus meals) than females with smaller abdomens.

Sperm competition may be more intense in these females; only 6% of my sampled females had no sperm in their spermatheca. Therefore, the benefit to males of mating fecund females with large inflated abdomens may be off-set by the potential costs of sperm competition, thereby weakening selection for inflated abdomen size. A final hypothesis is that female abdomen size may be constrained by male load- lifting capacity. Although the male empid is dorsal to the female during copulation, it is only the male that keeps the pair in flight during the initial pairing and copulatory positioning (Marden, 1989; Wheeler, personal observation). Male empids have a maximum mass that they can carry for a given flight muscle mass

(Marden, 1989). Thus, fecund females with large inflated abdomens may have a more limited pool of males able to carry them than females with small abdomens.

This hypothesis predicts size-assortative mating. Bussiere et ah (in press) did not find any significant correlation between male and female phenotypes of coupled R. longicauda; however, they did not include abdominal ornaments in these analyses.

The potential effect of female ornamentation on male load-lifting capacity is an intriguing area of further study.

In summary, R. longicauda females possess two ornaments: eversible abdominal sacs and pinnate leg scales. Females experience stabilizing selection on pinnate scale area; females with intermediate scale areas have the greatest mating success. Inflated abdominal area has a significant, positive relationship with both egg size and egg number, and thus may serve as a fecundity indicator. 51

3.6. Figures

Figure la. The thin-plate spline perspective-view visualization of the fitness surface on ml and m6. 52

Q -4 o

-0.5 0.0 0.5

Figure lb. The thin-plate spline contour map visualization of the fitness surface on ml and m6. 53

^6K *. 0

Figure 2a. The thin-plate spline perspective-view visualization of the fitness surface on m2 and m6. 54

! T T -1.5 -1.0' -0.5 0.0 0.5 1.0 1.5

Figure 2b. The thin-plate spline contour map visualization of the fitness surface on m2 and m6. 55

SS*

Figure 3a. The thin-plate spline perspective-view visualization of the fitness surface on ml and m2. 56

id N

o CM © I ©

if) T -0.5 0.0 0,5

Figure 3b. The thin-plate spline contour map visualization of the fitness surface on ml and m6. 57

CO o g °° ° «> %J« 0•LJ »**^ o eg 0 J>Q •&*%mo «o °o° ate o ..,>-"" CO o COO o 1 o oo o 1 ©00 o 1 i I 0.15 0.20 0.25

Log Thorax Length- (mm)

Figure 4. Linear regression of log scale length on log thorax length (p = 0.915, P < 0.001, R2 = 0.48). c» o, o 0 S,0 , O/' o E £> £ © I if) 35 Q 1 ? w J o"»"J o

0.15 0.20 0.25

Log. Thorax Length (mm)

Figure 5. Linear regression of log square root of scale area on log thorax length (p 0.963, P < 0.001, R2 = 0.77). 59

"St

Tg ° o

Q>' o 0.15 0.20 0,25

Log Thorax Length (mm)

Figure 6. Linear regression of log square root of inflated abdomen area on log thorax length (p = 0.940, P < 0.001, R2 = 0.27). UT5 o

O'o

o d o o o ft. QJ O -rtO .a «1 >° v^jn'a"^*:'? O w o o a* o1 o -a <^

o o

2.5 3,0' 3.5 4.0

Inflated Abdomen Area (mm)

Figure 7. Partial regression of egg size on inflated abdomen area ((3 0.001, R2 = 0.49). Inflated Abdomen Area fmm)

Figure 8. Partial regression of egg number on inflated abdomen area (p 0.01, R2 = 0.06). o o o 0 o 0 o -43 •o c £> 3 °

^ A, ©> ©"5

-0,5 0,0 0.5 Residual Mated Abdomen Area (mm)

Figure 9. Regression of egg size on residual inflated abdomen area (P 0.01, R2 = 0.18). 63

o o^ Q oo o $

-0,05 0.00 0,05

Residual inflated Ab Area (mm)

Figure 10. Regression of egg number on residual inflated abdomen area (p = 7.82, P = 0.004, R2 = 0.03). 64

3.7. Tables

Table 1. The vector of standardized linear selection (P) and the matrix of standardized quadratic and correlational selection gradients (y). Significance tests from logistic regressions. * P < 0.05. N = 264 Y_ P Wing Tibia Scale Thorax V Scale VAb Length Length Length Length Area Area Wing 0.161 1.202 Length Tibia -0.103 -0.819 0.243 Length Scale 0.080 -0.686 -0.592 -0.177 Length Thorax 0.092 -1.122 0.863 0.250 -0.114 Length V Scale -0.036 -0.006 0.037 *1.164 0.206 *-0.618 Area V Ab -0.062 -0.337 0.024 -0.116 0.177 0.414 -0.141 Area 65

Table 2. The M matrix of eigenvectors from the canonical analysis of y. The linear (0) and quadratic (X) gradients of selection along each eigenvector are given in the last two columns. Significance tests from logistic regressions: * P < 0.05. N = 264 M Selection Wing Tibia Scale Thorax V Scale VAb e k Length Length Length Length Area Area ml 0.850 -0.325 -0.156 -0.363 -0.069 -0.101 0.232 *1.682 m2 0.166 0.670 -0.626 0.137 -0.329 -0.066 0.013 *0.454 m3 0.460 0.411 0.345 0.376 0.505 0.321 0.029 0.000 m4 0.081 0.185 0.326 0.131 0.036 -0.913 0.157 -0.128 m5 -0.125 0.467 0.166 -0.831 0.219 0.032 -0.184 -0.496 m6 -0.125 -0.155 -0.575 0.009 0.764 -0.216 -0.199 *-1.116 66

Table 3. Separate least-square regressions of each ornament trait on thorax length (body size). Each trait was log transformed. N = 264. P Standard Error t-statistic P-value Scale Length 0.915 0.06 15.6 <0.01 V Scale Area 0.963 0.03 29.9 <0.01 V Ab Area 0.940 0.09 9.96 <0.01 67

Table 4. Multiple regression of all morphological traits on egg size. N = 224. P S.E. P Intercept -0.198 0.112 0.078 Wing Length 0.068 0.060 0.260 Tibia Length -0.119 0.111 0.285 Scale Length -0.338 0.283 0.233 Thorax Length -0.051 0.145 0.726 V Scale Area 0.172 0.265 0.518 V Abdomen Area 0.134 0.020 <0.001

F 6,217 12.16 <0.001 Multiple R2 0.252 Table 5. Multiple regression of all morphological traits on egg number. N = 228 P S.E. P Intercept -46.8 13.6 0.001 Wing Length 0.88 7.32 0.904 Tibia Length 9.30 13.5 0.490 Scale Length 48.4 34.2 0.159 Thorax Length 16.0 17.7 0.368 V Scale Area 30.0 32.3 0.355 V Abdomen Area 6.18 2.39 0.010

F 6,221 20.08 <0.001 Multiple R2 0.353 69

Table 6. Separate linear regressions of each residual ornament trait on egg number. N = 228 Differential Standard T-statistic P-value Error Scale Length 82.4 35.2 2.34 0.02 V Scale Area 77.4 25.6 3.02 <0.01 V Ab Area 7.82 2.75 2.84 <0.01 70

Table 7. Separate linear regressions of each residual ornament trait on egg size. N = 224 Differential Standard T-statistic P-value Error Scale Length -0.119 0.273 -0.434 0.67 V Scale Area 0.323 0.197 1.638 0.10 VAbArea 0.138 0.019 7.08 <0.01 71

Chapter 4: Conclusions

Female ornamentation may evolve via sexual selection if ornaments provide an advantage in the competition for mates, and thus enhance female mating success

(Darwin, 1871). However, it has been proposed that females may suffer a fecundity cost by trading off resources into ornament investment (Fitzpatrick et ah, 1995).

Chenoweth et ah (2006) accept that females may trade-off fecundity for ornaments but predict that in systems where males must rely on ornaments as indicators of fecundity there will be stabilizing selection for ornamentation. Thus, the influence of sexual selection on female ornamentation remains puzzling. Female long-tailed dance flies Rhamphomyia longicauda have two remarkable ornaments: inflatable abdominal sacs and pinnate tibial leg scales. Previous studies have shown that females may be subject to sexual selection (Funk & Tallamy, 2000; Bussiere et ah, in press), and thus this species provides the opportunity to study the role of sexual selection in the expression of female-specific ornaments.

In Chapter 2,1 explored male mating preferences for female ornaments of R. longicauda. I constructed two-dimensional models, keeping body size constant, but manipulating abdomen size within the natural range of females. I presented these large and small models simultaneously during the swarming period. Large models 72 were approached by males significantly more often than small models, suggesting males prefer females with large ornaments. This male preference persisted even when the swarms were male-biased. These results echo a previous study that found males approached large models more frequently than small models, although their models exceeded the natural size range of females (Funk & Tallamy, 2000).

In Chapter 3,1 investigated the hypothesis that the female ornaments of R. longicauda are sexually-selected and I looked at the pattern of that selection. I conducted a selection analysis comparing wild-caught mated and single females, and I also investigated the relationship ornament size and both body size and fecundity. The main result from the selection analysis was strong stabilizing selection for pinnate scale area. Furthermore, there was disruptive selection: females with either long wings and short tibiae and thoraxes, or vice versa, had the greatest mating success. Finally, inflated abdomen area was a reliable indicator of both egg size and number.

My selection analysis is one of the few studies to examine multivariate selection on female-specific ornaments, and the only study to incorporate canonical analyses. Canonical analyses not only yield more accurate estimates of quadratic selection (Blows & Brooks, 2003), but also show how selection affects more than one trait, particularly correlated traits (Phillips & Arnold, 1989). LeBas et al. (2003) found escalating quadratic selection on the female-specific pinnate leg scales of

Rhamphomyia tarsata, but did not include canonical analyses in their study.

Chenoweth et al. (2005) included canonical analyses in their selection analysis of 73

Drosophila serrata and showed stabilizing selection on female cuticular hydrocarbons

(CHCs). However, CHCs are not female-specific ornaments; males and females

express the same CHCs, although males and females differ in which CHCs

significantly affect reproductive success.

My results show that males prefer females with large ornaments, and inflated

abdomen is a reliable indicator of fecundity. However, there is only weak

stabilizing sexual selection on inflated abdomen area in contrast to strong stabilizing

sexual selection on scale area. This pattern may be explained by the fact that males

are responding to the shape of both female ornaments in the perceived female

silhouette. The two female ornaments—scaled legs and inflated abdomen—may act

together to exaggerate female size as perceived by males. Thus, males may not be selecting mates based on inflated abdomen size alone, but the composite shape produced by the two ornaments. An intriguing future study could photograph inflated mated and single females in flight and investigate the relationship between shape and mating success. There may also be weak selection on abdomen area due to intense sperm competition. Females require the nutrition from nuptial gifts to develop their eggs (Downes, 1970); thus egg size may increase with number of matings. Since inflated abdomen area is a reliable indicator of egg size, females with large abdomens may have mated multiple times. The benefit to males of mating fecund females with large abdomens may be traded off with intense sperm competition, thus males are going to be less likely to base mate choice on the abdomen ornament than the scale ornament. Moreover, fecund females with large abdomens presumably weigh more than females with small abdomens. Male empids face load lifting constraints relative to their size (Marden, 1989); thus small males may be unable to carry large, fecund females. Bussiere et al. (in press) did not find any evidence for size-assortative mating, but they were unable to include weight measures in their analyses. Thus, future work should investigate male load lifting constraints on selection for female abdomen size. Finally, my selection analysis studied relative mating success in one cross-section of time. Longitudinal studies of lifetime mating success may provide a more powerful analysis, and reveal stronger selection on inflated abdomen area.

My results are somewhat contradictory, since I have shown a male preference for large ornaments—which concurs with Funk and Tallamy's (2000) result of male preference for supernormal females—and yet there is only stabilizing selection on the scale ornaments. Other factors must be shaping female ornamentation through their mating success, and these factors must conflict with the positive directional selection for female ornaments exerted by male preference. It is possible that female-female competition for optimal swarm position may offer opposing selection to male preferences. Since males enter the courtship swarm from below, females at the bottom of the swarm may have greater opportunities to mate than females at the top. Bussiere et al. (in press) found that females at the bottom of swarms have longer tibiae than females at the top. In my canonical analysis, the major axes with the strongest loadings of the scale and abdomen ornaments contrasted the ornaments 75 with tibia length. Thus, female-female competition and male mate choice may pose conflicting selection on female ornamentation.

The trade-off between wing length versus tibia and thorax lengths revealed in my selection analysis suggests females may have alternative mating strategies.

Females with long wings and short tibiae and thoraxes may be able to display in courtship swarms longer and increase their mating opportunities. Although my post-hoc analyses did not show any differences in female phenotype from the start to the finish of swarming, this combination of traits may enhance flight performance and thus mating success. A future study could investigate female phenotype and display duration by marking females prior to swarming. The alternative combination of traits—short wings and long tibiae and thoraxes—may be advantageous to females since tibia length may be an important factor in female- female competition for optimal position in the swarm. The role of swarm position on mating success has yet to be investigated in this species. Additionally, future studies should focus on the influence of female ornamentation on female-female competition and swarm position. Literature Cited

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