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Mating Behavior of Two Populations of Drosophila melanogaster

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PAGE 1

MATING BEHAVIOR OF TWO POPULATIONS OF Drosophila melanogaster By KELLY MARIE JONES A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Kelly Marie Jones

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To my parents, family, and friends. Your l ove, support, encouragemen t, and belief in my potential helped me to grow and provided th e support I needed to achieve my dreams.

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iv ACKNOWLEDGMENTS This thesis would not be possible without the commitment, support, and encouragement of my supervisory committee chair, Dr Marta Wayne, and my committee members, Dr. Steven Phelps and Dr. Colette St Mary. All members greatly contributed to the development of different aspects of my work. I would like to also thank the past an d current members of the Wayne lab for feedback and assistance throughout my res earch study. Additionally, I would like to thank Arne Mooers for the Vancouver popul ation of flies and Matt Wallace for his assistance in collecting the Lees burg flies used in my study. Moreover, I also thank Dana Drake for all of her help, especially with the multiple-choice experiments. I am also grateful to the Zoology Department, for f unds received to purchase video recording equipment and financial support in the form of a teaching assistantship.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii ABSTRACT.....................................................................................................................vi ii INTRODUCTION...............................................................................................................1 Sexual Selection and Speciation...................................................................................1 Drosophila as a Model Organism.................................................................................2 Courtship Behavior of Drosophila melanogaster .........................................................3 Sexual Isolation and D. melanogaster ..........................................................................9 Motivation...................................................................................................................10 Objectives...................................................................................................................11 MATERIALS AND METHODS.......................................................................................13 Fly Populations...........................................................................................................13 Vancouver Population (V)...................................................................................13 Leesburg Population (L)......................................................................................13 Rearing Conditions.....................................................................................................14 Collection for Multiple-Choice Assays......................................................................14 Experimental Setup for Multiple-Choice Assays.......................................................14 Analysis of Multiple-Choice Assays..........................................................................15 Collection for No-Choice Behavior Assays...............................................................15 Experimental Setup for No -Choice Behavior Assays................................................16 Analysis of No-Choice-Behavior Assays...................................................................19 Copulation Latency.............................................................................................19 Body Size.............................................................................................................20 Composite Measures of Behavior.......................................................................20 Courtship Behavior..............................................................................................20 Discriminant Function Analysis..........................................................................21 Logistic Regression Analysis for Copulation Success........................................21 Multiple Regression Analysis for Copulation Latency.......................................22 RESULTS........................................................................................................................ ..23 Multiple-Choice Assays..............................................................................................23

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vi No-Choice Behavior Assays.......................................................................................23 Copulation Success..............................................................................................23 Copulation Latency.............................................................................................24 Body Size.............................................................................................................24 Composite Measures of Behavior.......................................................................25 Discriminant Function Analysis..........................................................................26 Logistic Regression Analysis for Copulation Success........................................27 Multiple Regression Analysis for Copulation Latency.......................................27 DISCUSSION....................................................................................................................3 6 LIST OF REFERENCES...................................................................................................41 BIOGRAPHICAL SKETCH.............................................................................................46

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vii LIST OF TABLES Table page 1 Copulation success for each mating type in the MC assays between the Vancouver and Leesburg populations......................................................................29 2 Copulation success of males and females from each population in the NC assays........................................................................................................................3 0 3: Copulation success and copulation latenc y of interacting individuals of the within and between population assays in the NC assays..........................................31 4 Mean body size ( SE) for each mating type for copulating (COP) and noncopulating (NO COP) individuals for the NC assays...............................................32 5 Composite measures of behavior of each sex for each mating type for copulating (COP) and non-copulating (NO COP) individuals in the NC assays.......................33 6 Courtship behavior measures for eac h mating type for copulating (COP) and non-copulating (NO COP) indivi duals in the NC assays.........................................34 7 Discriminant function analyses to de termine whether copulating cases can be discriminated from noncopulat ing cases for each mating type..............................35

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viii Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Masters of Science MATING BEHAVIOR OF TWO POPULATIONS OF Drosophila melanogaster By Kelly Marie Jones May 2006 Chair: Marta L Wayne Major Department: Zoology Drosophila melanogaster provides an unique opportunity to examine the important role of mating behavior in population dive rgence and isolation. Courtship of D. melanogaster is complex and involves many sens ory modalities. The basic pattern consists of males orientating toward a pot ential mate and perf orming species-specific displays before attempting to copulate with the female (Greenspan and Ferveur, 2000). Candidate traits responsible fo r behavioral isolation in and among species have begun to be identified. Isolation due to differences in mating preference has been detected for several different populations of D. melanogaster (Korol et al., 2000; Hollocher et al., 1997; Haerty et al., 2002). In this experiment multiple-choice and no-choice assays were performed with D. melanogaster to test for sexual isolation, mate discrimination for male courtship behavior, and evidence of differences in mati ng behavior in two ge ographically isolated populations wild-caught from Vanc ouver, B.C. and Leesburg, FL.

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ix Similar to other sexual isol ation studies, there was ev idence of nonrandom mating between the two populations. Vancouver males obtained significantly more matings than Leesburg males when interacting with females of either population. Female mate discrimination for male cour tship behavior within each population could not be determined with any confidence. This may be partly because of the low number of assays that did not result in mating (thereby reducing the power to detect differences among copulating and non-copulating indivi duals): but could also be because of differences in traits that were not measur ed in this study such as pheromone profile or courtship song. However, as in other studi es, male body size was found to be important in mating success, at least for the Leesburg males. The goal of this study was to determine if there was evidence of sexual isolation due to differences in mating behavior between two outbred populations of D. melanogaster Evidence of sexual isolation wa s not detected between the two populations (Vancouver and Leesburg). So me interesting mating patterns were uncovered and evidence of mate discriminati on was found, but the behaviors responsible could not be determined.

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1 INTRODUCTION Sexual Selection and Speciation Sexual selection is a form of natural selection, defined by Charles Darwin as “a struggle between individuals of one sex, gene rally the males, for the possession of the other sex”(Darwin, 1871). Darwin hypothesi zed two mechanisms underlying sexual selection: competition for mates and mate choice. Competition for mates is defined as any behavior within a sex that increases th e number of potential mates for the winner. Mate choice is defined as the behavi ors that reduce the number of mates by discriminating among the potential mates (W iley & Poston, 1996). Either sex may use mate choice or compete for mates, but generally females are more discriminating, as females generally invest more in their offspr ing, and their fitness is not increased by the number of mates acquired but by the number of viable offspring produced. Conversely, males usually invest less and produce more game tes than females. Therefore, the number of mates acquired increases male fitness. Fe male mate choice occurs when the female uses certain male attributes or traits to di scriminate one male over another. Females can therefore affect the fitn ess of males and the evol ution of certain male attributes or traits (Andersson, 1994). Consequently, sexual sele ction can drive intr apopulation evolution, and through female choice can cause sexua l isolation among populations, potentially leading to speciation. Speciation is a complex process that may involve numerous mechanisms. Speciation occurs through the evolution of prezygotic and/or postzygotic barriers that

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2 lead to reproductive isolati on (Turelli et al., 2001). Spec iation by sexual selection takes place when a change in female preference and a corresponding change in the sexually selected male traits in a population is th e primary cause of prezygotic isolation and consequently reproductive isolation between populations (Kelly & Noor, 1996; Panhuis et al., 2001; Servedio, 2001; Kirkpatrick & Ravigne, 2002). It has been shown through population genetic models that changes in th e way mates are acquired or chosen in a population can lead to rapid speciation because of the direct affects these changes have on gene flow (Lande, 1981;1982). A pattern that suggests speciation by sexual selection is within-s pecies variation in sexually selected traits and mate preferen ce. For instance, in complete reproductive isolation between populations could be the result of within-species, among population variation in sexually selected traits and mate preferences. Likewise, if the reproductive isolation between species is the result of differences in mating signals and mate preferences, and the species have little diffe rence in other traits, then speciation via sexual selection is suspected (Panhuis et al., 2001). Ther efore, it is important to understand whether sexual selection is pl aying a role in the difference between populations and/or species. Drosophila as a Model Organism The genus Drosophila is a good system to study sexua l selection because of the elaborate courtship displayed by males toward females. Courtship within Drosophila is complex and involves many sensory modalities. The basic courtship pattern consists of males orienting toward a potential mate and performing species-specific displays before attempting to copulate with the female (S pieth, 1974; Spieth & Ringo, 1983). Courtship behavior composes most of the social behavior s observed and is an intrinsic feature of the

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3 fly, in that a male fly raised in isolation is fully capable of performing all associated behaviors when presented with the corre ct stimuli (Greenspan & Ferveur, 2000). Drosophila is also a valuable system for sexua l isolation and speciation studies. Courtship behavior of related species groups in the genus ar e similar because of several behavioral elements of courtship originat ing in a non-sexual context (Spieth & Ringo, 1983); but within the order Diptera, courtship is widespread and diverse (Spieth, 1952). Many studies have already evaluated and descri bed differences in rela ted species of this genus that contribute to is olation and the possible mechanisms that contributed to isolation both behaviorally (Spieth, 1974; Spieth, 1952) a nd genetically (Greenspan & Ferveur, 2000; Sawamura & Tomaru, 2002). Courtship Behavior of Drosophila melanogaster In addition to classical genetic studies, aspects of courtship behavior are well studied in Drosophila melanogaster (Spieth, 1952; Bastoc k & Manning, 1955; Spieth, 1974). Drosophila melanogaster adults search for fermenting or decomposing plant matter as food sites, such as rotting fruits or flowers during a peri od in the morning and the late afternoon. In the cour se of time that the flies ar e at the food site, four major aspects of their life occur: feeding, cour tship, mating, and oviposition. After mating, the females oviposit on suitable feeding substrates When the offspring emerge from the pupal stage both sexes are immature. On emer gence, the offspring disperse to secluded areas but return to neighbori ng food sites. Once they return to feeding areas they are exposed to stimuli of hetero specifics and mature conspeci fics before becoming sexually mature. This allows immature individuals to learn to identify appropriate mates and receptive individuals (Spieth, 1974; Spieth & Ringo, 1983).

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4 Initial studies of courtship behavior in D. melanogaster examined the complex series of behaviors that occur before copulation (Spieth, 1952; Bastock & Manning, 1955; Spieth, 1974; Markow & Hanson, 1981). As previously stated, courtship occurs at feeding sites where males indiscriminately appr oach feeding females. Before mating, the male first aligns with a potential mate and be gins tapping the female with his front leg. After determining if the female is a potential mate, the male vibrates his wing toward her, producing a species-specific courtship song. He then circles around the female and contacts her genitalia using hi s proboscis (“licking”). After the genital licking, the male attempts copulation by bending his abdomen and thrusting his genitalia toward her. The male usually performs this series of beha viors repeatedly befo re copulation occurs (Greenspan & Ferveur, 2000). The female al so plays an active role in courtship by performing a variety of accepta nce and rejection behaviors. The acceptance behaviors are not especially overt and consist of slowing locomotor activity, preening, and genital spreading. Conversely, the rejection behavi ors are more apparent and may terminate courtship. These behaviors are ovipositor ex trusion; single or double wing flick; decamping (walking, flying, or jumping away); kicking; and abdominal elevation and depression (Spieth, 1952; Bast ock & Manning, 1955; Spiet h, 1974; Markow & Hanson, 1981). After the complex courtship behavior s, the pair may begin to copulate. Copulation lasts an average of 20 minutes, dur ing which the male transfers seminal fluid into the reproductive tract of the female. Th e seminal fluid contains ejaculate composed of sperm and accessory gland proteins (Acps) The seminal fluid components have an effect on sperm storage, sperm transfer, and sperm competition in the female reproductive tract. The accessory gland protei ns also have an effect on the female by

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5 decreasing her receptivity, increasing ovul ation rate, and affecting egg production (Wolfner, 2002). Additionally, the pheromone synthesis in females also changes, reducing her attractiveness and courtship elic itation from conspecifics. Post-mating changes that occur can last more than2 days (Ferveur, 1997). The courtship behaviors of the male and female produce visual, olfactory, tactile, and auditory stimuli. Additional studies of courtship behavior sought to identify the numerous signals exchanged between interacting individuals. Markow (1987) found that different sensory stimuli are important for successful courtship in each sex. In males, visual stimuli from the female are needed to correctly perform courtship behaviors; females need auditory and olfactory stimuli to become receptive to a courting male. The visual stimulus provided by the female is locomotion. The male must be able to stay in contact with the fe male and perform the correct behaviors; therefore, the male visual system is important. Additionally, an indicator of the female’s receptivity and acceptance behavior is a decrease in locomoti on, which the courting male must be able to perceive (Markow, 1987). Auditory stimuli provided during courtshi p are also important for successful courtship. Drosophila males generate two kinds of acous tic signals duri ng vibration of the wings: a pulse song and a sine song. These auditory signals consist of a species-specific pattern and presumably act in species recognition. The wing vibrations also function to stimulate females to b ecome receptive (Greenspan & Ferveur, 2000) and lack of these acoustic signals results in a not able reduction of courts hip success (Rybak et al., 2002).

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6 Moreover, courtship provides olfactor y stimuli that are important to both interacting individuals. Ph eromones and their function in courtship were recently determined (Ferveur, 1997). Sex pherom ones are chemical signals produced by both males and females and are presumably recognized in each sex by gustation during tapping and licking behaviors. Like the acoustic signals produced, pheromones or cuticular hydrocarbons function in species and mate recognition, and also in mate stimulation. For a given sex, strain, and age flies express a particular cuticular hydrocarbon pattern. For example, the cuticular hydrocarbons found on the female cuticle elicit precopulatory behaviors in ma les, and pheromones also enable males to discriminate potential from non-potential mate s, i.e. virgin or receptive females vs. unreceptive females and males (Ferveur, 1997; Greenspan & Ferveur, 2000). Initially it was assumed that D. melanogaster females mated indiscriminately, although after the work of Bateman (1948) a nd Petit and Ehrman (1969) sexual selection has been well establis hed for this species. Females of this species invest more in their offspring, and their fitness is not increased by the number of mates acquire d but by the number of viable offspring produced. Conversely, males usually invest le ss and produce more gametes than females, therefore their fitness is increased by the number of ma tes acquired (Bateman, 1948). Because of the increased investment in gamete production, females produce limited number of ova (nutritionally demandi ng) and males produce excess sperm (low nutritional demands). Therefore, females are expected to be more discriminating (Spieth & Ringo, 1983; Andersson, 1994). Female choi ce is also expected to evolve when mating is costly, e.g. when mating causes an incr ease in the rate of predation and risk of

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7 infection (Andersson, 1994). If females have the ability to choose their mates, then we expect female choice in this species becau se the act of mating reduces the female’s fitness causing an increased cost to ma ting. The females suffer a reduction in reproductive success and longevity because of the male accessory gland products of the seminal fluid (Chapman, 2001; Wolfner, 2002). Therefore, the cost of mating makes female choice adaptive. Furthermore, females are essentially in cont rol of whether or not they will mate with a particular male. Th ey vigorously avoid unde sirable copulations by performing a variety of rejecti on behaviors that usually term inates courtship (Bastock & Manning, 1955; Gromoko & Markow, 1993). An exception to this is newly emerged, teneral females who cannot perform rejection behaviors and consequently may be forced to mate (Markow, 2000). Although females gain no direct benefits from mates, an increase in fitness associated with the opportunity for female choice was found (Promislow et al., 1998). This indicates a benefit for female choi ce and non-random mating. Additionally, the signals produced by the males during courts hip may be energetically costly because courtship alone reduces longevi ty, therefore may provide info rmation about their quality (Cordts & Partridge, 1996). Now that sexual selection is known to occu r in this species, many studies have begun to examine its role in this system. Mo st of the studies performed do not allow for analysis of sexual selection into its component parts, interand intra sexual selection (i.e. male-male competition and female choice) (Spieth & Ringo, 1983). However a number of studies have been able to look solely at the intersexual selec tion component. Female discrimination for males with certain phenot ypes has been directly observed in many

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8 studies (Bennet-Clark & Ewing, 1967;1969; Kyriacou & Hall, 1982; Partridge & Farquhar, 1983; Partridge et al., 1987; Sc ott, 1994; Wu et al., 1995; Bangham et al., 2002; Rybak et al., 2002). These studies identif ied potential traits us ed in female mate discrimination. These traits include body size, courtship song, and pheromone profile of the male. There is evidence that these tra its influence the female’s mating decision, and there may be an interaction between them. All of these traits may be related, for instance large males may be able to visually attr act the female’s attention, produce a louder courtship song, and produce higher quanti ties of pheromones than smaller males (Partridge et al., 1987; Rybak et al., 2002). Such studies have provided information on female responses to males of different phe notypes and some rules they use in mate discrimination, but more work is needed. If all of the known potential tr aits are used in female mating decisions, which trait is more significant in her decision? Are there other unidentified traits that could influence her mating decision? The difficulty in pinpoi nting a single determin ant of female mate discrimination (or male courtship success) is the result of factors that can influence sexual selection in nature are constantly changing, for exampl e female mating status, age, size, and social environment, therefore a male trait that is a good predictor of courtship success in one environment may not be in another (Markow & Sawka, 1992). Although the courtship behaviors were fi rst described usi ng an ethological approach (Bastock & Manning, 1955), most of the stimuli and their role in mate discrimination were discovered using sensory deficient mutants, transgenic lines, or surgical manipulation of sensory structures Little attention has been given to the ethological approach of studyi ng courtship, i.e. studying natura l intraand interspecific

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9 variants or examining differences between natural populations. L ittle information is available for how much geographical and natu ral population variation exists in courtship behavior. Because of the natural social environment of flies differing greatly from the lab environment, results from most studies are not directly applicable to natural populations. Therefore, an important goa l should be to try to understand natural population variation in the most natural setting possible to better understand the complexity and implications of se xual selection in this species. Sexual Isolation and D. melanogaster Courtship behavior differenc es can play a role within or between species in preventing gene flow (Butlin & Richie, 1994). There is great interest in analysis of geographic variation within species in courtshi p signals because this variation contributes to population divergence. As previously stated, Drosophila is a good model for examining sexual isolation. Comparisons in traits related to courtshi p can be made within and between related species, because variation in courtship patte rns between species usually differ in the relative frequency of each behavior but not in the overall pattern observed (Spieth, 1952; Bastock, 1956; Spieth, 1974). Furthermore, ge netic dissection of species differences in courtship behaviors will aid in understandi ng how genetics and se xual selection are involved in reproductive isol ation leading to speciation (Hollocher, 1998; Sawamura & Tomaru, 2002). Many studies have already begun to gather evidence that factors affecting courtship are important in population divergence and isol ation in this genus. Candidate traits responsible for reproductive isol ation within and between sp ecies have been identified (Ewing, 1983; Coyne & Oyama, 1995; Savar it et al., 1999; Tomaru & Oguma, 2000;

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10 Sawamura & Tomaru, 2002) For example, in terspecific differences in pheromones and courtship song may play a role in the se xual isolation between two sibling species, D. melanogaster and D. simulans (Sawamura & Tomaru, 2002). Drosophila melanogaster provides a unique opportunity to examine the role of courtship related traits in sexual isola tion. Until recently, it was assumed that D. melanogaster had a uniform, world wide range a nd exhibited no evidence of sexual isolation (Henderson & Lambert, 1982). Ho wever, three cases of sexual isolation between populations have been found. First, many studies have reported that Zimbabwe populations of D. melanogaster are sexually isolated fr om populations on other continents. Female mate discrimination a nd pheromone composition was found to be the dominant components in the divergence of Zimbabwe populations from all other populations (Wu et al., 1995; Hollo cher et al., 1997; Takahashi et al., 2001). In another case, Korol et al. (2000) found that selection for stress to lerance resulted in behavioral divergence in female mate disc rimination in populations of D. melanogaster on the slopes of “Evolution Canyon” producing sexua l isolation between the populations. Lastly, Haerty et al (2002) found pre-ma ting isolation between two natural Congolese populations that may be because of a difference in pheromone composition. Motivation This study of sexual selection research was motivated because an understanding of the traits involved in sexual selection and reproductive isolation ma y provide insight into the forces that cause populations to diverge. Most approaches have been to manipulate a courtship signal (Greenspan & Ferveur, 2000) or summarize courtship behavior with the probabilities of transition between behavi ors (Markow & Hanson, 1981). This provides information about the signals involved in sexual communication that differs between

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11 species, but in order to determine the cause of sexual isolation, direct observation of individuals from one population interacting wi th individuals of another is necessary. Moreover, because most studies of courtshi p behavior in this species use sensory deficient mutants, transgenic lines, or su rgical manipulation, little attention has been given to studying natural variat ion in courtship behavior. Objectives My approach involved two analys es with outbred populations of D. melanogaster, to determine if there is evidence of sexual isolation because of differences in mating behavior between two populations from different ecological environments. The populations were wild-caught from Vancouver, B.C. and Leesburg, FL. The study is laboratory based because using natural population s in the lab will allow me to control for: quality of the environment, age at testi ng, reproductive status, and population density during rearing. The questions I address is this research include Question 1: Do females of each population prefer to mate with males from their own population or with males of the other population? Question 2: What is the role of male courts hip behavior and body size in female mate discrimination within each population? Question 3: What is the variation in fema le mate discrimination between populations? No-choice (NC) assays were performed in a sex population combination with four mating types possible (V V; L L; V L; L V; female and male respectively), as well as multiple-choice (MC) assays, to dete rmine if females from each population prefer to mate with males from their own populat ion. If the populations differ in mating behavior, I expect a decreas e in mating success (increased copulation latency or no copulation) between individuals from the different populations in the NC assays.

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12 Additionally, if the females pref er to mate with males from their own population, then I expect matings to deviate from random in th e MC assays, with more females mating with males from their own population than the othe r population. However, in multiple choice experiments male-male competition cannot be excluded as the potential cause for mating patterns observed. Previous research has suggested a possi ble role of body size, courtship song, and pheromone profile in mate discrimination for this species, but othe r traits involved in courtship have been overlooked. As stated prev iously, courtship in this species consists of elaborate and complex displays, and all of the behaviors invol ve visual, olfactory, tactile, and auditory stimuli. The behavior s involved in visual stimuli have not been thoroughly examined for a role in mate disc rimination. These behaviors include chasing, orienting toward the female, licki ng, etc. In this study, I am interested in determining the relative importance of these behaviors, as well as body size in mate discrimination for each population. Males in the NC assays were characterized for the behaviors, and female discrimination for these male beha viors and/or body size was determined using copulation success and copulation latency as an indicator of female mate discrimination. Using the data collected from the NC assa ys, I am able to determine the variation for males and females in the courtship relate d behaviors between th e populations, as well as variation in female mate di scrimination between the populations.

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13 MATERIALS AND METHODS Fly Populations Vancouver Population (V) The base population was collected (~50 mate d females) in the summer 2001 in East Vancouver, B.C. The popul ation maintained at 25 C, ~55% RH, on a 12:12 LD cycle. The larvae were raised at low density on a 14-day schedule. For more information contact Arne Mooers amooers@sfu.ca Forty mated females from the base popul ation (>71,000) were used to start the Vancouver population used in the behavioral as says. The emerging offspring were mixed randomly for at least one generation in large two liter bottles with standard Drosophila medium before setting up at a constant density. Leesburg Population (L) The population was collected in the summ er 2004 in Leesburg, FL. One hundred twenty mated females from the original population were used to start the Leesburg population used in the behavioral assa ys. The population maintained at 25 C, ~50% humidity, on a 12:12 LD cycle. The larv ae were raised at low density on a 14-day schedule. The emerging offspring were mixe d randomly for at least one generation in large two liter bottles with standard Drosophila medium before setting up at a constant density.

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14 Rearing Conditions After allowing for random mixing between al l flies within a population they were setup on a two-week schedule. The populations were setup in 1/2 pint bottles to avoid crowding (Markow & Hanson, 1981). Sixteen bottles per popul ation (8 originals and 8 backups) were setup at a constant density of 25 X 25 (females and males, respectively) with 50 mL of food (standard medium: co rnmeal, yeast, molasses, tegosept, and proprionic acid). Flies were mixed among all replicate population bottles with each generation. Flies were allowed to mate for 5 days, then cleared to avoid larvae overcrowding. On Day 14, the setup was repeated. Collection for Multiple-Choice Assays Flies used in the MC assays were collect ed within 12 hours of eclosion under light CO anesthesia. Collected individuals were kept until testing in vials containing ten individuals of the same sex. The food medi um of each vial was colored red or green using one drop of food coloring per vial in order to identify the populations during the mating assays. Color was alternated for each population between assays to control for the effect of coloring on behavior (Som & Singh, 2002). Experimental Setup for Multiple-Choice Assays MC assays were performed between 0700 and 0900 hours, because of a lower circadian rhythm effect and high mating ac tivity during this peri od (Sakai & Ishida, 2001). The mating assays were conducted in a temperature-controlled room. Multiple assays were performed each day simultaneously in 1/2-pint bottles with 5 mL of food in each. Males from each population were manuall y aspirated without anesthesia into the 1/2 pint bottle. Afterwards, females of each population were introduced, for at least 80 flies per mating bottle. The sex ratio was kept at 1:1, one male for every female for each

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15 assay. Scan sampling was used to identify c opulating individuals, which were aspirated out and identified based on sex and abdo men color (population source) using a microscope. Four mating types were possible: V V, V L, L V, and L L (female and male respectively). Assays were termin ated before 50% of a ll possible matings had occurred to control for possible differen ces in mating propens ity between the two populations (Casares et al ., 1998), or for one hour, whichever occurred first. Analysis of Multiple-Choice Assays Contingency chi-square tests were perfor med for each replicate assay to test for deviation from random mating (Table 1). In order to test for de viations from random mating for all replicate assays, a ( ) method was performed (Everitt, 1997; Panhuis et al., 2003). The square root of each was calculated for each a ssay; and its sign depends on the direction of the data. The sign di rection was obtained by calculating the cross product of homotypic (V V and L L) minus heterotypic (V L and L V) matings from the contingency tables. The signed values have a norma l distribution under the null hypothesis of random mating with a mean of zero and unit standard deviation. The standard normal variate was calculated for the MC assays (Table 1). Under the null hypothesis of random mating for all replicates, is the sum of all ( ) values divided by (n), where n is the number of replicate assays. A calculated 1.96 is significant at the 5% level. Collection for No-Choice Behavior Assays Flies used in the NC behavior assays we re collected within 12 hours of eclosion under light CO anesthesia. Ten males and ten females from each population were collected seven days before testing, by randomly choosing individuals from the

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16 population bottles. The collected individuals were ke pt until testing in vials containing five individuals of the same sex. All individua ls used in the assays were 7-day-old virgin flies. The benefits of using 7 day old indi viduals is that the di scrimination ability and receptivity is increased compared to younger flies (Spieth, 1974; Manning, 1967) Experimental Setup for No-Choice Behavior Assays Each mating assay was digitally video reco rded to allow for consistent behavior scoring. Assays were performed in a window less, temperature controlled room and the mating chambers placed on a light table. By being lit from underneath small movements could be easily discerned, such as wi ng vibrations, preening, and licking. A high resolution Sony DCR-HC85 MiniDV Handycam Camcorder, with a focal length of 47 cm for recording was used. Th e camera was setup on a tripod 82 cm from lens to light box. The manual focus of the camera was set at 0.8 m and the manual exposure (open aperture) set at 10. This se tup was repeated for all NC assays. Assays were performed between 080 0 and 1200 hours because of high mating activity and a decrease in the circadian rhyt hms effects during this time period (Sakai & Ishida, 2001). Assays were performed using mating chambers 35 mm 10 mm high containing 1 mL of standard Drosophila medium. Each mating chamber was used only for a single assay. For each assay, a single male and female were added simultaneously to a mating chamber by manual aspiration, immediately preceding the beginning of each assay. Video recording began after the introduction of the flies to the chamber and performed until copulation, or for 30 minutes as this is sufficient time for copulation to occur (Rybak et al., 2002; Manning 1967).

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17 Both within population (V V; L L) and between population (V L; L V) assays were performed each day. The order of the assays was randomized each day to avoid an effect of day and time, which is known to influence behavior. A total of 40 assays for each mating type were performed. Assays in which the male failed to court the female were excluded from analysis resulti ng in approximately 37 hours of behavioral observations per sex. Immediately after the assays, the male and female were measured for body size ( m). Measurements were made using an Olympus SZX9 dissecting microscope with a micrometer inserted into an eyepiece. Thorax length was measured, as this is a reliable estimate of body size for this species. An observer who did not know the populati on source of the flie s in the assays analyzed the videotapes. Focal animal sa mpling was performed to gather courtship behavior data for both the male and female using JWatcher 0.9 software (Altmann, 1974). This software was used as an event r ecorder that logs the time of each behavior when an assigned key is pressed. To char acterize the males and females, behavior elements were defined for each sex that encompass D. melanogaster courtship (Speith, 1974). For each assay, I recorded the time each individual was active, resting, or courting. When the male courted the female, 12 male and 8 female courtship behaviors were recorded. The following is a description of each courtship behavior recorded, along with an abbreviation for the behavior. Throughout the text, each behavior will be written using the abbreviation for brevity.

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18 Male Courtship Behaviors: Tap (TAP): Male taps the female tarsus with foreleg Orienting toward the female during courtship: o Orient-back (OB): Orienting to the back of the female o Orient-front (OF): Orienting to the front of the female Licking: Male extends the proboscis to the female’s geni talia while chasing (CHASE LICK) or orienting to the back (OB LICK) of the female Wing vibration of one or both wings by the male, based on position of the male to the female: o Chase + vibrate (CHASE WV): vibrat es wings at a short distance from female o Orient back + vibrate (O B WV): vibrates when or ienting toward the back of the female o Orient front + vibrate (O F WV): vibrates when orienting toward the front of the female o Attempted copulation + vibrate (AC WV ): vibrates while attempting to mount the female Chasing (CHASE): male follows the female at a short distance Attempted copulation (AC): uns uccessful copulation attempt Copulation (COP): successful mounting of male on female Female Courtship Behaviors: Wing flick (WF): Female f licks one or both wings Decamping (DECAMP): Female walks, flies, or jumps away from a courting male Kick (KICK): Female kicks courting male Ovipositor extrusion: Female extrudes ovi positor toward the head of the courting male while standing still (SS OVI) or decamping (DECAMP OVI) Abdominal elevation and depression (AED ): Female elevates and depresses abdomen

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19 Preening (PREEN): Female preens while male is courting Stand still (SS): Female ceases locomotion For all behaviors, I measured the freque ncy of occurrence or duration depending on whether the behavior is classi fied as an event (no duration) or a state (duration). By collecting data on frequency of occurrence or duration of each behavior, I was able to calculate: Proportion of time active, resting, or courting Copulation latency: period of time from the introduction of f lies into the mating chamber to the onset of successful copulation Proportion of time allocated to performing each behavior during courtship: total duration of the behavior/t otal courtship duration Frequency of each behavior during courtship: number of times the behavior occurred/total number of behaviors performed during courtship Analysis of No-Choice-Behavior Assays Copulation Success Chi-square contingency test s were performed to determin e if there was a difference in the number of assays that resulted in copulation for V and L females and also for V and L males (Table 2). Additional Chi-square contingency tests were performed to determine if there were differences in the number of assays that le d to copulation for: V males with V females and L males with L females; V females with V and L males; L females with L and V males; V males with V and L females; L males with L and V females (Table 3). Copulation Latency A Kruskal-Wallis analysis of variance was performed to determine if copulation latency was different across mating types. Ma nn-Whitney U tests were also performed to

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20 determine if there were differences in copul ation latency observed for: V males with V females and L males with L females; V fema les with V and L males; L females with L and V males; V males with V and L females; L males with L and V females (Table 3). Body Size Mean body size (thorax length) of each se x was calculated for each population. A Mann-Whitney U test was performed to determine if male or female body size differs between the populations. Body size of each sex was also calcul ated for copulating and non-copulating individuals of each mating type. Differe nces between non-copulating and copulating individuals in the mean body size for each sex was compared with Mann-Whitney U tests (Table 4). Composite Measures of Behavior The mean proportion of time each sex was active, resting, or courting (mutually exclusive categories) was cal culated for copulating and noncopulating individuals of each mating type. Differences between non-c opulating and copulating individuals in the proportion of time each sex was active, resting, or courting was compared with MannWhitney U tests (Table 5). Courtship Behavior Courtship behaviors that were observed in less than 10% of all assays were excluded from analysis. The behaviors excluded were OB LICK, SS OVI, DECAMP OVI, TAP, and KICK. These behaviors were di fficult to observe, therefore inconsistent scoring of the observer could further complicate the low occurrence of these behaviors. Additionally, for other behaviors many i ndividuals did not ex hibit the behavior, therefore the frequency dist ribution was skewed. These behaviors include: CHASE

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21 LICK, OB, OB WV, OF, OF WV AC WV, WF, and AED. Th erefore, these behaviors were considered in analyses as a binomial response variable, and scored as either performing the behavior (1) or not performing the behavior (0). The mean proportion of time allocated t o, or frequency of each behavior during courtship, as well as the mean frequency of occurrence (0,1) was calculated for each mating type for copulating and non-c opulating individuals (Table 6). Before statistical analyses, the non-binomial courtship behavior data were either: square root transformed (frequency data), arcsine-square root transformed (proportion data), or log transformed (time data) to adjust for deviations from normality. Discriminant Function Analysis Discriminant function analyses were pe rformed to determine whether copulating individuals could be discrimi nated from non-copulating indivi duals for each mating type based on male and female courtship behavior s and body size (Table 7). Both male and female behaviors and body size were included, since using only the behavior of one sex markedly reduced the percentage of cases co rrectly classified (resu lts not presented). Logistic Regression Analysis for Copulation Success The effects of male and female courtshi p behaviors and body size on whether or not copulation occurred for each mating type were analyzed using logistic regression. Behaviors examined in the logistic regr ession were ones that had a standardized coefficient with an absolute value greater th an 1 in the DF analysis. The standardized coefficients indicate the relative importance of each variable to the DF, however if none of the standardized coeffi cients were greater than 1 then all of the behaviors were examined for an effect. The logistic regression was performed beginning with all

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22 variables using backwards elimination to re move variables one at a time based on their significance to the model. Vari ables were eliminated if P 0.15. Multiple Regression Analysis for Copulation Latency Although there were no differences in copul ation latency across mating types (see Results), there may be a difference in the behaviors that are im portant for copulation latency for each mating type. Therefore, the effects of male and female courtship behaviors and body size on copulatio n latency for individuals th at did copulate for each mating type were determined using multiple regression analyses. As in the logistic regression, the multiple regression was perf ormed beginning with all variables using backwards elimination to remove variables one at a time based on their significance to the model. Variables were eliminated if P 0.15. All statistical procedures were perfor med using SPSS v. 12 (SPSS, 2003) or JMP IN v. 5.1 (SAS Institute, 2001).

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23 RESULTS Multiple-Choice Assays Five of the replicate MC assays chi-square contingency tests result ed in rejection of the null hypothesis of ra ndom mating among individuals (Table 1). The ( ) method performed to test for deviation from random ma ting for all replicate assays resulted in a Z value of 5.65, which led to rejection of th e null hypothesis of random mating (P < 0.001; Table 1). Overall, the number V V copulating pa irs accounted for 48% of all matings observed, whereas number of L L copulat ing pairs only accounted for 16.5%. The number V L copulating pairs accounted fo r 16%, similar to the percentage L L copulating pairs, and interestingly the numbe r of L V copulating pairs accounted for a greater percentage of all matings than L L with 19.5% of all matings observed. V males achieved 67% of all copulations observed, whereas L males only achieved 33% of all copulations observed across all replicate assays. No-Choice Behavior Assays Copulation Success There was no difference detected in the num ber of assays that led to copulation for V and L females (P < 0.32; Table 2); however there was a difference in the number of assays that led to copula tion between males of each popul ation, with V males achieving copulation in more assays than L males ( = 5.68, P < 0.02; Table 2).

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24 A difference was found in the number of assa ys that led to copulation for V males with V females and L males with L females, with V males with V females having a greater number of assays th at resulted in copulation ( = 5.48, P < 0.02; Table 3). There was no difference in the number of assays that led to copulation for V females with V and L males (P < 0.20; Tabl e 3), however there was a difference in the number of assays that led to copulation for L females with L and V males, with the L V mating combination more assays resul ting in copulation wh en L females were interacting with V males ( = 4.24, P < 0.04; Table 3). Nevertheless, there was no difference in the number of assays that led to copulation for V males with V and L females (P < 0.82; Table 3). Similarly, there was no difference in the number of assays that led to copulat ion for L males with L and V females (P < 0.28; Table 3). Copulation Latency Copulation latency was not significantly different across mating types in the Kruskal-Wallis analysis of variance ( = 1.23, P < 0.75). There was also no difference in copulation latency for: V males with V fe males and L males with L females (P < 0.52; Table 3); V females with V and L males (P < 0.63; Table 3); L females with L and V males (P < 0.38; Table 3); V males with V and L females (P < 0.99; Table 3); L males with L and V females (P < 0.30; Table 3). Body Size There was no difference between populations in female ( = 0.55, P < 0.46) or male body size ( = 0.99, P < 0.32).

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25 Female body size was also not signifi cantly different for non-copulating and copulating individuals of each mating type ( P 0.05; Table 4). Male body size was not significantly different for non-copulating and co pulating individuals of the V V and the L V mating type (P 0.05; Table 4). However, for the L L mating type, copulating males were larger than non-copulating males ( = 6.49, P < 0.01; Table 4). A similar trend was also observed for the V L ma ting type, although the difference was not significant (P 0.05; Table 4). This suggests that body size was used by females when assessing males for the Leesburg population, but not for ma les of the Vancouver population. Composite Measures of Behavior There was no difference in the mean propor tion of time active, resting, or courting of each sex for copulating and non-copulating individuals for the V V mating type (P 0.05; Table 5). However, for the V L mating type there was a difference in the proportion of time females were active ( = 9.66, P < 0.001; Table 5) and resting ( = 6.15, P < 0.01; Table 5) with non-copulating females spendi ng a greater proportion of time active and resting than copulating females; copulati ng females were found to spend a greater proportion of time courting ( = 9.87, P < 0.001; Table 5). Additionally, the males of this mating type exhibited the same patte rn as females, with non-copulating males spending a greater proportion of time active ( = 10.53, P < 0.001; Table 5) and resting ( = 7.34, P < 0.01; Table 5) than copulating males and copulating males spending more time courting than non-copulating males ( = 10.31, P < 0.001; Table 5).

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26 For the L V mating type, there was no evidence of differences in the proportion of time copulating and non-copulating fema les and males were active and courting, however non-copulating females and males we re found to spend a greater proportion of time resting ( = 14.47, P < 0.001 and = 8.71, P < 0.001 respectively; Table 5) than copulating females and males. Comparisons for the L L mating type between copulating and non-copulating males indicate no significant difference in the proportion of time males were active, resting, and courting, and there was no difference in the propor tion of time females were active and courting (P 0.05; Table 5), however non-copul ating females were found to spend a greater proportion of time resting than copulating females ( = 4.82, P < 0.05; Table 5). These results suggest that males of bo th populations, when participating in unsuccessful courtship interactions with females from their own population have difficulty assessing the female interest; yet when interacting with a female of a different population, they tend to spend more time resting or active if the courtship interactions are unsuccessful. This pattern is also shown fo r V females. However, if the courtship interactions are unsuc cessful, regardless of the interacting male’s population, L females are more likely to spend time resting. Discriminant Function Analysis DF analyses performed to determine whether copulating cases could be discriminated from non-copulating cases for each mating type based on male and female courtship behaviors were able to correctly classify greater than 83% of non-copulating cases and greater than 83% of copulating cases for each mating type with an overall

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27 percentage of greater than 85% cases correc tly classified (Table 7). These results indicate that copulating and non-copulating cas es can be distinguished based on male and female behavior for all mating types. Logistic Regression Analysis for Copulation Success In the V V mating type assays, the only c ourtship behavior that predicted whether or not copulation would occur was whether or not the males exhibited OB (Logistic regression: OB (0,1): = 5.68, P < 0.02; Table 6). However, for all other mating types none of the measures for male and female courtship behaviors or body size predicted whether or not copulation would occur (L L; V L; L V; Table 6). However, there was a trend for non-copul ating males to exhibit OB, OF WV, and AC WV more often than c opulating individuals (Table 6). Moreover, a trend was observed for CHASE WV, with copulating male s spending a greater proportion of time performing the behavior for all mating type s except V V, in which case the opposite trend was observed (Table 6). A trend wa s also observed for copulating females to exhibit the WF behavior more than non-copul ating females for all mating types except V V, in which case the opposite trend was observed (Table 6). Multiple Regression Analysis for Copulation Latency Three male behaviors, CHASE, OB ( 0,1), and AC, and one female behavior, PREEN, as well as female body size were signi ficantly related to copulation latency for the V V mating type (Multiple regression: r2 adj = 0.39, F6,16 = 3.37, P < 0.02; CHASE: = -0.671, t = -2.24, P < 0.04; OB (0,1): = 0.614, t = 3.14, P < 0.006; AC: = -0.511, t = -2.67, P < 0.02; PREEN: = 0.723, t = 3.73, P < 0.002; FEMALE BS: = -0.61, t = 2.99, P < 0.009). As the proportion of time allocat ed to CHASE and the frequency of AC

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28 increased, copulation latency decreased. Si milarly, as female body size increased, copulation latency decreased. However, as the proportion of time allocated to PREEN increased, copulation latency increased. Males that did not exhibit the OB (0,1) behavior were more likely to have a decreased time to copulation than males that exhibited the behavior. Conversely, there was no evidence that ma le or female behaviors or body size was related to copulation latenc y for L L mating type. For the V L mating type, only the female behavior WF (0,1) was significantly related to copulating latenc y (Multiple regression: r2 adj = 0.183, F1,16 = 4.820, P < 0.04; WF (0,1): = 0.481, t = 2.20, P < 0.04). Females that did not exhibit th e WF behavior were more likely to have a decreased time to copulation than females that did exhibit the behavior. For the L V mating type, male body size al ong with the male behavior OF (0,1), as well as female behaviors, AED (0,1), PR EEN, and SS, were significantly related to copulation latency (Multiple regression: r2 adj = 0.422, F6,16 = 3.67, P < 0.017; MALE BS: = 0.788, t = 3.36, P < 0.004; OF (0,1): = -0.579, t = -2.75, P < 0.014; AED (0,1): = 0.465, t = 2.66, P < 0.017; PREEN: = -0.836, t = -3.95, P < 0.001; SS: = 0.563, t = 2.92, P < 0.01). As male body size and the propor tion of time allocated to SS increased, copulation latency increased. Conversely, as the proportion of time allocated to PREEN increased, copulation latency decreased. Male s that did exhibit the OF behavior were more likely to have an increased time to copulation than males that did exhibit the behavior. Similarly, females th at did not exhibit the AED be havior were more likely to have a decreased time to copulation than females that did exhibit the behavior.

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29 Table 1: Copulation succe ss for each mating type in th e MC assays between the Vancouver and Leesburg populations (N = number of males and number of females from each population). Assays were terminated before 50% of all possible matings had occurred. Results from the contingency tests determinin g deviation from random mating, ( ) for each assay, and the value (= / (n)). Significance level of: *P 0.05, **P 0.01, ***P < 0.001 indicates a significant de parture from random mating. Male Replicate N Female V L V 42 11 1 80 L 14 3 0.08 0.28 V 46 11 2 80 L 11 12 8.65** 2.94 V 21 12 3 60 L 14 11 0.35 0.59 V 34 16 4 80 L 20 8 0.10 0.32 V 16 10 5 40 L 9 3 0.66 0.81 V 39 16 6 80 L 16 9 0.38 0.62 V 31 9 7 80 L 18 23 9.56** 3.09 V 40 12 8 80 L 12 17 10.23** 3.20 V 61 15 9 120 L 23 21 10.40*** 3.22 V 54 20 10 120 L 22 24 7.73** 2.78 5.65***

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30 Table 2: Copulation success of males and females from each population in the NC assays; results for the Chi-square contingency tests to determine if there was a difference in the number of assays that led to copulation for V and L females and for V and L males. *P 0.05, **P 0.01, ***P < 0.001 Copulated (N/Y) N Y V 18 57 L 22 48 Female = 1.0 P < 0.32 N Y V 14 60 L 26 45 Male = 5.68 P < 0.02*

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31 Table 3: Copulation success and copulation la tency of interacting individuals of the within and between population assays in the NC assays; results for MannWhitney U tests and Chi-square continge ncy tests to determine if there were differences in copulation latency and in the number of assays that led to copulation; *P 0.05, **P 0.01, ***P < 0.001 Mating Type Copulated (N/Y) N Y V V 7 32 L L 15 20 Copulation Latency= 0.41 Copulation Success= 5.48 P < 0.52 P < 0.02* N Y V V 7 32 V L 11 25 Copulation Latency= 0.23 Copulation Success= 1.63 P < 0.63 P < 0.20 N Y L L 15 20 L V 7 28 Copulation Latency= 0.77 Copulation Success= 4.24 P < 0.38 P < 0.04* N Y V V 7 32 L V 7 28 Copulation Latency= 0.00 Copulation Success= 0.05 P < 0.99 P < 0.82 N Y L L 15 20 V L 11 25 Copulation Latency= 1.06 Copulation Success= 1.16 P < 0.30 P < 0.28

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32Table 4: Mean body size ( SE) for each mating type for copulating (COP) a nd non-copulating (NO COP) individuals for the NC assays, with differences compared with Mann-Whitney U tests. N = 39, V V; N = 36, V L; N = 35, L V; N = 35, L L; *P < 0.05. **P < 0.01, ***P < 0.001. Body Size V V NO COP COP V L NO COP COP L V NO COP COP L L NO COP COP Male 40.83 (0.54) 40.96 (0.44) 0.03 38.90 (0.62) 40.35 (0.81) 2.84 41.00 (1.02) 41.08 (0.45) 0.01 39.92 (0.43) 42.00 (0.57) 6.49** Female 48.86 (1.34) 46.92 (0.55) 2.51 46.20 (0.84) 47.39 (0.51) 1.37 45.50 (1.78) 47.44 (0.65) 2.15 46.43 (0.83) 46.13 (0.75) 0.04

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33Table 5: Composite measures of behavior of each sex for eac h mating type for copulating (C OP) and non-copulating (NO COP) individuals in the NC assays; di fferences in the mean proportion ( SE) of time active, resting, and courting compared with Mann-Whitney U tests. N = 39, V V; N = 36, V L; N = 35, L V; N = 35, L L; *P < 0.05. **P < 0.01, ***P < 0.001. Proportion of Time V V NO COP COP V L NO COP COP L V NO COP COP L L NO COP COP Female Active 0.34 (0.09) 0.36 (0.03) 0.26 0.51 (0.08) 0.23 (0.03) 9.66*** 0.33 (0.08) 0.32 (0.03) 0.00 0.34 (0.04) 0.32 (0.03) 0.19 Female Resting 0.01 (0.00) 0.00 (0.00) 1.46 0.01 (0.00) 0.00 (0.00) 6.15** 0.07 (0.04) 0.01 (0.00) 14.47*** 0.05 (0.02) 0.00 (0.00) 4.82* Female Courting 0.66 (0.09) 0.64 (0.03) 0.11 0.47 (0.08) 0.77 (0.03) 9.87*** 0.60 (0.09) 0.68 (0.03) 0.61 0.61 (0.04) 0.67 (0.04) 2.17 Male Active 0.32 (0.08) 0.36 (0.03) 0.34 0.51 (0.07) 0.23 (0.03) 10.53*** 0.37 (0.08) 0.31 (0.03) 0.38 0.37 (0.04) 0.33 (0.03) 1.27 Male Resting 0.01 (0.01) 0.00 (0.00) 2.11 0.02 (0.00) 0.00 (0.00) 7.34** 0.02 (0.01) 0.00 (0.00) 8.71*** 0.01 (0.00) 0.00 (0.00) 0.73 Male Courting 0.67 (0.08) 0.64 (0.03) 0.16 0.48 (0.08) 0.77 (0.03) 10.31*** 0.61 (0.09) 0.69 (0.03) 0.29 0.62 (0.04) 0.66 (0.03) 1.27

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34Table 6: Courtship behavior measures for each mating type for copulating (COP) and non-copulating (NO COP) individuals in the N C assays; mean proportion of time allocated to performing each behavior during courtship ( SE), mean frequency of each behavior performed during courtship ( SE), or mean frequency of occurrence (0,1; SE). Courtship Behaviors V V NO COP COP V L NO COP COP L V NO COP COP L L NO COP COP Chase Proportion 0.14 (0.04) 0.16 (0.03) 0.27 (0.05) 0.14 (0.03) 0.22 (0.10) 0.17 (0.02) 0.23 (0.03) 0.18 (0.03) Chase WV Proportion 0.77 (0.04) 0.76 (0.03) 0.62 (0.05) 0.73 (0.04) 0.58 (0.12) 0.71 (0.04) 0.62 (0.05) 0.66 (0.05) Chase Lick Frequency (0,1) 0.86 (0.14) 0.59 (0.09) 0.73 (0.14) 0.76 (0.09) 0.86 (0.14) 0.64 (0.09) 0.87 (0.09) 0.80 (0.09) OB Frequency (0,1) 0.43 (0.20) 0.31 (0.08) 0.82 (0.12) 0.64 (0.10) 0.71 (0.18) 0.39 (0.09) 0.87 (0.09) 0.70 (0.11) OB WV Frequency (0,1) 0.57 (0.20) 0.59 (0.09) 0.45 (0.16) 0.40 (0.10) 0.57 (0.20) 0.46 (0.10) 0.20 (0.11) 0.60 (0.11) OF Frequency (0,1) 0.43 (0.20) 0.38 (0.09) 0.45 (0.16) 0.48 (0.10) 0.43 (0.20) 0.50 (0.10) 0.60 (0.13) 0.55 (0.11) OF WV Frequency (0,1) 0.86 (0.14) 0.56 (0.09) 0.73 (0.14) 0.52 (0.10) 0.43 (0.20) 0.64 (0.09) 0.73 (0.12) 0.65 (0.11) AC Frequency 0.09 (0.03) 0.07 (0.01) 0.11 (0.03) 0.13 (0.02) 0.11 (0.02) 0.09 (0.01) 0.10 (0.02) 0.11 (0.02) AC WV Frequency (0,1) 0.29 (0.18) 0.06 (0.04) 0.36 (0.15) 0.24 (0.09) 0.00 (0.00) 0.07 0.05) 0.27 (0.12) 0.20 (0.09) Decamp Proportion 0.80 (0.05) 0.82 (0.03) 0.85 (0.04) 0.76 (0.04) 0.78 (0.04) 0.74 (0.04) 0.79 (0.04) 0.74 (0.05) WF Frequency (0,1) 0.71 (0.18) 0.59 (0.09) 0.27 (0.14) 0.60 (0.10) 0.57 (0.20) 0.64 (0.09) 0.47 (0.13) 0.70 (0.11) AED Frequency (0,1) 0.14 (0.14) 0.38 (0.09) 0.27 (0.14) 0.36 (0.10) 0.14 (0.14) 0.39 (0.09) 0.20 (0.11) 0.30 (0.11) Preen Proportion 0.06 (0.02) 0.08 (0.02) 0.04 (0.01) 0.11 (0.03) 0.16 (0.05) 0.08 (0.02) 0.09 (0.02) 0.08 (0.02) SS Proportion 0.06 (0.01) 0.11 (0.02) 0.08 (0.03) 0.11 (0.02) 0.10 (0.05) 0.13 (0.03) 0.09 (0.03) 0.13 (0.03)

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35Table 7: Discriminant function analyses to determine whether copulating cases can be discriminated from noncopulating cases for each mating type, with the percentage of cases correctly classified for non-copulati ng (NO COP) and copulating (COP) individual s, along with the overall percent of cases correctly classified for the NC assays. Mating Type NO COP Cases Correctly Classified (%) COP Cases Correctly Classified (%) Overall Correct Classification (%) V V 83.3 95.7 93.1 V L 88.9 94.4 92.6 L V 100.0 83.3 96.6 L L 83.3 87.5 85.7

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36 DISCUSSION One objective of this study was to determin e if females from each population prefer to mate with males from th eir own population, or with male s of the other population. If the populations are sexually isol ated or differ in courtship behavior, then females should mate more often with individuals from their own population, a nd there should be a decrease in mating success in interactions of individuals from different populations. Results from the MC tests, indicate that V females prefer to mate with V males. Though, male-male competition cannot be exclud ed as a potential cause for the observed mating patterns, since in this setup males have the opportunity to interact. However, in the NC assays the percentage of NC assays that led to copulation for V females with V and L males was not different. Therefore, Vanc ouver females may in fact prefer to mate with males from their own population, however they are equally likely to mate with males from the other population when not given a choice. The MC results, along with the observati on that L males achie ved copulation in fewer NC assays than V males, and that a gr eater percentage of a ssays led to copulation for L females with V males than with L males indicate that L females are more likely to mate with V males more than with males of their own population. Therefore, L females may prefer individuals from the Vancouver population. Similar to other sexual isol ation studies, there is ev idence of non-random mating between the two populations (Wu et al., 1995; Korol et al., 2000; H aerty et al., 2002). However, the pattern seems to be driven by the high number of V V matings. This may

PAGE 46

37 be because of asymmetrical isolation fo r females of the Vancouver population, however additional experiments would need to be conducted to confirm this hypothesis. Moreover, the increased copulation success of V males compared to L males for L females may be because of a novel male effect or the observed pattern could be because of L males being less “attractive” than V males, since L males obtained significantly fewer matings than V males in both the MC and NC tests. Copulation latency was used in this st udy as an indicator of mating success, however for individuals that di d copulate the time it took fo r these individuals to mate was not different across mating types, between the within population mating types, nor when interacting with individuals from the same or a different population. Therefore, copulation latency may not be a good indica tor of mating success. For example, once females assess the male and decide either to copulate or not, the time it takes to mate may be related to a certain stimulus threshold requ ired to successfully produce eggs or related to investment in egg production. The second objective of this study was to determine the role of male courtship behaviors and body size, if any, in female ma te discrimination within each population. Little information is known about the role these behaviors may play in female mate discrimination. Initially, it was assumed that visual cues were unimportant for mating success since D. melanogaster females could mate in the dark; yet it was found that mating success is significantly decreased when visual cues are lacking (Cobb & Ferveur, 1996). Furthermore, the lack of detailed studies may be partly because of the time required to make detailed observations of courts hip interactions, increased interest in the role of pheromones and courtship so ng, and a negative publication bias.

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38 In this study, when V males were interact ing with V females, behaviors that were significantly or to some extent related to copulation success could not be identified; however there was evidence of a behavior that if performed indicated that copulation was unlikely to occur. Yet when V males were in teracting with L females, copulation success was slightly correlated with the proportion of time alloca ted to CHASE WV. Similarly, when L males were interacting with L or V females, copulation success was slightly correlated with the proportion of time allocated to CHASE WV, and also correlated to male body size. Similar to other studies, male body size was found to be important in mating success, at least for the Leesburg males (Par tridge & Farquhar, 1983; Partridge et al., 1987). However, female mate discrimination for male courtship be havior within each population could not be determined with any confidence. This may be because of low number of assays that did not result in copul ation, thereby reducing the power to detect differences among copulating and non-copulating indivi duals; but could also be because of differences in traits that were not measur ed in this study, such as pheromone profile or courtship song. For assays in which copulation did occur, the proportion of time males spent in CHASE, the frequency of AC, and whether or not males exhibited OB was significantly related to copulation latency when V males were interacting with V females; however when interacting with L females, only body si ze and whether or not males exhibited OF was related to copulation latency. Convers ely, there was no evidence that courtship behavior or body size was relate d to copulation latency when L males were interacting with L or V females.

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39 The contradictions between behaviors id entified as potentially important for whether or not copulation will occur and the behaviors related to copulation latency, may be because of once females assess the male a nd decide either to copulate or not, the time it takes to mate may be related to a certain stimulus threshold required to successfully produce eggs or related to investment in egg production. The third objective of this study was to de termine if there is variation between the populations in female mate discrimination. Since female discrimination for male courtship behaviors within each population c ould not be determined, it is difficult to determine the variation between the populations. The only consistent pattern observed was for body size, in which females of both populations interacting with L males mated more often with larger males. Many studies have already begun to gather evidence that factors that affect courtship are important in population dive rgence and isolation in this genus. Drosophila melanogaster provides a unique opportunity to examine the role of courtship related traits in sexual isolation since three cases of pr ezygotic isolation between populations have been found (Wu et al., 1995; Hollocher et al ., 1997; Takahashi, 2001; Korol et al.,. 2000; Haerty et al., 2002). The goal of this study was to determine if there is evidence of sexual isolation because of differences in mating behavior between two outbred populations of D. melanogaster from different ecological environm ents. Although, evidence of sexual isolation was not detected between the two populations, Vancouver and Leesburg, there were some interesting mating patterns uncove red and the exact mechanism driving these patterns remains to be elucidated.

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40 Sexual selection can drive intrapopulation evolution, and through female choice or male-male competition can cause sexual isol ation among populations, potentially leading to speciation. Population genetic models ha ve shown that changes in the way mates are acquired or chosen in a populati on can lead to rapid speciation due to the direct effects these changes have on gene flow (Lande, 1981; 1982). Therefore, it is important to understand whether sexual selection is pl aying a role in the difference between populations and/or species. Courtship behavior differenc es can play a role within or between species in preventing gene flow (Butlin & Richie, 1994). Therefore, there is great interest in analysis of geographic variati on within species in courtship signals, because this variation contributes to population diverg ence. However, scarce inform ation is available for how much geographical variation exists for D. melanogaster as well as for many other species. Furthermore, genetic dissection of species differences in courtship behaviors will aid in understanding how genetics and sexu al selection are involved in reproductive isolation leading to speciation (Hollo cher, 1998; Sawamura & Tomaru, 2002).

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41 LIST OF REFERENCES Altmann, J. 1974. Observationa l study of behavior: samp ling methods. Behaviour, 48, 227-65. Andersson, M. 1994. Sexual Selection Princeton, NJ: Princeton University Press. Bangham, J., Chapman, T. & Partridge, L. 2002. Effects of body size, accessory gland and testis size on prean d postcopulatory success in Drosophila melanogaster Animal Behaviour, 64, 915-921. Bastock, M. 1956. A gene mutation which ch anges a behavior pattern. Evolution, 10, 421-439. Bastock, M. & Manning, A. 1955. The courtship of Drosophila melanogaster Behaviour, 8, 85-111. Bateman, A. 1948. Intrasexual selection in Drosophila Heredity, 2, 349-368. Bennet-Clark, H. & Ewing, A. 1969. Pulse interval as a critical parameter in the courtship song of Drosophila melanogaster Animal Behaviour, 17, 755-759. Bennet-Clark, H. C., and Ewing, A.W. 1967. Stimuli provided by courtship of male Drosophila melanogaster Nature, 215, 669-671. Butlin, R.K. & Ritchie, M.G. 1994. Mating beha viour and speciation. In: Behaviour and Evolution (Ed. By Slater, P.J.B. & Halliday, T.R.), pp. 43-79. Cambridge University Press, Cambridge. Casares, P., Carracedo, M.C., del Rio, B., Pine iro, R., Garcia-Florez, L., & Barros, A.R. 1998. Disentangling the effects of mati ng propensity and mating choice in Drosophila. Evolution, 52, 126-133. Chapman, T. 2001. Seminal fluid-me diated fitness traits in Drosophila Heredity, 87, 511-521. Cobb, M. & Ferveur, J. 1996. Evolution and ge netic control of mate recognition and and stimulation in Drosophila Behavioural Processes, 35, 35-54. Cordst, R. & Partridge, L. 1996. Cour tship reduces longevity of male Drosophila melanogaster Animal Behaviour, 52, 269-278.

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42 Coyne, J. A., and Oyama, R. 1995. Localizat ion of pheromonal sexual dimorphism in Drosophila melanogaster and its effect on sexual is olation. Proceedings of the National Academy of Sciences, 92, 9505-9509. Darwin, C. 1871. The Descent of Man, and Selection in Relation to Sex London: Murray. Everitt, B.S. 1977. The analysis of con tingency tables, pp. 26-31. Chapman and Hall, London. Ewing, A. 1983. Functional Aspects of Drosophila courtship. Biological Review, 58, 275-292. Ferveur, J. 1997. The pheromonal ro le of cuticular hydrocarbons in Drosophila melanogaster BioEssays, 19, 353-358. Greenspan, R. & Ferveur, J. 2000. Courtship in Drosophila Annual Review of Genetics, 34, 205-232. Gromoko, M. & Markow, T. 1993. Courtship and remating in field populations of Drosophila Animal Behaviour, 45, 253-262. Haerty, W., Jallon, J., Rouault, J., Bazin, C. & Capy, P. 2002. Reproductive isolation in natural populations of Drosophila melanogaster from Brazzaville (Congo). Genetica, 116, 215-224. Henderson, N.R. & Lambert, D.M. 1982. No significant deviation from random mating worldwide populations of Drosophila melanogaster Nature, 300, 437-440. Hollocher, H. 1998. Reproductive isolation in Drosophila : how close are we to untangling the genetics of speciatio n? Current Opinion in Genetics and Development, 8, 709-714. Hollocher, H., Ting, C., Pollack, F. & W u, C. 1997. Incipient Speciation by Sexual Isolation in Drosophila melanogaster : Variation in Mating Preference and Correlation Between the Sexes. Evolution, 51, 1175-1181. Kelly, J. & Noor, M. 1996. Speciation by Rein forcement: A Model Derived from Studies of Drosophila. Genetics, 143, 1485-1497. Kirkpatrick, M. & Ravigne, V. 2002. Speciati on by Natural and Sexual Selection: Models and Experiments. Ameri can Naturalist, 159, 22-35. Korol, A., Rashkovetsky, E., Iliadi, K ., Michalak, P., Ronin, Y. & Nevo, E. 2000. Nonrandom mating in Drosophila melanogaster laboratory popul ations derived from closely adjacent contrasting slopes at "Evolution Canyon." Proceedings of the National Academy of Sciences, 97, 12637-12642.

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43 Kyriacou, C. & Hall, J. 1982. The functi on of courtship song rhythms in Drosophila Animal Behaviour, 30, 794-801. Lande, R. 1981. Models of speciation by sexual selection on polygenic traits. Proceedings of the National Academy of Sciences 78, 3721-3725. Lande, R. 1982. Rapid origin of sexual isolatio n and character divergence in a cline. Evolution, 36, 213-223. Manning, A. 1967. The control of sexual receptivity in female Drosophila Animal Behaviour, 15, 239-250. Markow, T. 1987. Behavioral and sensory basis of courtship success in Drosophila melanogaster Proceedings of the National Academy of Sciences, 84, 6200-6204. Markow, T. 2000. Forced Matings in Natural Populations of Drosophila American Naturalist, 156, 100-103. Markow, T. & Hanson, S. 1981. Multivariate analysis of Drosophila courtship. Proceedings of the National Academy of Sciences, 78, 430-434. Markow, T. & Sawka, S. 1992. Dynamics of Mating Success in Experimental Groups of Drosophila melanogaster (Diptera: Drosophilidae). Journal of Insect Behavior, 5, 375-383. Panhuis, T., Butlin, R., Zuk, M. & Tregenza, T. 2001. Sexual selection and speciation. Trends in Ecology and Evolution, 16, 364-371. Panhuis, T.M., Swanson, W.J., & Nunney, L. 2003. Population genetics of accessory gland proteins and sexual behavior in Drosophila melanogaster populations from Evolution Canyon. Evolution, 57, 2785-2791. Partridge, L., Ewing, A. & Chandler, A. 1987. Male size and mating success in Drosophila melanogaster : the roles of male and female behaviour. Animal Behaviour, 35, 555-562. Partridge, L., and Farquhar, M. 1983. Li fetime mating success of male fruitflies ( Drosophila melanogaster ) is related to their size Animal Behaviour, 31, 871-877. Petit, C. & Ehrman, L. 1969. Sexual Selection in Drosophila In: Evolutionary Biology (Ed. by Steere, W.), pp. 157-191. New Yo rk: Appleton-Century-Crofts. Promislow, D.E., Smith, E.A., & Pearse, L. 1998. Adult fitness cons equences of sexual selection in Drosophila melanogaster Proceedings of the National Academy of Sciences, 95, 10687-10692.

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44 Rybak, F., Sureau, G. & Aubin, T. 2002. Func tional coupling of acoustic and chemical signals in the courtship behavior of the male Drosophila melanogaster Proceedings of the Royal Society of London-B, 269, 695-701. Sakai, T. & Ishida, N. 2001. Circadian r hythms of female mating activity governed by clock genes in Drosophila Proceedings of the National Academy of Sciences, 98, 9221-9225. Savarit, F., Sureau, G., Cobb, M. & Ferveu r, J. 1999. Genetic elimination of known pheromones reveals the fundamental chemi cal bases of mating and isolation in Drosophila Proceedings of the National Academy of Sciences, 96, 9015-9020. Sawamura, K. & Tomaru, M. 2002. Biol ogy of reproductive isolation in Drosophila : toward a better understanding of speciation. Population Ecology, 44, 209-219. Scott, D. 1994. Genetic Variation fo r Female Mate Discrimination in Drosophila melanogaster Evolution, 48, 112-121. Servedio, MR. 2001. Beyond reinforcement: The evolution of premating isolation by direct selection on preferences and po stmating, prezygotic incompatibilities. Evolution, 55, 1909-1920. Spieth, H. 1952. Mating behavior within the genus Drosophila (Diptera). Bulletin of the American Museum of Natural History, 99, 395-374. Spieth, H. 1974. Courtship behavior in Drosophila Annual Review of Entomology, 19, 385-405. Spieth, H. & Ringo, J. 1983. Mating Be havior and Sexual Isolation in Drosophila In: The Genetics and Biology of DROSOPHILA (Ed. by Thompson, J.), pp. 223-284. London: Academic Press. Som, A. & Singh, B.N. 2002. No evidence for minority male mating advantage in wild type strains of Drosophila ananassae tested in multiple-choice experiments. Genetics and Molecula r Research, 1, 317-326. Takahashi, A., Tsaur, S., Coyne, J. and Wu, C. 2001. The nucleotide changes governing cuticular hydrocarbon variati on and their evolution in Drosophila melanogaster Proceedings of the National Academy of Sciences, 98, 3920-3925. Tomaru, M. & Oguma, Y. 2000. Mate choice in Drosophila melanogaster and D. sechellia : criteria and their va riation depending on c ourtship song. Animal Behaviour, 60, 797-804. Turelli, M., Barton, N.H. and Coyne, J.A. 2001. Theory and speciation. Trends in Ecology and Evolution, 16, 330-343.

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45 Wiley, R. & Poston, J. 1996. Indirect ma te choice, competition for mates, and coevolution of the sexes. Evolution, 50, 1371-1381. Wolfner, M. 2002. The gifts that keep on giving: physiological functions and evolutionary dynamics of male seminal proteins in Drosophila Heredity, 88, 8593. Wu, C., Hollocher, H., Begun, D., Aquadro, C. & Xu, Y. 1995. Sexual isolation in Drosophila melanogaster : A possible case of incipien t speciation. Proceedings of the National Academy of Sciences, 92, 2519-2523.

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46 BIOGRAPHICAL SKETCH Kelly Marie Jones was born on November 13, 1979, Fort Walton Beach, FL. She is one of three children of Marsha A. J ones and Wesley H. Jones. She followed her passion for biological sciences and enrolled at the University of Florida, where she earned her bachelor’s degree in zoology. During her underg raduate education, she found an opportunity to perform research on the behavi or of fruit flies, with Dr. Laura Higgins. After graduating, she enrolled in graduate courses at the Un iversity of Florida as a postbaccalaureate student. In 2003 she was acc epted into the graduate program in the Department of Zoology at the University of Florida. She was awarded a Master of Science degree in May 2006.


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Title: Mating Behavior of Two Populations of Drosophila melanogaster
Physical Description: Mixed Material
Copyright Date: 2008

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MATINTG BEHAVIOR OF TWO POPULATIONS OF Drosophila melan2oga~ster


By

KELLY MARIE JONES

















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

UNIVERSITY OF FLORIDA


2006
































Copyright 2006

by

Kelly Marie Jones




























To my parents, family, and friends. Your love, support, encouragement, and belief in my
potential helped me to grow and provided the support I needed to achieve my dreams.
















ACKNOWLEDGMENTS

This thesis would not be possible without the commitment, support, and

encouragement of my supervisory committee chair, Dr Marta Wayne, and my committee

members, Dr. Steven Phelps and Dr. Colette St Mary. All members greatly contributed

to the development of different aspects of my work.

I would like to also thank the past and current members of the Wayne lab for

feedback and assistance throughout my research study. Additionally, I would like to

thank Arne Mooers for the Vancouver population of flies and Matt Wallace for his

assistance in collecting the Leesburg flies used in my study. Moreover, I also thank Dana

Drake for all of her help, especially with the multiple-choice experiments. I am also

grateful to the Zoology Department, for funds received to purchase video recording

equipment and financial support in the form of a teaching assistantship.




















TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. .................... iv


LI ST OF T ABLE S ................. ................. vii........ ....


AB S TRAC T ......_ ................. ..........._..._ viii..


INTRODUCTION .............. ...............1.....


Sexual Selection and Speciation............... ...............
Drosophila as a Model Organism............... ... ..............
Courtship Behavior of Drosophila melan2oga~ster. ................. ....._._. ...............3
Sexual Isolation and D. melan2oga~ster ................. .........._. .......9...... ...
Motivation............... ...............1
Obj ectives ................. ...............11........ ......

MATERIALS AND METHODS ........._....._ ....__.. ...............13....


Fly Populations ........._.. ... .......__ .. ...............13.....
Vancouver Population (V) ................. ...............13......__.. ....
Leesburg Population (L) ....._.__................. ...............13......
Rearing Conditions ................. ........... ................. 14....
Collection for Multiple-Choice Assays .............. ...............14....
Experimental Setup for Multiple-Choice Assays .............. ...............14....
Analysis of Multiple-Choice Assays ................. ......... ......... ............1
Collection for No-Choice Behavior Assays .............. ...............15....
Experimental Setup for No-Choice Behavior Assays .............. .....................1
Analysis of No-Choice-Behavior Assays ................ ...............19................
Copulation Latency .............. ...............19....
Body Size............... ........... ...........2
Composite Measures of Behavior .............. ...............20....
Courtship Behavior............... ...............2
Discriminant Function Analysis............... ... ..............2
Logistic Regression Analysis for Copulation Success .............. ....................21
Multiple Regression Analysis for Copulation Latency .............. ...................22

RE SULT S .............. ...............23....


Multiple-Choice As say s............... ...............23.












No-Choice Behavior Assays............... ...............23.
Copulation Success ................. ...............23.................
Copulation Latency .............. ...............24....
Body Size............... ........... ...........2
Composite Measures of Behavior .............. ...............25....
Discriminant Function Analysis............... ... ..............2
Logistic Regression Analysis for Copulation Success .............. ....................27
Multiple Regression Analysis for Copulation Latency .............. ...................27


DI SCUS SSION ........._.___..... .___ ...............36.....


LIST OF REFERENCES ........._.___..... .___ ...............41....


BIOGRAPHICAL SKETCH ............. ..............46.....

















LIST OF TABLES


Table pg

1 Copulation success for each mating type in the MC assays between the
Vancouver and Leesburg populations. ............. ...............29.....

2 Copulation success of males and females from each population in the NC
as say s............... ...............3 0

3: Copulation success and copulation latency of interacting individuals of the
within and between population assays in the NC assays............... .................3

4 Mean body size (+ SE) for each mating type for copulating (COP) and non-
copulating (NO COP) individuals for the NC assays............... ...............32.

5 Composite measures of behavior of each sex for each mating type for copulating
(COP) and non-copulating (NO COP) individuals in the NC assays.......................33

6 Courtship behavior measures for each mating type for copulating (COP) and
non-copulating (NO COP) individuals in the NC assays ................. ................ ...34

7 Discriminant function analyses to determine whether copulating cases can be
discriminated from non- copulating cases for each mating type ........._...................35
















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

MATINTG BEHAVIOR OF TWO POPULATIONS OF Drosophila melan2oga~ster

By

Kelly Marie Jones

May 2006

Chair: Marta L Wayne
Major Department: Zoology

Drosophila melan2oga~ster provides an unique opportunity to examine the important

role of mating behavior in population divergence and isolation. Courtship of

D. melan2oga~ster is complex and involves many sensory modalities. The basic pattern

consists of males orientating toward a potential mate and performing species-specific

displays before attempting to copulate with the female (Greenspan and Ferveur, 2000).

Candidate traits responsible for behavioral isolation in and among species have begun to

be identified. Isolation due to differences in mating preference has been detected for

several different populations ofD. melan2oga~ster (Korol et al., 2000; Hollocher et al.,

1997; Haerty et al., 2002).

In this experiment multiple-choice and no-choice assays were performed with

D. melan2oga~ster to test for sexual isolation, mate discrimination for male courtship

behavior, and evidence of differences in mating behavior in two geographically isolated

populations wild-caught from Vancouver, B.C. and Leesburg, FL.









Similar to other sexual isolation studies, there was evidence of nonrandom mating

between the two populations. Vancouver males obtained significantly more matings than

Leesburg males when interacting with females of either population.

Female mate discrimination for male courtship behavior within each population

could not be determined with any confidence. This may be partly because of the low

number of assays that did not result in mating (thereby reducing the power to detect

differences among copulating and non-copulating individuals): but could also be because

of differences in traits that were not measured in this study such as pheromone profile or

courtship song. However, as in other studies, male body size was found to be important

in mating success, at least for the Leesburg males.

The goal of this study was to determine if there was evidence of sexual isolation

due to differences in mating behavior between two outbred populations of

D. melan2oga~ster. Evidence of sexual isolation was not detected between the two

populations (Vancouver and Leesburg). Some interesting mating patterns were

uncovered and evidence of mate discrimination was found, but the behaviors responsible

could not be determined.
















INTTRODUCTION

Sexual Selection and Speciation

Sexual selection is a form of natural selection, defined by Charles Darwin as "a

struggle between individuals of one sex, generally the males, for the possession of the

other sex"(Darwin, 1871). Darwin hypothesized two mechanisms underlying sexual

selection: competition for mates and mate choice. Competition for mates is defined as

any behavior within a sex that increases the number of potential mates for the winner.

Mate choice is defined as the behaviors that reduce the number of mates by

discriminating among the potential mates (Wiley & Poston, 1996). Either sex may use

mate choice or compete for mates, but generally females are more discriminating, as

females generally invest more in their offspring, and their fitness is not increased by the

number of mates acquired but by the number of viable offspring produced. Conversely,

males usually invest less and produce more gametes than females. Therefore, the number

of mates acquired increases male fitness. Female mate choice occurs when the female

uses certain male attributes or traits to discriminate one male over another. Females can

therefore affect the fitness of males and the evolution of certain male attributes or traits

(Andersson, 1994). Consequently, sexual selection can drive intrapopulation evolution,

and through female choice can cause sexual isolation among populations, potentially

leading to speciation.

Speciation is a complex process that may involve numerous mechanisms.

Speciation occurs through the evolution of prezygotic and/or postzygotic barriers that









lead to reproductive isolation (Turelli et al., 2001). Speciation by sexual selection takes

place when a change in female preference and a corresponding change in the sexually

selected male traits in a population is the primary cause of prezygotic isolation and

consequently reproductive isolation between populations (Kelly & Noor, 1996; Panhuis

et al., 2001; Servedio, 2001; Kirkpatrick & Ravigne, 2002). It has been shown through

population genetic models that changes in the way mates are acquired or chosen in a

population can lead to rapid speciation because of the direct affects these changes have

on gene flow (Lande, 1981;1982).

A pattern that suggests speciation by sexual selection is within-species variation in

sexually selected traits and mate preference. For instance, incomplete reproductive

isolation between populations could be the result of within-species, among population

variation in sexually selected traits and mate preferences. Likewise, if the reproductive

isolation between species is the result of differences in mating signals and mate

preferences, and the species have little difference in other traits, then speciation via

sexual selection is suspected (Panhuis et al., 2001). Therefore, it is important to

understand whether sexual selection is playing a role in the difference between

populations and/or species.

Drosophila as a Model Organism

The genus Drosophila is a good system to study sexual selection because of the

elaborate courtship displayed by males toward females. Courtship within Drosophila is

complex and involves many sensory modalities. The basic courtship pattern consists of

males orienting toward a potential mate and performing species-specific displays before

attempting to copulate with the female (Spieth, 1974; Spieth & Ringo, 1983). Courtship

behavior composes most of the social behaviors observed and is an intrinsic feature of the










fly, in that a male fly raised in isolation is fully capable of performing all associated

behaviors when presented with the correct stimuli (Greenspan & Ferveur, 2000).

Drosophila is also a valuable system for sexual isolation and speciation studies.

Courtship behavior of related species groups in the genus are similar because of several

behavioral elements of courtship originating in a non-sexual context (Spieth & Ringo,

1983); but within the order Diptera, courtship is widespread and diverse (Spieth, 1952).

Many studies have already evaluated and described differences in related species of this

genus that contribute to isolation and the possible mechanisms that contributed to

isolation both behaviorally (Spieth, 1974; Spieth, 1952) and genetically (Greenspan &

Ferveur, 2000; Sawamura & Tomaru, 2002).

Courtship Behavior of Drosophila melanogaster

In addition to classical genetic studies, aspects of courtship behavior are well

studied in Drosophila melan2oga~ster (Spieth, 1952; Bastock & Manning, 1955; Spieth,

1974). Drosophila melan2oga~ster adults search for fermenting or decomposing plant

matter as food sites, such as rotting fruits or flowers during a period in the morning and

the late afternoon. In the course of time that the flies are at the food site, four maj or

aspects of their life occur: feeding, courtship, mating, and oviposition. After mating, the

females oviposit on suitable feeding substrates. When the offspring emerge from the

pupal stage both sexes are immature. On emergence, the offspring disperse to secluded

areas but return to neighboring food sites. Once they return to feeding areas they are

exposed to stimuli of heterospecifics and mature conspecifics before becoming sexually

mature. This allows immature individuals to learn to identify appropriate mates and

receptive individuals (Spieth, 1974; Spieth & Ringo, 1983).









Initial studies of courtship behavior in D. melan2oga~ster examined the complex

series of behaviors that occur before copulation (Spieth, 1952; Bastock & Manning,

1955; Spieth, 1974; Markow & Hanson, 1981). As previously stated, courtship occurs at

feeding sites where males indiscriminately approach feeding females. Before mating, the

male first aligns with a potential mate and begins tapping the female with his front leg.

After determining if the female is a potential mate, the male vibrates his wing toward her,

producing a species-specific courtship song. He then circles around the female and

contacts her genitalia using his proboscis ("licking"). After the genital licking, the male

attempts copulation by bending his abdomen and thrusting his genitalia toward her. The

male usually performs this series of behaviors repeatedly before copulation occurs

(Greenspan & Ferveur, 2000). The female also plays an active role in courtship by

performing a variety of acceptance and rejection behaviors. The acceptance behaviors

are not especially overt and consist of slowing locomotor activity, preening, and genital

spreading. Conversely, the rej section behaviors are more apparent and may terminate

courtship. These behaviors are ovipositor extrusion; single or double wing flick;

decamping (walking, flying, or jumping away); kicking; and abdominal elevation and

depression (Spieth, 1952; Bastock & Manning, 1955; Spieth, 1974; Markow & Hanson,

1981). After the complex courtship behaviors, the pair may begin to copulate.

Copulation lasts an average of 20 minutes, during which the male transfers seminal fluid

into the reproductive tract of the female. The seminal fluid contains ejaculate composed

of sperm and accessory gland proteins (Acps). The seminal fluid components have an

effect on sperm storage, sperm transfer, and sperm competition in the female

reproductive tract. The accessory gland proteins also have an effect on the female by









decreasing her receptivity, increasing ovulation rate, and affecting egg production

(Wolfner, 2002). Additionally, the pheromone synthesis in females also changes,

reducing her attractiveness and courtship elicitation from conspecifics. Post-mating

changes that occur can last more than days (Ferveur, 1997).

The courtship behaviors of the male and female produce visual, olfactory, tactile,

and auditory stimuli. Additional studies of courtship behavior sought to identify the

numerous signals exchanged between interacting individuals. Markow (1987) found that

different sensory stimuli are important for successful courtship in each sex. In males,

visual stimuli from the female are needed to correctly perform courtship behaviors;

females need auditory and olfactory stimuli to become receptive to a courting male.

The visual stimulus provided by the female is locomotion. The male must be able

to stay in contact with the female and perform the correct behaviors; therefore, the male

visual system is important. Additionally, an indicator of the female's receptivity and

acceptance behavior is a decrease in locomotion, which the courting male must be able to

perceive (Markow, 1987).

Auditory stimuli provided during courtship are also important for successful

courtship. Drosophila males generate two kinds of acoustic signals during vibration of

the wings: a pulse song and a sine song. These auditory signals consist of a

species-specific pattern and presumably act in species recognition. The wing vibrations

also function to stimulate females to become receptive (Greenspan & Ferveur, 2000) and

lack of these acoustic signals results in a notable reduction of courtship success (Rybak et

al., 2002).









Moreover, courtship provides olfactory stimuli that are important to both

interacting individuals. Pheromones and their function in courtship were recently

determined (Ferveur, 1997). Sex pheromones are chemical signals produced by both

males and females and are presumably recognized in each sex by gustation during

tapping and licking behaviors. Like the acoustic signals produced, pheromones or

cuticular hydrocarbons function in species and mate recognition, and also in mate

stimulation. For a given sex, strain, and age flies express a particular cuticular

hydrocarbon pattern. For example, the cuticular hydrocarbons found on the female

cuticle elicit precopulatory behaviors in males, and pheromones also enable males to

discriminate potential from non-potential mates, i.e. virgin or receptive females vs.

unreceptive females and males (Ferveur, 1997; Greenspan & Ferveur, 2000).

Initially it was assumed that D. melan2oga~ster females mated indiscriminately,

although after the work of Bateman (1948) and Petit and Ehrman (1969) sexual selection

has been well established for this species.

Females of this species invest more in their offspring, and their fitness is not

increased by the number of mates acquired but by the number of viable offspring

produced. Conversely, males usually invest less and produce more gametes than females,

therefore their fitness is increased by the number of mates acquired (Bateman, 1948).

Because of the increased investment in gamete production, females produce limited

number of ova (nutritionally demanding) and males produce excess sperm (low

nutritional demands). Therefore, females are expected to be more discriminating (Spieth

& Ringo, 1983; Andersson, 1994). Female choice is also expected to evolve when

mating is costly, e.g. when mating causes an increase in the rate of predation and risk of









infection (Andersson, 1994). If females have the ability to choose their mates, then we

expect female choice in this species because the act of mating reduces the female' s

fitness causing an increased cost to mating. The females suffer a reduction in

reproductive success and longevity because of the male accessory gland products of the

seminal fluid (Chapman, 2001; Wolfner, 2002). Therefore, the cost of mating makes

female choice adaptive. Furthermore, females are essentially in control of whether or not

they will mate with a particular male. They vigorously avoid undesirable copulations by

performing a variety of rej section behaviors that usually terminates courtship (Bastock &

Manning, 1955; Gromoko & Markow, 1993). An exception to this is newly emerged,

general females who cannot perform rejection behaviors and consequently may be forced

to mate (Markow, 2000).

Although females gain no direct benefits from mates, an increase in fitness

associated with the opportunity for female choice was found (Promislow et al., 1998).

This indicates a benefit for female choice and non-random mating. Additionally, the

signals produced by the males during courtship may be energetically costly because

courtship alone reduces longevity, therefore may provide information about their quality

(Cordts & Partridge, 1996).

Now that sexual selection is known to occur in this species, many studies have

begun to examine its role in this system. Most of the studies performed do not allow for

analysis of sexual selection into its component parts, inter- and intra sexual selection (i.e.

male-male competition and female choice) (Spieth & Ringo, 1983). However a number

of studies have been able to look solely at the intersexual selection component. Female

discrimination for males with certain phenotypes has been directly observed in many









studies (Bennet-Clark & Ewing, 1967; 1969; Kyriacou & Hall, 1982; Partridge &

Farquhar, 1983; Partridge et al., 1987; Scott, 1994; Wu et al., 1995; Bangham et al.,

2002; Rybak et al., 2002). These studies identified potential traits used in female mate

discrimination. These traits include body size, courtship song, and pheromone profile of

the male. There is evidence that these traits influence the female's mating decision, and

there may be an interaction between them. All of these traits may be related, for instance

large males may be able to visually attract the female's attention, produce a louder

courtship song, and produce higher quantities of pheromones than smaller males

(Partridge et al., 1987; Rybak et al., 2002). Such studies have provided information on

female responses to males of different phenotypes and some rules they use in mate

discrimination, but more work is needed.

If all of the known potential traits are used in female mating decisions, which trait

is more significant in her decision? Are there other unidentified traits that could influence

her mating decision? The difficulty in pinpointing a single determinant of female mate

discrimination (or male courtship success) is the result of factors that can influence

sexual selection in nature are constantly changing, for example female mating status, age,

size, and social environment, therefore a male trait that is a good predictor of courtship

success in one environment may not be in another (Markow & Sawka, 1992).

Although the courtship behaviors were first described using an ethological

approach (Bastock & Manning, 1955), most of the stimuli and their role in mate

discrimination were discovered using sensory deficient mutants, transgenic lines, or

surgical manipulation of sensory structures. Little attention has been given to the

ethological approach of studying courtship, i.e. studying natural intra- and interspecific









variants or examining differences between natural populations. Little information is

available for how much geographical and natural population variation exists in courtship

behavior. Because of the natural social environment of flies differing greatly from the

lab environment, results from most studies are not directly applicable to natural

populations. Therefore, an important goal should be to try to understand natural

population variation in the most natural setting possible to better understand the

complexity and implications of sexual selection in this species.

Sexual Isolation and D. melanogaster

Courtship behavior differences can play a role within or between species in

preventing gene flow (Butlin & Richie, 1994). There is great interest in analysis of

geographic variation within species in courtship signals because this variation contributes

to population divergence.

As previously stated, Drosophila is a good model for examining sexual isolation.

Comparisons in traits related to courtship can be made within and between related

species, because variation in courtship patterns between species usually differ in the

relative frequency of each behavior but not in the overall pattern observed (Spieth, 1952;

Bastock, 1956; Spieth, 1974). Furthermore, genetic dissection of species differences in

courtship behaviors will aid in understanding how genetics and sexual selection are

involved in reproductive isolation leading to speciation (Hollocher, 1998; Sawamura &

Tomaru, 2002).

Many studies have already begun to gather evidence that factors affecting courtship

are important in population divergence and isolation in this genus. Candidate traits

responsible for reproductive isolation within and between species have been identified

(Ewing, 1983; Coyne & Oyama, 1995; Savarit et al., 1999; Tomaru & Oguma, 2000;









Sawamura & Tomaru, 2002) For example, interspecific differences in pheromones and

courtship song may play a role in the sexual isolation between two sibling species, D.

melan2oga~ster and D. simulan2s (Sawamura & Tomaru, 2002).

Drosophila melan2oga~ster provides a unique opportunity to examine the role of

courtship related traits in sexual isolation. Until recently, it was assumed that D.

melan2oga~ster had a uniform, world wide range and exhibited no evidence of sexual

isolation (Henderson & Lambert, 1982). However, three cases of sexual isolation

between populations have been found. First, many studies have reported that Zimbabwe

populations of D. melan2oga~ster are sexually isolated from populations on other

continents. Female mate discrimination and pheromone composition was found to be the

dominant components in the divergence of Zimbabwe populations from all other

populations (Wu et al., 1995; Hollocher et al., 1997; Takahashi et al., 2001). In another

case, Korol et al. (2000) found that selection for stress tolerance resulted in behavioral

divergence in female mate discrimination in populations ofD. melan2oga~ster on the

slopes of "Evolution Canyon" producing sexual isolation between the populations.

Lastly, Haerty et al (2002) found pre-mating isolation between two natural Congolese

populations that may be because of a difference in pheromone composition.

Motivation

This study of sexual selection research was motivated because an understanding of

the traits involved in sexual selection and reproductive isolation may provide insight into

the forces that cause populations to diverge. Most approaches have been to manipulate a

courtship signal (Greenspan & Ferveur, 2000) or summarize courtship behavior with the

probabilities of transition between behaviors (Markow & Hanson, 1981). This provides

information about the signals involved in sexual communication that differs between










species, but in order to determine the cause of sexual isolation, direct observation of

individuals from one population interacting with individuals of another is necessary.

Moreover, because most studies of courtship behavior in this species use sensory

deficient mutants, transgenic lines, or surgical manipulation, little attention has been

given to studying natural variation in courtship behavior.

Obj ectives

My approach involved two analyses with outbred populations of D. melan2oga~ster,

to determine if there is evidence of sexual isolation because of differences in mating

behavior between two populations from different ecological environments. The

populations were wild-caught from Vancouver, B.C. and Leesburg, FL. The study is

laboratory based because using natural populations in the lab will allow me to control for:

quality of the environment, age at testing, reproductive status, and population density

during rearing. The questions I address is this research include

* Question 1: Do females of each population prefer to mate with males from their
own population or with males of the other population?

* Question 2: What is the role of male courtship behavior and body size in female
mate discrimination within each population?

* Question 3: What is the variation in female mate discrimination between
populations?

No-choice (NC) assays were performed in a sex population combination with

four mating types possible (V V; L L; V L; L V; female and male respectively), as

well as multiple-choice (MC) assays, to determine if females from each population prefer

to mate with males from their own population. If the populations differ in mating

behavior, I expect a decrease in mating success (increased copulation latency or no

copulation) between individuals from the different populations in the NC assays.









Additionally, if the females prefer to mate with males from their own population, then I

expect matings to deviate from random in the MC assays, with more females mating with

males from their own population than the other population. However, in multiple choice

experiments male-male competition cannot be excluded as the potential cause for mating

patterns observed.

Previous research has suggested a possible role of body size, courtship song, and

pheromone profile in mate discrimination for this species, but other traits involved in

courtship have been overlooked. As stated previously, courtship in this species consists

of elaborate and complex displays, and all of the behaviors involve visual, olfactory,

tactile, and auditory stimuli. The behaviors involved in visual stimuli have not been

thoroughly examined for a role in mate discrimination. These behaviors include chasing,

orienting toward the female, licking, etc. In this study, I am interested in determining the

relative importance of these behaviors, as well as body size in mate discrimination for

each population. Males in the NC assays were characterized for the behaviors, and

female discrimination for these male behaviors and/or body size was determined using

copulation success and copulation latency as an indicator of female mate discrimination.

Using the data collected from the NC assays, I am able to determine the variation

for males and females in the courtship related behaviors between the populations, as well

as variation in female mate discrimination between the populations.
















MATERIALS AND METHODS

Fly Populations

Vancouver Population (V)

The base population was collected (~50 mated females) in the summer 2001 in East

Vancouver, B.C. The population maintained at 250C, ~55% RH, on a 12: 12 LD cycle.

The larvae were raised at low density on a 14-day schedule. For more information

contact Arne Mooers amooers@sfu.ca.

Forty mated females from the base population (>71,000) were used to start the

Vancouver population used in the behavioral assays. The emerging offspring were mixed

randomly for at least one generation in large two liter bottles with standard Drosophila

medium before setting up at a constant density.

Leesburg Population (L)

The population was collected in the summer 2004 in Leesburg, FL. One hundred

twenty mated females from the original population were used to start the Leesburg

population used in the behavioral assays. The population maintained at 250C, ~50%

humidity, on a 12: 12 LD cycle. The larvae were raised at low density on a 14-day

schedule. The emerging offspring were mixed randomly for at least one generation in

large two liter bottles with standard Drosophila medium before setting up at a constant

density.









Rearing Conditions

After allowing for random mixing between all flies within a population they were

setup on a two-week schedule. The populations were setup in 1/2 pint bottles to avoid

crowding (Markow & Hanson, 1981). Sixteen bottles per population (8 originals and 8

backups) were setup at a constant density of 25 X 25 (females and males, respectively)

with 50 mL of food (standard medium: cornmeal, yeast, molasses, tegosept, and

proprionic acid). Flies were mixed among all replicate population bottles with each

generation. Flies were allowed to mate for 5 days, then cleared to avoid larvae

overcrowding. On Day 14, the setup was repeated.

Collection for Multiple-Choice Assays

Flies used in the MC assays were collected within 12 hours of eclosion under light

CO2 anOSthesia. Collected individuals were kept until testing in vials containing ten

individuals of the same sex. The food medium of each vial was colored red or green

using one drop of food coloring per vial in order to identify the populations during the

mating assays. Color was alternated for each population between assays to control for

the effect of coloring on behavior (Som & Singh, 2002).

Experimental Setup for Multiple-Choice Assays

MC assays were performed between 0700 and 0900 hours, because of a lower

circadian rhythm effect and high mating activity during this period (Sakai & Ishida,

2001). The mating assays were conducted in a temperature-controlled room. Multiple

assays were performed each day simultaneously in 1/2-pint bottles with 5 mL of food in

each. Males from each population were manually aspirated without anesthesia into the

1/2 pint bottle. Afterwards, females of each population were introduced, for at least 80

flies per mating bottle. The sex ratio was kept at 1:1, one male for every female for each









assay. Scan sampling was used to identify copulating individuals, which were aspirated

out and identified based on sex and abdomen color (population source) using a

microscope. Four mating types were possible: V V, V L, L V, and L L (female

and male respectively). Assays were terminated before 50% of all possible matings had

occurred to control for possible differences in mating propensity between the two

populations (Casares et al., 1998), or for one hour, whichever occurred first.

Analysis of Multiple-Choice Assays

Contingency chi-square tests were performed for each replicate assay to test for

deviation from random mating (Table 1). In order to test for deviations from random

mating for all replicate assays, a 9(X2) method was performed (Everitt, 1997; Panhuis et

al., 2003). The square root of each X2 was calculated for each assay; and its sign depends

on the direction of the data. The sign direction was obtained by calculating the cross

product of homotypic (V V and L L) minus heterotypic (V L and L V) matings

from the contingency tables. The signed X values have a normal distribution under the

null hypothesis of random mating with a mean of zero and unit standard deviation. The

standard normal variate Z was calculated for the MC assays (Table 1). Under the null

hypothesis of random mating for all replicates, Z is the sum of all 9(~X2) values divided

by -\(n), where n is the number of replicate assays. A calculated Z > 1.96 is significant at

the 5% level.

Collection for No-Choice Behavior Assays

Flies used in the NC behavior assays were collected within 12 hours of eclosion

under light CO2 anOSthesia. Ten males and ten females from each population were

collected seven days before testing, by randomly choosing individuals from the









population bottles. The collected individuals were kept until testing in vials containing

five individuals of the same sex. All individuals used in the assays were 7-day-old virgin

flies. The benefits of using 7 day old individuals is that the discrimination ability and

receptivity is increased compared to younger flies (Spieth, 1974; Manning, 1967).

Experimental Setup for No-Choice Behavior Assays

Each mating assay was digitally video recorded to allow for consistent behavior

scoring. Assays were performed in a windowless, temperature controlled room and the

mating chambers placed on a light table. By being lit from underneath small movements

could be easily discerned, such as wing vibrations, preening, and licking.

A high resolution Sony DCR-HC85 MiniDV Handycam@ Camcorder, with a focal

length of 47 cm for recording was used. The camera was setup on a tripod 82 cm from

lens to light box. The manual focus of the camera was set at 0.8 m and the manual

exposure (open aperture) set at 10. This setup was repeated for all NC assays.

Assays were performed between 0800 and 1200 hours because of high mating

activity and a decrease in the circadian rhythms effects during this time period (Sakai &

Ishida, 2001). Assays were performed using mating chambers 35 mm 10 mm high

containing 1 mL of standard Drosophila medium. Each mating chamber was used only

for a single assay.

For each assay, a single male and female were added simultaneously to a mating

chamber by manual aspiration, immediately preceding the beginning of each assay.

Video recording began after the introduction of the flies to the chamber and performed

until copulation, or for 30 minutes as this is sufficient time for copulation to occur

(Rybak et al., 2002; Manning 1967).









Both within population (V V; L L) and between population (V L; L V)

assays were performed each day. The order of the assays was randomized each day to

avoid an effect of day and time, which is known to influence behavior. A total of 40

assays for each mating type were performed. Assays in which the male failed to court the

female were excluded from analysis resulting in approximately 37 hours of behavioral

observations per sex.

Immediately after the assays, the male and female were measured for body size

(Clm). Measurements were made using an Olympus SZX9@ dissecting microscope with a

micrometer inserted into an eyepiece. Thorax length was measured, as this is a reliable

estimate of body size for this species.

An observer who did not know the population source of the flies in the assays

analyzed the videotapes. Focal animal sampling was performed to gather courtship

behavior data for both the male and female using JWatcherTM 0.9 software (Altmann,

1974). This software was used as an event recorder that logs the time of each behavior

when an assigned key is pressed. To characterize the males and females, behavior

elements were defined for each sex that encompass D. melan2oga~ster courtship (Speith,

1974).

For each assay, I recorded the time each individual was active, resting, or courting.

When the male courted the female, 12 male and 8 female courtship behaviors were

recorded. The following is a description of each courtship behavior recorded, along with

an abbreviation for the behavior. Throughout the text, each behavior will be written

using the abbreviation for brevity.









Male Courtship Behaviors:

* Tap (TAP): Male taps the female tarsus with foreleg

* Orienting toward the female during courtship:

0 Orient-back (OB): Orienting to the back of the female

0 Orient-front (OF): Orienting to the front of the female

* Licking: Male extends the proboscis to the female's genitalia while chasing
(CHASE LICK) or orienting to the back (OB LICK) of the female

* Wing vibration of one or both wings by the male, based on position of the male to
the female:

0 Chase + vibrate (CHASE WV): vibrates wings at a short distance from
female

0 Orient back + vibrate (OB WV): vibrates when orienting toward the back
of the female

0 Orient front + vibrate (OF WV): vibrates when orienting toward the front
of the female

0 Attempted copulation + vibrate (AC WV): vibrates while attempting to
mount the female

* Chasing (CHASE): male follows the female at a short distance

* Attempted copulation (AC): unsuccessful copulation attempt

* Copulation (COP): successful mounting of male on female

Female Courtship Behaviors:

* Wing flick (WF): Female flicks one or both wings

* Decamping (DECAMP): Female walks, flies, or jumps away from a courting male

* Kick (KICK): Female kicks courting male

* Ovipositor extrusion: Female extrudes ovipositor toward the head of the courting
male while standing still (SS OVI) or decamping (DECAMP OVI)

* Abdominal elevation and depression (AED): Female elevates and depresses
abdomen










* Preening (PREEN): Female preens while male is courting

* Stand still (SS): Female ceases locomotion

For all behaviors, I measured the frequency of occurrence or duration depending on

whether the behavior is classified as an event (no duration) or a state (duration). By

collecting data on frequency of occurrence or duration of each behavior, I was able to

calculate:

* Proportion of time active, resting, or courting

* Copulation latency: period of time from the introduction of flies into the mating
chamber to the onset of successful copulation

* Proportion of time allocated to performing each behavior during courtship: total
duration of the behavior/total courtship duration

* Frequency of each behavior during courtship: number of times the behavior
occurred/total number of behaviors performed during courtship

Analysis of No-Choice-Behavior Assays

Copulation Success

Chi-square contingency tests were performed to determine if there was a difference

in the number of assays that resulted in copulation for V and L females and also for V

and L males (Table 2).

Additional Chi-square contingency tests were performed to determine if there were

differences in the number of assays that led to copulation for: V males with V females

and L males with L females; V females with V and L males; L females with L and V

males; V males with V and L females; L males with L and V females (Table 3).

Copulation Latency

A Kruskal-Wallis analysis of variance was performed to determine if copulation

latency was different across mating types. Mann-Whitney U tests were also performed to









determine if there were differences in copulation latency observed for: V males with V

females and L males with L females; V females with V and L males; L females with L

and V males; V males with V and L females; L males with L and V females (Table 3).

Body Size

Mean body size (thorax length) of each sex was calculated for each population. A

Mann-Whitney U test was performed to determine if male or female body size differs

between the populations.

Body size of each sex was also calculated for copulating and non-copulating

individuals of each mating type. Differences between non-copulating and copulating

individuals in the mean body size for each sex was compared with Mann-Whitney U tests

(Table 4).

Composite Measures of Behavior

The mean proportion of time each sex was active, resting, or courting (mutually

exclusive categories) was calculated for copulating and non-copulating individuals of

each mating type. Differences between non-copulating and copulating individuals in the

proportion of time each sex was active, resting, or courting was compared with Mann-

Whitney U tests (Table 5).

Courtship Behavior

Courtship behaviors that were observed in less than 10% of all assays were

excluded from analysis. The behaviors excluded were OB LICK, SS OVI, DECAMP

OVI, TAP, and KICK. These behaviors were difficult to observe, therefore inconsistent

scoring of the observer could further complicate the low occurrence of these behaviors.

Additionally, for other behaviors many individuals did not exhibit the behavior,

therefore the frequency distribution was skewed. These behaviors include: CHASE









LICK, OB, OB WV, OF, OF WV, AC WV, WF, and AED. Therefore, these behaviors

were considered in analyses as a binomial response variable, and scored as either

performing the behavior (1) or not performing the behavior (0).

The mean proportion of time allocated to, or frequency of each behavior during

courtship, as well as the mean frequency of occurrence (0,1) was calculated for each

mating type for copulating and non-copulating individuals (Table 6).

Before statistical analyses, the non-binomial courtship behavior data were either:

square root transformed (frequency data), arcsine-square root transformed (proportion

data), or log transformed (time data) to adjust for deviations from normality.

Discriminant Function Analysis

Discriminant function analyses were performed to determine whether copulating

individuals could be discriminated from non-copulating individuals for each mating type

based on male and female courtship behaviors and body size (Table 7). Both male and

female behaviors and body size were included, since using only the behavior of one sex

markedly reduced the percentage of cases correctly classified (results not presented).

Logistic Regression Analysis for Copulation Success

The effects of male and female courtship behaviors and body size on whether or not

copulation occurred for each mating type were analyzed using logistic regression.

Behaviors examined in the logistic regression were ones that had a standardized

coefficient with an absolute value greater than 1 in the DF analysis. The standardized

coefficients indicate the relative importance of each variable to the DF, however if none

of the standardized coefficients were greater than 1 then all of the behaviors were

examined for an effect. The logistic regression was performed beginning with all









variables using backwards elimination to remove variables one at a time based on their

significance to the model. Variables were eliminated ifP > 0.15.

Multiple Regression Analysis for Copulation Latency

Although there were no differences in copulation latency across mating types (see

Results), there may be a difference in the behaviors that are important for copulation

latency for each mating type. Therefore, the effects of male and female courtship

behaviors and body size on copulation latency for individuals that did copulate for each

mating type were determined using multiple regression analyses. As in the logistic

regression, the multiple regression was performed beginning with all variables using

backwards elimination to remove variables one at a time based on their significance to

the model. Variables were eliminated if P > 0.15.

All statistical procedures were performed using SPSS v. 12 (SPSS, 2003) or JMP

IN v. 5.1 (SAS Institute, 2001).
















RESULTS

Multiple-Choice Assays

Five of the replicate MC assays chi-square contingency tests resulted in rej section of

the null hypothesis of random mating among individuals (Table 1). The 9(7X2) method

performed to test for deviation from random mating for all replicate assays resulted in a Z

value of 5.65, which led to rej section of the null hypothesis of random mating (P < 0.001;

Table 1).

Overall, the number V V copulating pairs accounted for 48% of all matings

observed, whereas number oft L L copulating pairs only accounted for 16.5%. The

number V L copulating pairs accounted for 16%, similar to the percentage L L

copulating pairs, and interestingly the number of L V copulating pairs accounted for a

greater percentage of all matings than L L with 19.5% of all matings observed.

V males achieved 67% of all copulations observed, whereas L males only achieved

33% of all copulations observed across all replicate assays.

No-Choice Behavior Assays

Copulation Success

There was no difference detected in the number of assays that led to copulation for

V and L females (P < 0.32; Table 2); however, there was a difference in the number of

assays that led to copulation between males of each population, with V males achieving

copulation in more assays than L males (X2 = 5.68, P < 0.02; Table 2).









A difference was found in the number of assays that led to copulation for V males

with V females and L males with L females, with V males with V females having a

greater number of assays that resulted in copulation (X2 = 5.48, P < 0.02; Table 3).

There was no difference in the number of assays that led to copulation for V

females with V and L males (P < 0.20; Table 3), however there was a difference in the

number of assays that led to copulation for L females with L and V males, with the L *

V mating combination more assays resulting in copulation when L females were

interacting with V males (X2 = 4.24, P < 0.04; Table 3).

Nevertheless, there was no difference in the number of assays that led to copulation

for V males with V and L females (P < 0.82; Table 3). Similarly, there was no difference

in the number of assays that led to copulation for L males with L and V females (P <

0.28; Table 3).

Copulation Latency

Copulation latency was not significantly different across mating types in the

Kruskal-Wallis analysis of variance (X2 = 1.23, P < 0.75). There was also no difference

in copulation latency for: V males with V females and L males with L females (P < 0.52;

Table 3); V females with V and L males (P < 0.63; Table 3); L females with L and V

males (P < 0.38; Table 3); V males with V and L females (P < 0.99; Table 3); L males

with L and V females (P < 0.30; Table 3).

Body Size

There was no difference between populations in female (X2 = 0.55, P < 0.46) or

male body size (X2 = 0.99, P < 0.32).









Female body size was also not significantly different for non-copulating and

copulating individuals of each mating type ( P > 0.05; Table 4). Male body size was not

significantly different for non-copulating and copulating individuals of the V V and the

L V mating type (P > 0.05; Table 4). However, for the L L mating type, copulating

males were larger than non-copulating males (X2 = 6.49, P < 0.01; Table 4). A similar

trend was also observed for the V L mating type, although the difference was not

significant (P > 0.05; Table 4).

This suggests that body size was used by females when assessing males for the

Leesburg population, but not for males of the Vancouver population.

Composite Measures of Behavior

There was no difference in the mean proportion of time active, resting, or courting

of each sex for copulating and non-copulating individuals for the V V mating type (P >

0.05; Table 5).

However, for the V L mating type there was a difference in the proportion of time

females were active (X2 = 9.66, P < 0.001; Table 5) and resting (X2 = 6.15, P < 0.01;

Table 5) with non-copulating females spending a greater proportion of time active and

resting than copulating females; copulating females were found to spend a greater

proportion of time courting (X2 = 9.87, P < 0.001; Table 5). Additionally, the males of

this mating type exhibited the same pattern as females, with non-copulating males

spending a greater proportion of time active (X2 = 10.53, P < 0.001; Table 5) and resting

(X2 = 7.34, P < 0.01; Table 5) than copulating males and copulating males spending more

time courting than non-copulating males (X2 = 10.31, P < 0.001; Table 5).









For the L V mating type, there was no evidence of differences in the proportion

of time copulating and non-copulating females and males were active and courting,

however non-copulating females and males were found to spend a greater proportion of

time resting (X2 = 14.47, P < 0.001 and X2 = 8.71, P < 0.001 respectively; Table 5) than

copulating females and males.

Comparisons for the L L mating type between copulating and non-copulating

males indicate no significant difference in the proportion of time males were active,

resting, and courting, and there was no difference in the proportion of time females were

active and courting (P > 0.05; Table 5), however non-copulating females were found to

spend a greater proportion of time resting than copulating females (X2 = 4.82, P < 0.05;

Table 5).

These results suggest that males of both populations, when participating in

unsuccessful courtship interactions with females from their own population have

difficulty assessing the female interest; yet when interacting with a female of a different

population, they tend to spend more time resting or active if the courtship interactions are

unsuccessful. This pattern is also shown for V females. However, if the courtship

interactions are unsuccessful, regardless of the interacting male' s population, L females

are more likely to spend time resting.

Discriminant Function Analysis

DF analyses performed to determine whether copulating cases could be

discriminated from non-copulating cases for each mating type based on male and female

courtship behaviors were able to correctly classify greater than 83% of non-copulating

cases and greater than 83% of copulating cases for each mating type with an overall









percentage of greater than 85% cases correctly classified (Table 7). These results

indicate that copulating and non-copulating cases can be distinguished based on male and

female behavior for all mating types.

Logistic Regression Analysis for Copulation Success

In the V V mating type assays, the only courtship behavior that predicted whether

or not copulation would occur was whether or not the males exhibited OB (Logistic

regression: OB (0,1i): X2 = 5.68, P < 0.02; Table 6). However, for all other mating types

none of the measures for male and female courtship behaviors or body size predicted

whether or not copulation would occur (L L; V L; L V; Table 6).

However, there was a trend for non-copulating males to exhibit OB, OF WV, and

AC WV more often than copulating individuals (Table 6). Moreover, a trend was

observed for CHASE WV, with copulating males spending a greater proportion of time

performing the behavior for all mating types except V V, in which case the opposite

trend was observed (Table 6). A trend was also observed for copulating females to

exhibit the WF behavior more than non-copulating females for all mating types except V

* V, in which case the opposite trend was observed (Table 6).

Multiple Regression Analysis for Copulation Latency

Three male behaviors, CHASE, OB (0,1), and AC, and one female behavior,

PREEN, as well as female body size were significantly related to copulation latency for

the V *" V mating type (Multiple regression: r2 adj = 0.39, F6,16 = 3.37, P < 0.02; CHASE:

P = -0.671, t = -2.24, P < 0.04; OB (0,1): P = 0.614, t = 3.14, P < 0.006; AC: P = -0.511, t

= -2.67, P < 0.02; PREEN: P = 0.723, t = 3.73, P < 0.002; FEMALE BS: P = -0.61, t = -

2.99, P < 0.009). As the proportion of time allocated to CHASE and the frequency of AC









increased, copulation latency decreased. Similarly, as female body size increased,

copulation latency decreased. However, as the proportion of time allocated to PREEN

increased, copulation latency increased. Males that did not exhibit the OB (0,1) behavior

were more likely to have a decreased time to copulation than males that exhibited the

behavior.

Conversely, there was no evidence that male or female behaviors or body size was

related to copulation latency for L L mating type.

For the V L mating type, only the female behavior WF (0, 1) was significantly

related to copulating latency (Multiple regression: r2 adj = 0. 183, F1,16 = 4.820, P < 0.04;

WF (0,1i): P = 0.481, t = 2.20, P < 0.04). Females that did not exhibit the WF behavior

were more likely to have a decreased time to copulation than females that did exhibit the

behavior.

For the L V mating type, male body size along with the male behavior OF (0,1),

as well as female behaviors, AED (0,1), PREEN, and SS, were significantly related to

copulation latency (Multiple regression: r2 adj = 0.422, F6,16 = 3.67, P < 0.017; MALE

BS: P = 0.788, t = 3.36, P < 0.004; OF (0,1): P = -0.579, t = -2.75, P < 0.014; AED (0,1):

P = 0.465, t = 2.66, P < 0.017; PREEN: P = -0.836, t = -3.95, P < 0.001; SS: P = 0.563, t

= 2.92, P < 0.01). As male body size and the proportion of time allocated to SS increased,

copulation latency increased. Conversely, as the proportion of time allocated to PREEN

increased, copulation latency decreased. Males that did exhibit the OF behavior were

more likely to have an increased time to copulation than males that did exhibit the

behavior. Similarly, females that did not exhibit the AED behavior were more likely to

have a decreased time to copulation than females that did exhibit the behavior.
























































2.78


Table 1: Copulation success for each mating type in the MC assays between the
Vancouver and Leesburg populations (N = number of males and number of
females from each population).
Male


IV IV


Assays were terminated before 50% of all possible matings had occurred. Results from
the X2 contingency tests determining deviation from random mating, 9(~X2) for each
assay, and the Z value (- CX/9(n)). Significance level of: *P < 0.05, **P < 0.01, ***P <
0.001 indicates a significant departure from random mating.


Replicate


1


Female


V


r2


0.08


Z


5.65***


0.28


L

80 V

L

60 V

L

80 V

L

40 V

L

80 V

L

80 V

L

80 V

L

120 V

L

120 V

L


8.65**


2.94


0.35


0.59


0.10


0.32


0.66


0.81


0.38


0.62


9.56**


3.09


10.23**


3.20


10.40***


3.22


7.73**










Table 2: Copulation success of males and females from each population in the NC assays;
results for the Chi-square contingency tests to determine if there was a
difference in the number of assays that led to copulation for V and L females
and for V and L males.



Copulated (N/Y)

N Y

Female V 18 57

L 22 48

X2 = 1.0
P < 0.32

N Y

Male V 14 60

L 26 45

X2 = 5.68
P < 0.02*
*P < 0.05, **P < 0.01, ***P < 0.001










Table 3: Copulation success and copulation latency of interacting individuals of the
within and between population assays in the NC assays; results for Mann-
Whitney U tests and Chi-square contingency tests to determine if there were
differences in copulation latency and in the number of assays that led to
copulation; *P < 0.05, **P < 0.01, ***P < 0.001


Mating Type


Copulated (N/Y)


V*V
L*L


Copulation Latency- X2 = 0.41
P < 0.52


Copulation Success- X2 = 5.48
P < 0.02*



32
25

Copulation Success- X2 = 1.63
P < 0.20


V *V
V*L


Copulation Latency- X2 = 0.23
P < 0.63


L*L
L*V


Copulation Latency- X2 = 0.77
P < 0.38


Copulation Success- X2 = 4.24
P < 0.04*



32

28

Copulation Success- X2 = 0.05
P < 0.82

Y

20
25

Copulation Success- X2 = 1.16
P < 0.28


V *V

L *V


Copulation Latency- X2 = 0.00
P < 0.99

N


L*L
V*L


Copulation Latency- X2 = 1.06
P < 0.30












Table 4: Mean body size (+ SE) for each mating type for copulating (COP) and non-copulating (NO COP) individuals for the NC
assays, with differences compared with Mann-Whitney U tests.
V*V V*L L*V L*L

Body Size NO COP COP X2 NO COP COP X2 NO COP COP X2 NO COP COP X2

Male 40.83 40.96 0.03 38.90 40.35 2.84 41.00 41.08 0.01 39.92 42.00 6.49**
(0.54) (0.44) (0.62) (0.81) (1.02) (0.45) (0.43) (0.57)


Female 48.86 46.92 2.51 46.20 47.39 1.37 45.50 47.44 2.15 46.43 46.13 0.04
(1.34) (0.55) (0.84) (0.51) (1.78) (0.65) (0.83) (0.75)

N = 39, V V; N = 36, V L; N = 35, L V; N = 35, L L; *P < 0.05. **P < 0.01, ***P < 0.001.












Table 5: Composite measures of behavior of each sex for each mating type for copulating (COP) and non-copulating (NO COP)
individuals in the NC assays; differences in the mean proportion (+ SE) of time active, resting, and courting compared with
Mann-Whitney U tests.
V*V V*L L*V L*L
Proportion of
Time NO COP COP X2 NO COP COP X2 NO COP COP X2 NO COP COP X2


Female Active


0.34 0.36 0.26 0.51 0.23 9.66*** 0.33 0.32


0.00


0.34


0.32


0.19


(0.09) (0.03)


(0.08) (0.03)


(0.08) (0.03)


(0.04) (0.03)


Female Resting



Female Courting



Male Active


0.01
(0.00)

0.66
(0.09)

0.32
(0.08)

0.01
(0.01)

0.67
(0.08)


0.00
(0.00)

0.64
(0.03)

0.36
(0.03)

0.00
(0.00)

0.64
(0.03)


1.46 0.01
(0.00)

0.11 0.47
(0.08)

0.34 0.51
(0.07)

2.11 0.02
(0.00)

0.16 0.48
(0.08)


0.00
(0.00)

0.77
(0.03)

0.23
(0.03)

0.00
(0.00)

0.77
(0.03)


6.15** 0.07
(0.04)

9.87*** 0.60
(0.09)

10.53*** 0.37
(0.08)

7.34** 0.02
(0.01)

10.31*** 0.61
(0.09)


0.01
(0.00)

0.68
(0.03)

0.31
(0.03)

0.00
(0.00)

0.69
(0.03)


14.47*** 0.05
(0.02)


0.00
(0.00)

0.67
(0.04)

0.33
(0.03)

0.00
(0.00)

0.66
(0.03)


4.82*


0.61



0.38


0.61
(0.04)

0.37
(0.04)


2.17



1.27


Male Resting



Male Courting


8.71*** 0.01
(0.00)


0.73


0.29


0.62
(0.04)


1.27


N = 39, V V; N


36, V L; N = 35, L V; N = 35, L L; *P < 0.05. **P < 0.01, ***P < 0.001.

















Courtship Behaviors
Chase ]Proportion

Chase WV Proportion

Chase Lick Frequency (0,1)

OB Frequency (0,1)

OB WV Frequency (0,1i)

OF Frequency (0,1)

OF WV Frequency (0,1)

AC Frequency

AC WV Frequency (0,1i)

Decamp Proportion

WF Frequency (0,1)

AED Frequency (0,1i)

Preen Proportion

SS 1Proporticut


Table 6: Courtship behavior measures for each mating type for copulating (COP) and non-copulating (NO COP) individuals in the NC
assays; mean proportion of time allocated to performing each behavior during courtship (f SE), mean frequency of each
behavior performed during courtship (f SE), or mean frequency of occurrence (0,1 i, SE).


V*
NO COP
0.:27
(0.05)
0.62
(0.05)
0.73
(0.14)
0.82
(0.12)
0.45
(0.16)
0.45
(0.16)
0.73
(0.14)
0.11
(0.03)
0.36
(0.15)
0.85
(0.04)
0.27
(0.14)
0.27
(0.14)
0.04
(0.01)
0.D8
(0.03)


L"
NO COP
0.:22
(0.10)
0.58
(0.12)
0.86
(0.14)
0.71
(0.18)
0.57
(0.20)
0.43
(0.20)
0.43
(0.20)
0.11
(0.02)
0.00
(0.00)
0.78
(0.04)
0.57
(0.20)
0.14
(0.14)
0.16
(0.05)
0.10
(0.05)


L*L
NO COP
0.23
(0.03)
0.62
(0.05)
0.87
(0.09)
0.87
(0.09)
0.20
(0.11)
0.60
(0.13)
0.73
(0.12)
0.10
(0.02)
0.27
(0.12)
0.79
(0.04)
0.47
(0.13)
0.20
(0.11)
0.09
(0.02)
0.09
(0.03)


NO COP
0.14
(0.04)
0.77
(0.04)
0.86
(0.14)
0.43
(0.20)
0.57
(0.20)
0.43
(0.20)
0.86
(0.14)
0.09
(0.03)
0.29
(0.18)
0.80
(0.05)
0.71
(0.18)
0.14
(0.14)
0.06
(0.02)
0.D6
(0.01)


COP
0.16
(0.03)
0.76
(0.03)
0.59
(0.09)
0.31
(0.08)
0.59
(0.09)
0.38
(0.09)
0.56
(0.09)
0.07
(0.01)
0.06
(0.04)
0.82
(0.03)
0.59
(0.09)
0.38
(0.09)
0.08
(0.02)
0.11
(0.02)


COP
0.14
(0.03)
0.73
(0.04)
0.76
(0.09)
0.64
(0.10)
0.40
(0.10)
0.48
(0.10)
0.52
(0.10)
0.13
(0.02)
0.24
(0.09)
0.76
(0.04)
0.60
(0.10)
0.36
(0.10)
0.11
(0.03)
0.11
(0.02)


COP
0.17
(0.02)
0.71
(0.04)
0.64
(0.09)
0.39
(0.09)
0.46
(0.10)
0.50
(0.10)
0.64
(0.09)
0.09
(0.01)
0.07
0.05)
0.74
(0.04)
0.64
(0.09)
0.39
(0.09)
0.08
(0.02)
0.13
(0.03)


COP
0.18
(0.03)
0.66
(0.05)
0.80
(0.09)
0.70
(0.11)
0.60
(0.11)
0.55
(0.11)
0.65
(0.11)
0.11
(0.02)
0.20
(0.09)
0.74
(0.05)
0.70
(0.11)
0.30
(0.11)
0.08
(0.02)
0.13
(0.03)


*V


L


V*V













Table 7: Discriminant function analyses to determine whether copulating cases can be discriminated from non- copulating cases for
each mating type, with the percentage of cases correctly classified for non-copulating (NO COP) and copulating (COP) individuals,
along with the overall percent of cases correctly classified for the NC assays.


Mating Type NO COP Cases COP Cases Overall Correct
Correctly Classified (%) Correctly Classified (%) Classification (%)


V *V


V *L


L *V


L *L


83.3


88.9


100.0


83.3


95.7


94.4


83.3


87.5


93.1


92.6


96.6


85.7
















DISCUSSION

One obj ective of this study was to determine if females from each population prefer

to mate with males from their own population, or with males of the other population. If

the populations are sexually isolated or differ in courtship behavior, then females should

mate more often with individuals from their own population, and there should be a

decrease in mating success in interactions of individuals from different populations.

Results from the MC tests, indicate that V females prefer to mate with V males.

Though, male-male competition cannot be excluded as a potential cause for the observed

mating patterns, since in this setup males have the opportunity to interact. However, in

the NC assays the percentage of NC assays that led to copulation for V females with V

and L males was not different. Therefore, Vancouver females may in fact prefer to mate

with males from their own population, however they are equally likely to mate with

males from the other population when not given a choice.

The MC results, along with the observation that L males achieved copulation in

fewer NC assays than V males, and that a greater percentage of assays led to copulation

for L females with V males than with L males indicate that L females are more likely to

mate with V males more than with males of their own population. Therefore, L females

may prefer individuals from the Vancouver population.

Similar to other sexual isolation studies, there is evidence of non-random mating

between the two populations (Wu et al., 1995; Korol et al., 2000; Haerty et al., 2002).

However, the pattern seems to be driven by the high number of V V matings. This may









be because of asymmetrical isolation for females of the Vancouver population, however

additional experiments would need to be conducted to confirm this hypothesis.

Moreover, the increased copulation success of V males compared to L males for L

females may be because of a novel male effect or the observed pattern could be because

ofL males being less "attractive" than V males, since L males obtained significantly

fewer matings than V males in both the MC and NC tests.

Copulation latency was used in this study as an indicator of mating success,

however for individuals that did copulate the time it took for these individuals to mate

was not different across mating types, between the within population mating types, nor

when interacting with individuals from the same or a different population. Therefore,

copulation latency may not be a good indicator of mating success. For example, once

females assess the male and decide either to copulate or not, the time it takes to mate may

be related to a certain stimulus threshold required to successfully produce eggs or related

to investment in egg production.

The second obj ective of this study was to determine the role of male courtship

behaviors and body size, if any, in female mate discrimination within each population.

Little information is known about the role these behaviors may play in female mate

discrimination. Initially, it was assumed that visual cues were unimportant for mating

success since D. melan2oga~ster females could mate in the dark; yet it was found that

mating success is significantly decreased when visual cues are lacking (Cobb & Ferveur,

1996). Furthermore, the lack of detailed studies may be partly because of the time

required to make detailed observations of courtship interactions, increased interest in the

role of pheromones and courtship song, and a negative publication bias.









In this study, when V males were interacting with V females, behaviors that were

significantly or to some extent related to copulation success could not be identified;

however there was evidence of a behavior that if performed indicated that copulation was

unlikely to occur. Yet when V males were interacting with L females, copulation success

was slightly correlated with the proportion of time allocated to CHASE WV. Similarly,

when L males were interacting with L or V females, copulation success was slightly

correlated with the proportion of time allocated to CHASE WV, and also correlated to

male body size.

Similar to other studies, male body size was found to be important in mating

success, at least for the Leesburg males (Partridge & Farquhar, 1983; Partridge et al.,

1987). However, female mate discrimination for male courtship behavior within each

population could not be determined with any confidence. This may be because of low

number of assays that did not result in copulation, thereby reducing the power to detect

differences among copulating and non-copulating individuals; but could also be because

of differences in traits that were not measured in this study, such as pheromone profile or

courtship song.

For assays in which copulation did occur, the proportion of time males spent in

CHASE, the frequency of AC, and whether or not males exhibited OB was significantly

related to copulation latency when V males were interacting with V females; however

when interacting with L females, only body size and whether or not males exhibited OF

was related to copulation latency. Conversely, there was no evidence that courtship

behavior or body size was related to copulation latency when L males were interacting

with L or V females.









The contradictions between behaviors identified as potentially important for

whether or not copulation will occur and the behaviors related to copulation latency, may

be because of once females assess the male and decide either to copulate or not, the time

it takes to mate may be related to a certain stimulus threshold required to successfully

produce eggs or related to investment in egg production.

The third obj ective of this study was to determine if there is variation between the

populations in female mate discrimination. Since female discrimination for male

courtship behaviors within each population could not be determined, it is difficult to

determine the variation between the populations. The only consistent pattern observed

was for body size, in which females of both populations interacting with L males mated

more often with larger males.

Many studies have already begun to gather evidence that factors that affect

courtship are important in population divergence and isolation in this genus. Drosophila

melan2oga~ster provides a unique opportunity to examine the role of courtship related traits

in sexual isolation since three cases of prezygotic isolation between populations have

been found (Wu et al., 1995; Hollocher et al., 1997; Takahashi, 2001; Korol et al.,. 2000;

Haerty et al., 2002). The goal of this study was to determine if there is evidence of sexual

isolation because of differences in mating behavior between two outbred populations of

D. melan2oga~ster from different ecological environments. Although, evidence of sexual

isolation was not detected between the two populations, Vancouver and Leesburg, there

were some interesting mating patterns uncovered and the exact mechanism driving these

patterns remains to be elucidated.










Sexual selection can drive intrapopulation evolution, and through female choice or

male-male competition can cause sexual isolation among populations, potentially leading

to speciation. Population genetic models have shown that changes in the way mates are

acquired or chosen in a population can lead to rapid speciation due to the direct effects

these changes have on gene flow (Lande, 1981; 1982). Therefore, it is important to

understand whether sexual selection is playing a role in the difference between

populations and/or species.

Courtship behavior differences can play a role within or between species in

preventing gene flow (Butlin & Richie, 1994). Therefore, there is great interest in

analysis of geographic variation within species in courtship signals, because this variation

contributes to population divergence. However, scarce information is available for how

much geographical variation exists for D. melan2oga~ster, as well as for many other

species. Furthermore, genetic dissection of species differences in courtship behaviors

will aid in understanding how genetics and sexual selection are involved in reproductive

isolation leading to speciation (Hollocher, 1998; Sawamura & Tomaru, 2002).

















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BIOGRAPHICAL SKETCH

Kelly Marie Jones was born on November 13, 1979, Fort Walton Beach, FL. She

is one of three children of Marsha A. Jones and Wesley H. Jones. She followed her

passion for biological sciences and enrolled at the University of Florida, where she

earned her bachelor's degree in zoology. During her undergraduate education, she found

an opportunity to perform research on the behavior of fruit flies, with Dr. Laura Higgins.

After graduating, she enrolled in graduate courses at the University of Florida as a

postbaccalaureate student. In 2003 she was accepted into the graduate program in the

Department of Zoology at the University of Florida. She was awarded a Master of

Science degree in May 2006.