MECHANISMS THAT DRIVE VARIATION in FEMALE MATING PREFERENCES in XIPHOPHRUS MALINCHE a Thesis Presented to the Faculty of the Co

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MECHANISMS THAT DRIVE VARIATION IN FEMALE MATING

PREFERENCES IN XIPHOPHRUS MALINCHE

A thesis presented to

the faculty of

the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree

Master of Science

M. Scarlett Tudor

August 2007 2

M. Scarlett Tudor

This thesis titled

MECHANISMS THAT DRIVE VARIATION IN FEMALE MATING

PREFERENCES IN XIPHOPHRUS MALINCHE

M. SCARLETT TUDOR

has been approved for

the Department of Biological Sciences

and the College of Arts and Sciences by

______

Molly R. Morris

Associate Professor of Biological Sciences

______

Benjamin M. Ogles

Dean, College of Arts and Sciences

Abstract

TUDOR, M. SCARLETT M.S., August 2007, Biological Sciences

MECHANISMS THAT DRIVE VARIATION IN FEMALE MATING

PREFERENCES IN XIPHOPHRUS MALINCHE (65 pp.)

Director of Thesis: Molly R. Morris

In Xiphophorus malinche, larger, older females prefer asymmetrical males while smaller, younger females prefer symmetrical males. Mating experience has been shown to alter female preferences. I examined whether copulatory experience as well as experience with males of different symmetry affected female preference for symmetry in X. malinche. Male aggressive behaviors and male preferences may affect female experience, thus influencing female preference. I examined whether male aggression and male preferences for female size varies with symmetry. Female size at maturity and female experience affected female preference for symmetry suggesting a genetic and environmental component exists for preference for symmetry. Male mating preference for female size did not vary with symmetry and is not likely to affect female preferences. The less aggressive response by symmetrical males to the symmetrical image and their overall lower frequency in the field could play a role if females perceive symmetrical males as less aggressive.

Approved: ______

Molly R. Morris

Associate Professor of Biological Sciences 5

Acknowledgments

I will be forever indebted to Molly Morris for being such a great advisor and most importantly a great friend. I can barely verbalize what Molly has done for me over the past four years. Molly has been a strong woman role model, which has influenced my academic career as well as my person life. I am very grateful for having someone to fully share my interest in science as well as the natural world.

Through her support in many contexts Molly has allowed me to develop to my fullest potential as a scientist. The world can be very scary and I take comfort in knowing that Molly will be there for me even when I am no longer her student. I also want to thank Kevin de Queiroz for often getting me to think about things in a different perspective and for always giving good career advice.

Everyone in the Morris’ lab has been extremely supportive. I feel very fortunate to have been able to work with such a great group of people and to call these people my friends. Oscar Rios-Cardenas was the first person to get me more involved in the lab as an undergraduate and got me involved in a project that became my first paper. Natalie Dubois has been like big sister to me, she has always had time to answer my many questions about science and life. Carla Gutiérrez-Rodríguez showed me that genetics could actually answer some really cool questions. Donelle Robinson answered my endless questions about statistics. Andre Fernandez has been a supportive lab-mate. Susan Lyons took great care of the fish in the lab. Jason Brewer does a great job at taking care of lab logistics. 6

I want to thank everyone on my thesis committee, Don Miles, Mark Waters and Matt White for taking the time to give me great feedback. Don Miles has made me feel more confident talking about science and on occasion allowed me to use his phone. Matt White took me on my first fish-collecting trip and introduced me to the seine and most importantly the electroshocker.

Regina Macedo, Dan Wiegmann, Hugh Drummond and Colette St. Mary all made me feel welcome in a broader community. Regina Macedo could not have been more supportive of me; she came to see me give the same talk three times. Dan

Wiegmann is a constant reminder that scientist can be smart and cool.

I want to thank all of the students that helped collect data, Cari Moss, Marcie

Ryder and Jason Williams.

I would not have been able to accomplish this without the support of my family. My mom has been my “personal assistant” since I started college, which allowed me to spend more time in the lab. My grandparents and my Aunt Cathy have often helped me financially. My friends Mandy and Karl have also been there for me in ways that I can never repay them. Luna and Guthrie have given me the “doggy” attention that I needed to be able to get through the rough patches.

Table of Contents

Page

Abstract...... 4

Acknowledgments ...... 5

List of Tables...... 8

List of Figures...... 9

CHAPTER 1: Experience plays a role in female preferenece for symmetry in the swordtail fish Xiphophorus malinche...... 10

INTRODUCTION...... 10 METHODS...... 12 Preference Test:...... 13 Experiment 1: Virgin preference for symmetry ...... 15 Experiment 2: Mating experience and preference for symmetry ...... 15 Analysis:...... 17 RESULTS...... 18 Experiment 1: Virgin preference for symmetry ...... 18 Experiment 2: Mating experience and preference for symmetry ...... 19 DISCUSSION...... 23 LITERATURE CITED...... 30

CHAPTER 2: Differences between symmetrical and asymmetrical males; how male behavior could be influencing female preference for symmetry in the swordtail Xiphophorus malinche...... 40

INTRODUCTION...... 40 METHODS...... 43 Experiment 1: Male Aggression and Response to Symmetry...... 43 Experiment 2: Male Preference for Female Size...... 45 RESULTS...... 47 Experiment 1: Male Aggression and Response to Symmetry...... 47 Experiment 2: Male Preference for Female Size...... 49 DISCUSSION...... 52 LITERATURE CITED...... 57

List of Tables

Table Page

1.1: Female strength of preference, female mortality and male aggression for each replicate aquaria…………………………………………………. 39

List of Figures

Figures Page

1.1: Frequencies of symmetrical and asymmetrical males………………………..34

1.2: Comparison of the preference strengths across female age………………….35

1.3: Comparison of the preference strengths across three treatments groups for when females where a) young and b) old…………………………………….36

1.4: Correlation between standard length and preference for both young (A) and old (B) age classes……………………………………………………….37

1.5: Correlation between SL and preference for the young age class split by treatment group……………………………………………………………...38

2.1: Mean number of bites by symmetrical and asymmetrical males during a five-minute mirror-image stimulus trial…………………………………...... 61

2.2: Comparison of the time spent with small vs. large females for both symmetrical and asymmetrical males………………………………………..62

2.3: The relationship between male size and strength of preference (difference in time spent with small and large females) during the interaction test...... 63

2.4: Comparison of male behaviors directed towards small verse large females during the interaction test……………………………………………………64

2.5: The relationship between male isolation period and strength of male preference for female size during the choice tests………………………….65

CHAPTER 1

EXPERIENCE PLAYS A ROLE IN FEMALE PREFERENCE FOR SYMMETRY IN THE SWORDTAIL FISH XIPHOPHORUS MALINCHE

INTRODUCTION

Variability in female mating preference has been historically overlooked.

Most prior studies focused primarily on measuring mean preferences across populations or species as the main interest has been the role of female preference in species recognition or the consequences of female preference for the evolution of male traits (for review see Jennions & Petrie 1997). Recently, however, variability in female mating preference has been detected in a variety of species by testing the same females repeatedly (Godin & Dugatkin 1995; Wagner et al. 1995; Gerhardt et al. 2000;

Morris et al. 2003). Variability in female preference can lead to the evolution of complex male displays (Coleman et al. 2004) and even the maintenance of alternative mating strategies (Rios-Cardenas et al. in press). Thus a better understanding of female preference variability can give insight into the evolution and maintenance of complex male attributes.

Understanding variability in female preference could also provide insight into the evolution of female preference itself. Even female preferences that are variable can evolve if females alter their preferences in a predictable way (Fordyce 2006) and this variability could then be incorporated into the genome through genetic assimilation (for review see Pigliucci & Murren 2003) creating an evolvable genetic 11 component to the preference variability. Genetic models have been used to suggest mechanisms for which variability in a quantitative trait can be controlled by the genome (for review see Via et al. 1995). For example, female guppies from natural populations that experience high predation reduced their preferences for male coloration in the presence of predators (Godin & Briggs 1996). This suggests that there are costs associated with female mating preference and plasticity of those preferences could potentially increase a female’s fitness by allowing a female to be flexible about the circumstances in which a preference is exhibited. Mating experience has also been shown to alter female preferences (Marler et al. 1997; Hebets

2003; Eakley & Houde 2004) and could be another mechanism that drives the evolution of variability in female mating preference.

Many species of Xiphophorus fish exhibit the pigment pattern “vertical bars” that functions in mate attraction and male-male competition (Morris et al. 1995;

Morris and Ryan 1996). During courtship males use a display known as a ‘figure- eight’ display (Ryan & Causey 1989) in which a male swims back and forth in front of a female, providing the female with an opportunity to assess bilateral bar symmetry.

In a previous study, Xiphophorus malinche females exhibited a correlation between preference for vertical bar number symmetry and size where small females preferred symmetrical males while large females preferred asymmetrical males. Female

X. malinche grow after sexual maturity (Kallman 1989; Marcus & McCune 1999;

Morris et al. 2006); therefore variability in preference could potentially be due to experience. The X. malinche population located in the Rio Claro, Mexico is 12 comprised of both symmetrical and asymmetrical males, as measured by the difference in the number of vertical bars on left and right sides (23.3% symmetrical males and 76.7% asymmetrical males; Morris et al. 2006). Females in this population could potentially gain experience with both symmetrical and asymmetrical males.

Therefore to determine if experience could influence female preference for the bars in

X. malinche, I conducted two experiments. First, virgin females were tested to determine female preference prior to copulatory experience. Second, females were given experience with males of differing symmetry to examine how sexual experience, and more specifically experience with males that differed in bar number symmetry can alter female preference for symmetry. For both experiments females were tested at two different age classes (young and old age class) to assess the effects of these treatments on preference for symmetry over time.

METHODS

X. malinche juveniles, all less than 26 mm standard length (SL), which is the distance from the tip of the snout to the base of the caudal peduncle, were collected from the Rio Claro in the state of Hidalgo, Mexico (2004 and 2005), and reared in the lab in 37.9 l communal tanks until sexual maturity. Thus juveniles could have some experience with vertical body bars, but not in a mating context. Fish were fed

Tetramin commercial fish food daily and maintained on a 12h light/12h dark photoperiod at a constant room temperature of 22 C. Once sexual characteristics started to develop, gonopodium (males) or brood spot (females), fish were moved to 13 single sex aquaria to insure that individuals had no copulatory experience. All length measurements were taken using the SL. Sexually mature males collected in 2004 and

2005 were scored for vertical bar symmetry as well as the males reared in the laboratory that were collected as juveniles in 2005.

Preference Test:

Females were isolated for one week prior to the preference tests in 18.9 l aquaria. Female mating preference for symmetry was assessed using standard dichotomous preference tests. Association time in these tests is a reliable indicator of female mating preference in the closely related swordtail X. nigrensis (Ryan et al.

1990; Morris et al. 1992). Each female was tested twice at each age class (young and old classes), for a total of 4 tests, so that a measure of repeatability could be calculated for each age class. Time between tests was 2-4 days for experiment 1 (virgin females) and 3-13 days for experiment 2 (females housed with males). I examined the possibility that time between tests might influence the strength of preference that females exhibited, and found no relationship at the young age for either experiment

2 (linear regression: experiment 1: r = 0.105, N = 9, F1,8 = 0.822; P = 0.395;

2 experiment 2 (all treatments combined): r =0.043, N = 31, F1,30 = 1.290; P = 0.265).

Preference tests were conducted in a 37.6 l aquarium that was visually divided into three equal sections by drawing lines on the outside of the aquarium. Two Sony

Trinitron high-resolution monitors (model PVM-9L3) connected to two DVD players were placed at either end of the aquarium. The two animations used were created 14 using LIGHTWAVE 3D v. 5.6 (Newtek, details about the creation of these animations can be found in Morris et al. 2003). The two stimulus animations were “symmetrical”

(7 bars on the left and 7 bars on the right) and “asymmetrical” (6 bars on the left and 8 bars on the right). Using this bar number configuration keeps the total number of bars equal. A mirror was angled at the top of the aquarium allowing the females movements to be observed. The female was placed in a clear Plexiglass tube in the middle section and allowed to acclimate to the aquarium for 10 minutes prior to playing the videos. Each video contained 3 minutes of blank screen, followed by 9 min. 40 sec. of male animation. While still in the tube, the female was allowed to acclimate to the blank video screen for 3 minutes and to the animations of the males for 1 minute. After this time she was released into the aquarium and allowed to swim freely among all three sections. The time that the female spent in the sections adjacent to each monitor was recorded (denoted as association time) for the duration of the video (8 min. 40 sec.). In order to account for any side bias that the female may exhibit, the videos were switched to the opposite monitors. A second trial was conducted starting with a ten-minute acclimation period. The total time the female associated with each animation was summed across all four trials. Tests where the female that did not spent time on both sides of the aquarium during the observation were considered to be a side bias and the female was retested on a different day.

Experiment 1: Virgin preference for symmetry

Virgin females were used to assess preferences for symmetry for females that had no experience with males. In addition, these females were tested at two different ages (denoted as age class throughout) to determine if their preferences changed with age independent of mating experience. The size of the females in the first test ranged from 33-37.6 mm (denoted as young age class) and all females were larger than 37.6 mm in the second test (denoted as old age class). Females were housed in single sex communal aquaria until they reach 33 mm, at which time they were isolated prior to testing. Once testing was complete, females were returned to their single sex communal aquaria until they reached sizes > 37.6 mm, then they were re-isolated for the second test. I chose 37.6 mm as the size by which to separate the two age classes as this is the size identified as the “switch” point for females showing preference for symmetry as compared to asymmetry in Morris et al. (2006). A total of 8 females were tested at the young age class, 2 of these females died before the tests at the old age class thus 6 females were tested at both age classes.

Experiment 2: Mating experience and preference for symmetry

Females were given experience with males of differing bar symmetry to assess the effects of experience with a given bar pattern on female preference for this trait.

Virgin females were randomly assigned to three treatment groups at the time they reached sexual maturity. In treatment one, females were housed with two symmetrical males (“symmetrical” treatment). Treatment two females were housed with one 16 symmetrical and one asymmetrical male (“mixed” treatment), and treatment three females were housed with two barless males (“barless” treatment). X. malinche males are polymorphic for the presence of vertical bars. Treatment three was included to assess the preference for symmetry of females that have had mating experience, but no experience with vertical bars. There were three replicates for each treatment group for a total of nine 37.9 l aquaria, for a total of 31 females (2-5 females per tank).

As in experiment 1, females were tested twice at two different ages to determine if females would exhibit a switch in preference over time. The first tests were conducted 11-13 days after the females had been placed with the males (mean =

11.6 days) and this is denoted as young age class. The second tests were conducted when the females had reached sizes equal to or greater than 37.6 mm. At this time, the females had been housed with the males for 145-182 days (mean = 151.3 days) and is denoted as the old age class. During the second part of the experiment, one of the barless replicate tanks was lost due to mortality of all individuals. In addition, 11 females died before the second test across the various tanks. Therefore, a total of 20 females of the 31 were tested in the old age class tests, and the number of females per replicate ranged from 1 to 4 per tank.

Aggression levels were recorded for each replicate for all treatments after all females had been tested the first time, except for the one barless replicate. Focal observations were conducted on each male in a replicate aquarium for a period of 5 minutes. The number of bites that the focal male directed toward the other male and 17 the females were recorded, and the total number of bites by the focal males over the 10 minutes of observation were summed to give an aggression score for each tank.

Analysis:

The association times from the first and second test for each female at each age class were summed to give a total association time for both the asymmetrical and symmetrical animation for the two age classes. The time females associated with each animation were compared with paired t tests to determine if females had a significant preference for symmetry/asymmetry at both age classes, and within treatment groups for experiment 2. If no significant preference was found, we examined the variation in strength of response across the two preference tests with a one-way ANOVA (Becker

1992; Lessells & Boag 1987; Boake 1989). A significant ANOVA in addition to some females spending more time with the symmetrical treatment while some spending more time with the asymmetrical will indicate a polymorphism in preference for symmetry (Morris et al. 2001).

Strength of preference for asymmetry was calculated for each female by subtracting the total association time with the symmetrical male from the total association time with the asymmetrical male; a positive number reflects a preference for the asymmetrical male and a negative number reflects a preference for the symmetrical male. For all treatment groups at both ages the strength of preference was averaged across all females in that group to obtain the average strength of preference. Paired t tests were used to compare the average strength of preference 18 when young as compared to old, to determine if the strength of preference had changed over time.

For experiment 2, a Nested ANOVA (mixed procedure in SAS version 9.1) was also used to examine any differences in the average strength of preference among the three treatment groups. The treatment type was considered a fixed factor, while the replicate tank number was nested within the treatment type. Tukey’s post hoc test was used for the multiple comparisons.

RESULTS

There were higher percentages of asymmetrical males than symmetrical males for both years as well as for males that were collected in 2005 as juveniles and reared to sexual maturity in the lab (figure 1.1). There was no significant difference in the percentage of symmetrical/asymmetrical males caught as adults in 2004 and 2005

(Fisher’s exact test: P = 0.475; figure 1.1). There was also no significant difference between the males collected as adults in 2005 and males collected as juveniles in 2005

(Fisher’s exact test: P = 1.00; figure 1.1).

Experiment 1: Virgin preference for symmetry

Virgin females did not spend significantly more time with either the symmetrical or asymmetrical video at either age class (mean association time ± se: young age class: symmetrical male = 1038.4 ± 120.5s, asymmetrical male = 1040.4 ±

126.3s; paired t test: t 7 = -0.008, P = 0.994; old age class: symmetrical male = 1255.3 19

± 294.8s, asymmetrical male = 852.7 ± 298.0s; paired t test: t 5 = 0.679, P = 0.527).

This lack of overall preference does not appear to be due to a polymorphism in preference, as the variation in response to symmetry was not significantly greater across females than within females for both age classes (young age class: F7,8 = 4.267,

P = 0.078; old age class: F4,5 = 2.981, P = 0.159). Although females at the young age class had a close to significant ANOVA, the trend was for a negative correlation between test 1 and test 2 (Spearman’s rank test: rs = -0.517, N= 9, P = 0.154), suggesting that young females were more likely to switch than be consistent across tests.

Only females that were tested at both age classes were used to assess a shift in preference across the age classes. There was no significant difference in the average strength of preference across age classes, (paired t test: t 5 = 1.050, P = 0.342; figure

1.2). In addition, there was also no correlation in the strength of female mating preference across the age classes (Spearman’s rank test: rs = 0.257, N= 6, P = 0.623).

These results suggest that virgin females did not exhibit a shift in preference for symmetry/asymmetry over time.

Experiment 2: Mating experience and preference for symmetry

There was a significant difference in the average strength of preference among the three treatment groups and the replicate tanks at a young age class (Nested

ANOVA: treatment effect: F = 8.61, df = 2, P < 0.002; tank effect: F = 6.03, df = 6, P

<0.001; Fig 1.3a) where the barless treatment was significantly different from the 20 mixed and symmetrical treatments (Tukey’s post hoc: P < 0.05). The barless treatment had a significant preference for asymmetry (mean association time ± se: symmetrical male = 639.4 ± 60.6s, asymmetrical male = 1324.8 ± 60.8s; paired t test: t10 = -4.723, P < 0.001). There was no significant preference for either symmetry or asymmetry in the symmetrical treatment (mean association time ± se: symmetrical male = 1052.7 ± 152.2s, asymmetrical male = 925.5 ± 159.5s; paired t test: t 9 = 0.408,

P = 0.693) or the mixed treatment (mean association time ± se: symmetrical male =

979.3 ± 88.3s, asymmetrical male = 987.5 ± 81.2s; paired t test: t9 = -0.049, P =

0.962). The variation in response to symmetry was not significantly greater across females than within females in either the symmetrical or the mixed treatments

(symmetrical treatment: F9,10 = 1.486, P = 0.258; mixed treatment: F9,10 = 2.643, P =

0.143) suggesting that the lack of preference in these treatment groups was not due to a polymorphism in preference for symmetry.

Due to the significant tank effect in the Nested ANOVA a one-way ANOVA was used to examine the differences among replicate tanks within each treatment.

There was a significant difference among the replicate tanks in the symmetry treatment (One-way ANOVA: F = 15.54, df = 2, P < 0.004; Table 1.1) where replicate tank one was significantly different from both tank two (Tukey’s post hoc: P < 0.02) and three (Tukey’s post hoc: P < 0.01). Females in replicate tank one had a mean preference for asymmetry while the female in the other two replicate tanks had a mean preference for symmetry. There was also a significant difference among the replicate tanks in the barless treatment (one-way ANOVA: F = 4.516, df = 2, P < 0.05; Table 21

1.1) where replicate tank three was significantly different from replicate tank two

(Tukey’s post hoc: P < 0.05). However, females from all replicate tanks have a mean preference of asymmetry, thus the difference is due to a stronger preference for asymmetry in replicate tank three than in two. There was no significant difference among the replicate tanks in the mixed treatment group (one-way ANOVA: F = 0.166, df = 2, P = 0.850; Table 1.1)

There was no significant difference in the average strength of preference among the three treatment groups or the replicate tanks at the older age class (Nested

ANOVA: treatment effect: F = 2.67, df = 2, P = 0.110, tank effect: F = 1.55, df = 5, P

> 0.25; Fig 1.3b). The lack of significance may be due to the small sample size for the barless treatment (N= 4), or that the mean preference for the symmetrical treatment females was positive in the older age class (more time with asymmetric animation).

However, as in the young age class, females in the barless treatment had a significant preference for asymmetry when tested in the old age class (mean association time ± se: symmetrical male = 515.5 ± 65.2s, asymmetrical male = 1395.0 ± 130.7s; paired t test: t3 = -4.499, P < 0.03), whereas there was no significant preference for either symmetry or asymmetry in the symmetrical treatment (mean association time ± se: symmetrical male = 799.3 ± 195.5s, asymmetrical male = 1187.3 ± 193.5s; paired t test: t6 = -0.998, P = 0.357) or the mixed treatment (mean association time ± se: symmetrical male = 1023.4 ± 135.0s, asymmetrical male = 867.7 ± 136.5s; paired t test: t8 = 0.588, P = 0.572). The variation in response to symmetry was not significantly greater across females than within females in either the symmetrical or 22

the mixed treatments (symmetrical treatment: F6,7 = 0.481, P = 0.507; mixed treatment:

F8,9 = 0.006, P = 0.940) suggesting that the lack of preference in these treatment groups was not due to a polymorphism in preference for symmetry.

Twenty of the 31 females were tested at both ages and were used to assess a shift in preference across the age classes. Females in all three treatment groups did not differ in their average strength of preference across the two age classes (mean strength of preference ± se: symmetrical treatment: young = -127.2 ± 331.5s, old =

388.0 ± 388.9s; paired t test: t6 = -1.08, P = 0.322; barless treatment: young = 685.5 ±

145.1s, old = 879.5 ± 195.5s; paired t test: t3 = 0.317, P = 0.772; mixed treatment: young = 8.2 ± 186.9s, old = -155.8 ± 264.7s; paired t test: t8 = 0.259, P = 0.795).

Instead, in the young age class, all treatments combined, I detected a significant negative correlation between female size and the strength of preference (linear

2 regression: r = 0.175, N = 31, F1,29 = 6.169; P< 0.02; figure 1.4). In young age class separating the females by treatment the negative relationship between female size and the strength of preference was also significant for the females in the barless treatment

2 (linear regression: r = 0.513; F1,10 = 9.49; P < 0.02; figure 1.5). There was not a significant relationship between female size and the strength of preference for the symmetrical or the mixed treatments (symmetrical treatment: linear regression: r2 =

2 0.215 ; F1,9 = 2.19; P = 0.177; mixed treatment: linear regression: r = 0.007; F1,9 =

0.056 ; P = 0.819; figure 1.5), however the relationship in the symmetrical treatment had a trend that was similar to the relationship in the barless treatment (figure 1.5). 23

The relationship between female size and the strength of preference was not found in

2 the older size class (linear regression: r = 0.016, N = 20, F1,19 = 0.299; P > 0.6; figure

1.4). A repeated measures ANOVA, in R, was used to examine the effects of SL, age class (young/old), and the interaction between the SL and age class on the strength female preference. The strength of preference for all females from both age classes was used in this analysis. The mixed effect model included individual female number as a random factor to account for the same females occurring in both young and old age classes. Although there were no significant results, several of the factors were suggestive, but not definitive, including the interaction between SL and age class

(Repeated measures ANOVA: t = 1.85, df = 17, P = 0.08), age class and SL (Repeated measures ANOVA: age class: t = -1.74, df = 17, P = 0.10; SL: t= -1.97, df = 17, P =

0.06).

There was a higher rate of mortality in the barless treatment (67%, 7 females) as compared to the symmetrical treatment (27%, 2 females) or the mixed treatment

(10%, 1 females, Fisher’s exact test: P < 0.02; Table 1.1). However, there were no significant correlations between mortality, aggression scores, and mean female strength of preference across replicates (Table 1.1).

DISCUSSION

Female experience with males alters female mating preferences for vertical bar number symmetry in X. malinche. First, while wild-caught adult females had a significant preference for symmetry when small and asymmetry when large (Morris et 24 al. 2006), virgin females in the current study did not exhibit a preference for either symmetry or asymmetry. Virgin females of other poeciliid fishes do exhibit preferences for male traits (Clark et al. 1954; Kodric-Brown & Nicoletto 2001) and therefore, the lack of preference for symmetry in virgin X. malinche females suggests that at least copulatory experience is important for the development of this preference.

Second, females given copulatory experience with males of differing barring patterns exhibited significantly different preferences, suggesting that the barring pattern of males can influence female mating preference for symmetry. However, the patterns of preference detected across treatments did not match a priori predictions of how the barring patterns of the males would influence female preferences, and the females did not appear to switch their preferences as they got older in the direction predicted from the preferences of small and large wild-caught females. I discuss the results in relation to these differences and identify some patterns that would be interesting to study further below.

There were significant differences in female preferences across the replicate tanks within the treatment where females only saw males with symmetrical bars; the females from one replicate preferred asymmetry while the females from the other two replicates exhibit a preference for symmetry. These results suggest that a factor other than bar symmetry influenced female mating preferences. One factor that could have influenced female preference for bar symmetry is male aggression. If there was a relationship between male aggression and vertical bar symmetry, females could use symmetry as a signal to help them avoid aggressive males. In a closely related 25 species, X. cortezi, barless males are known to be more aggressive than barred males

(Moretz 2005). In this study barless males were not statistically more aggressive than symmetrical males, however the barless males were more aggressive than at least two of the symmetrical replicates (Table 1.1). As the X. malinche females housed with barless males exhibited a preference for asymmetry, this could mean that females assess barlessness as a state of symmetry (males have zero bars on the left and zero bars on the right), and used this trait to avoid aggressive “symmetrical” males.

Differences in preference within the symmetry treatment may also be correlated with differences in male aggression. In this treatment, the replicate that exhibited the highest level of male aggression was the replicate in which females had a significant preference for asymmetry, while in the replicates that had lower levels of aggression the females preferred symmetry (Table 1.1). Studies in other taxa have shown that females modify their preferences due to male aggression (Ophir & Galef 2003). For example in Japanese quail, females with mating experience used information attained by eavesdropping to avoid aggressive males, while virgin females exhibit a preference for the more aggressive males given the same information (Ophir and Galef 2004).

Females may learn which males will act aggressively towards them through mating experience and then use male phenotypic traits to avoid aggressive male phenotypes in future mating interactions.

High levels of aggression could be beneficial to males in the context of male- male competition, thus females could gain indirect benefits by mating with aggressive males as they would produce more competitive offspring. However, females that 26 associate with aggressive males could incur costs such as injury and potentially death.

In experiment 2, females in the barless treatment experienced significantly higher mortality rates than females in the other two treatments. If the barless males in X. malinche are more aggressive than barred males, as has been shown for males of the closely related species X. cortezi (Moretz 2005), this could suggest that swordtail females can incur high costs from associating with the barless, more aggressive males.

Females that have more than one reproductive event should be more concerned about their own direct fitness than any indirect benefits gained through their offspring’s fitness to realize maximum lifetime reproductive success (Warner 1998). In X. malinche, females continue to grow after sexual maturity and fecundity increases with female size (Morris et al. 2006). Therefore, smaller females may be even less willing to associate with aggressive males that incur costs to future reproductive potential.

The cost/benefit ratio for associating with aggressive males could change over time in other ways as well, and potentially cause variation in female preference. For example, larger females may be better able to handle more aggressive males, thus reducing the costs of associating with aggressive males for older, larger females.

The difference in preference for bar symmetry detected between small and large wild caught females suggested that females may shift their preference from symmetry to asymmetry as they age (Morris et al. 2006). However, only in the case of females in the symmetry treatment was there any indication that female preference shifted in the predicted direction as females got older and this was only a trend. It is possible that the experience necessary for a shift in preference from symmetry to 27 asymmetry over time was either not present or was too diluted by the experimental design to be detected. It is also possible that the shift in preference was not apparent by looking at the mean female preferences across the two age classes. The estimated size for females to switch their preference was calculated by examining wild-caught female preferences, however when females reach sizes over 37.6mm in the laboratory they are likely to be older than females in the field of the same size. A comparison of growth patterns in the laboratory, semi-natural, and field conditions in a closely related species (X. nigrensis) detected a reduction in growth rate in the laboratory as compared to field conditions (Morris and Ryan 1990), thus this difference in growth rate is also likely to occur in X. malinche. This difference in growth rate between laboratory and field conditions could have shifted the SL at which females would exhibit the shift in preference in the laboratory. To test this hypothesis I determined if there was a relationship between number of days in the laboratory and shift in preference (scored as having a positive strength of preference at one age class and negative in another, not considering the direction in which the female shifted). If I had simply mis-calculated the size at which females switched, I would have expected a positive correlation between number of days in the lab and switching, but this relationship was not significant (Spearman’s rank test: rs = -0.073, N = 20, P = 0.729).

The age and thus size at which males reach sexual maturity in several of the

Xiphophorus species is known to be controlled by different alleles at the Pituitary locus (P) on the Y chromosome (Kallman 1989). While in several species there is no variation in the alleles at the P locus on the X chromosome, all females carrying the s 28 allele, thus females reach sexual maturity at a small size, however there is some indication that females of other species carry different P alleles, thus females reach sexual maturity as different sizes (Kallman 1989). While it is not known if there is more than one P allele on the X chromosome in X. malinche, females in this study reached sexual maturity at sizes ranging from 26.9 mm to 34.5 mm. However, regardless of whether or not the size at sexual maturity was controlled by variation at the P allele in females, the significant relationship between SL and strength of preference in the young age class and the differences in this relationship among the treatment groups at the young age class as well as the suggestive, but not definitive, interaction between age class and SL on the strength of preference suggests that there may be some genetic and environmental component to the female preference function.

The close to significant interaction between the effects of SL and age class on the strength of preference suggests that the relationship between female size and preference is different in the old age class as compared to the young, it appears that the change in a female’s preference over time maybe much more complex than a linear relationship where all females prefer symmetry when young and asymmetry when old.

The complexity of this function may have influenced our inability to detect the switch in preference.

A better understanding of variation in female preference will have significant impacts on our understanding of the evolution of male traits as well our understanding of the evolution of female preference. The theory of reaction norms has been used to explain the evolution of phenotypic plasticity (for review see Via et al. 1995), however 29 this has mainly been applied to morphological and behavioral plasticity in the context of environmental fluctuations (Nylin & Gotthard 1998; Van 'T Land et al. 1999;

Trussell 2000; Van Buskirk & Schmidt 2000; Relyea 2001; Seigel & Ford 2001;

Bouton et al. 2002). When the evolutionary consequences of phenotypic plasticity were applied to sexual selection, however the focus was on plasticity of male traits

(Price 2006). As we learn more about how female preferences vary and what causes this variation, we can apply these theories to the evolution female preference as well. 30

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Figure 1.1 Frequencies of symmetrical and asymmetrical males for 2004 (n = 21), 2005 (n= 16), and males reared in the lab collected as juveniles in 2005(n = 29).

Figure 1.2 Comparison of the association strengths across female age. Negative scores indicate a preference for symmetrical males and positive scores indicate a preference for asymmetrical males. Bars represent mean value (± SE) of the association strengths.

Figure 1.3 Comparison of the association strengths across three treatments groups for when females where a) young and b) old. Negative scores indicate a preference for symmetrical males and positive scores indicate a preference for asymmetrical males. Bars represent mean value (± SE) of the association strengths. * P < 0.01

Figure 1.4 Correlation between SL and preference for both young (A) and old (B) age classes.

Figure 1.5 Correlation between SL and preference for the young age class split by treatment group. * P < 0.02

Table 1.1 Female strength of preference, female mortality and male aggression for each replicate aquaria.

CHAPTER 2

DIFFERENCES BETWEEN SYMMETRICAL AND ASYMMETRICAL MALES; HOW MALE BEHAVIOR COULD BE INFLUENCING FEMALE PREFERNCE FOR SYMMETRY IN THE SWORDTAIL XIPHOPHORUS MALINCHE.

INTRODUCTION

While it has often been argued that sexual selection via female mate choice would reduce variation in male traits (Kokko et al. 2007), this argument assumes that female preferences are consistent both across individuals and time. Recent studies argue that variation in female mate preferences can help maintain variation in male traits (Alonso & Warner 2000; Rios-Cardenas et al. in press). Several mechanisms have been suggested for how experience can alter female mating preferences (Marler et al. 1997; Patricelli et al. 2004; Ophir & Galef 2004; Eakley & Houde 2004; Chapter

1). Experience in multiple contexts can produce variation in preference both across and within females if females do not always respond to the same experience in the same way, or if the experience is different for different females.

Male mating behavior has been shown to play an important role in producing variation in female mating preferences (Marler et al. 1997; Patricelli et al. 2004). For example, differences in male display rates can result in a shift in female mating preferences in Amazon mollies, Poecilia formosa, where females switch their preference between two different heterospecifics to the one with a higher display rate after an interaction period (Marler et al. 1997). Male-male aggression that females 41 observe through eavesdropping (Ophir & Galef 2003) or aggression that males direct toward females (Ophir & Galef 2004) can also induce variation in female mating preference, as not all females exhibit a similar respond to male aggression. Other factors such as female age are correlated with tolerance of male aggression in satin bowerbirds, Ptilonorhynchus violaceus (Patricelli et al. 2004), which leads to females of different ages assessing different male traits (Coleman et al. 2004). In addition, variation in male aggression has been shown to correlate with differences in male phenotype (Barlow 1983; Pryke et al. 2001; Moretz 2005), and therefore these phenotypic traits can be used as signals of aggression level. While the way males use these traits during male-male competition has been studied extensively (for review see

Anderson 1994), females may also use these traits to assess the aggressive nature of potential mates.

Variation among males’ mating preferences could also produce variation in female mating preferences. Although females are thought of as the choosier sex

(Darwin 1871) this isn’t to say that males lack mating preferences. Males in a wide variety of taxa exhibit mating preferences for female traits such as size, which has been attributed to the increased fecundity (for review see Andersson 1994). Studies in livebearing fishes (family: Poeciliidae) have shown that male mating preference for larger females (Houde 1997; Ptacek & Travis 1997; Basolo 2004; Herdman et al.

2004) was attributed to the increased fecundity of larger females (Abrahams 1993;

Reznick et al. 1993; Herdman et al. 2004). However, male mating preference for female size varies with male size in Brachyrhaphis rhabdophora, where small males 42 prefer small females and large males prefer large females (Basolo 2004). Variation in male mating preference could give females of different sizes different experiences in that males of a particular phenotype may spend more time associating with females of a certain size. Therefore, variation in male mating preferences could alter female preference for male phenotypes, if the male phenotypes are correlated with male mating preferences.

In a previous study, Xiphophorus malinche females exhibited a correlation between preference for vertical bar number symmetry and size where small/young females preferred symmetrical males while large/older females preferred asymmetrical males (Morris et al. 2006). Females in this species continue to grow after sexual maturity (Kallman 1989; Marcus & McCune 1999; Morris et al. 2006), and therefore the correlation between female size and preference suggests females may change their mating preferences over their lifetime. In addition, mating experience influenced female preference for symmetry in this species (Chapter 1); female mating preference was conditional on mating experience with vertical bar number symmetry, female preference for bar symmetry varied depending on whether females were virgins, housed with symmetrical males, males without bars or both symmetrical and asymmetrical males.

In the current study, I consider aspects of male behavior that could influence differences in preference for vertical bar symmetry between large and small

X. malinche females. I investigated the possibility that variation in male aggression and male mating preference are correlated with differences in vertical bar number 43 symmetry, and therefore could influence variation in female mating preference for bar number symmetry. For example, if smaller females are less tolerant of aggressive male behaviors, then I predicted that symmetrical males would be less aggressive than asymmetrical males. In addition, if females are more likely to prefer males that prefer them, then I predicted that symmetrical males would be more likely to prefer small females while asymmetrical males would prefer larger females. I used mirror-image stimuli to examine whether male aggressive behavior varied with bar symmetry. I also examined whether male mating preferences for female size varied with vertical bar symmetry.

METHODS

Experiment 1: Male Aggression and Response to Symmetry

Xiphophorus malinche males and juveniles were collected from the Rio Claro in the state of Hidalgo, Mexico in 2005. Juveniles were reared in single sex aquaria allowing them to experience both symmetrical and asymmetrical males. Sexually mature males were individually housed in 18.9 l tanks and visually isolated from one another. All individuals were fed daily and maintained on a 12h light/ 12h dark photoperiod at a constant room temperature of 22C. All males were isolated for at least two weeks before testing to reduce the influence of prior experience.

Vertical bar symmetry was manipulated using a freeze branding technique

(Raleigh et al. 1973). This technique has been used in other Xiphophorus species and 44 does not alter male behavior or background coloration (Morris et al. 1995; Moretz &

Morris 2003). Technique allowed us to expose all males to their own symmetrical and asymmetrical image so that any differential response to the barring pattern would not confound measures of overall aggression, and so that the response to bar symmetry could be examined for both types of males. Symmetrical males were first branded at the caudal end past the last bar to control for branding and tested with their symmetrical image as stimuli. Then symmetrical males had two bars removed from one side, and were tested with their asymmetrical image as stimuli. Asymmetrical males were first branded to make all males asymmetrical by 2 bars and tested with their asymmetrical image as stimuli. This branding made the degree to which males were asymmetrical consistent across all males, as naturally asymmetrical males can be asymmetrical by up to at least 3 bars (personal observation). Then asymmetrical males were branded to remove two bars to make bar number the same on both sides, and tested with their symmetrical image as stimuli.

Standard mirror image stimulation (MIS) tests were used to assess the overall aggressive responses of symmetrical and asymmetrical males as well as their relative responses towards symmetrical and asymmetrical barring patterns. The testing procedure uses a mirror attached to the end of the individual’s 19 l aquarium, followed by a five-minute observation. Interaction time was recorded, and was defined as the time the male spent within 10 cm of the mirror. Bites directed towards the mirror image, chasing the mirror image and lateral displays were also recorded. 45

Although MIS has been used for many years, the validity of this procedure to accurately predict contest outcome and group hierarchies has been questioned

(Ruzzante 1992; but see Holtby 1992). Here I was less interested in contest outcome per se, but more interested in the signal since the signal may contain information about male aggression that could be used by females during mate choice decisions (Swaddle

1999). The actual winner and loser of a contest are thus less important than the information a female may gain by eavesdropping and/or associating male phenotypes with aggression. The benefits associated with MIS are that they provide balanced contests and instantaneous feed back, whereas using live fish or dummies as stimuli may have added confounding factors associated with contest dynamics and perceptions of asymmetry (Rowland 1999). In addition, MIS tests using the same manipulation methods are repeatable across treatments (Moretz and Morris 2003), providing a good measure of intrinsic aggression levels that can then be compared across individuals (Earley et al. 2000).

Experiment 2: Male Preference for Female Size

Male and female X. malinche were collected from the Rio Claro in the state of

Hidalgo, Mexico in 2005 and 2007. All individuals were fed daily and maintained on a 12h light/ 12h dark photoperiod at a constant room temperature of 22C. All individuals were measured for standard length (SL), which is the distance from the tip of the snout to the base of the caudal peduncle. Males were also scored for the 46 number of vertical bars on each side, where symmetry indicates equal bar number on both the left and right sides and asymmetry indicates a difference of one bar or greater between the left and right sides. A total of 9 female pairs were used, large females ranged from 43.7 mm to 56.9 mm SL (mean ± SE = 48.8 ± 1.51 mm), and small females ranged from 33.1 mm to 38.8 mm SL (mean ± SE = 36.1 ± 0.62 mm).

Prior studies of male mating preferences in livebearing fish using visual dichotomous choice tests did not detect male mate preference for females based on size (guppy: Herdman et al. 2004; swordtails: Brooks and Morris, unpublished data).

Therefore, I used a two part choice test to determine if I could detect male mate preference if males were allowed to assess more cues that just visual cues, and to determine the best method for examining male mate preference in these fishes. In the first test, I allowed a male to freely interact with a large and small female recording the behavior of all three individuals. Immediately following the interaction test, I tested each male’s preference to associate with the same two females in a dichotomous choice test. Thus, all males were tested for preferences for female size in two tests; an interaction test, where males could directly assess olfactory cues as well as visual cues, and a dichotomous choice test where the potentially confounding influence of female-female competition was removed.

Both preference tests were conducted in a 208 l aquarium divided into five equal sections. The two end sections were divided by removable Plexiglass sheets with small holes to allow transmission of visual and olfactory cues, while the inner three sections were visually divided by lines drawn on the outside of the aquarium. 47

Females were placed into the end sections while the male was placed into a clear

Plexiglass tube in the center section of the observation aquarium and allowed to acclimate for 10 minutes. After the acclimation period all individuals were released into the observation tank. Interactions between the females and the male were recorded for 12 minutes. I recorded the following agonistic and courtship behaviors by all three individuals, and when appropriate, to whom they directed these behaviors: chase, display, approach, expression of vertical bars, copulations, and bites. I also recorded the time that the male spent within one body length of each of female.

After the interaction period, each female was re-confined to the end sections of the aquarium with a Plexiglass divider with holes. The male was placed back into a clear Plexiglass tube in the center section of the tank for a 10-minute acclimation period. The male was then released allowing him to swim freely among the three inner sections. The time the male spent in the two sections adjacent to the sections containing the females was recorded for 10 minutes. The females were then placed on the opposite ends of the aquarium and the test was repeated in order to control for any side biases the male might exhibit.

RESULTS

Experiment 1: Male Aggression and Response to Symmetry

There was no significant difference between symmetrical and asymmetrical males in their overall levels of aggression, measured as the total number of bites 48 towards their symmetrical and asymmetrical mirror images (mean number of bites ±

SE: symmetrical males: 20.5 ± 6.3 bites, asymmetrical males: 27.5 ± 5.6 bites; Mann-

Whitney U: U = 59.5, P = 0.397). However, symmetrical and asymmetrical males differed significantly in the aggression they directed at their symmetrical image.

Asymmetrical males were more aggressive to their symmetrical image (mean number of bites ± SE: 16.2 ± 3.8) than symmetrical males (mean number of bites ± SE: 4.2 ±

2.4; Mann-Whitney U: U = 34.5, P < 0.03). There was no difference in the aggression symmetrical and asymmetrical males directed to their asymmetrical image (mean number of bites ± SE: towards asymmetrical image: symmetrical males = 16.3 ± 5.6 bites, asymmetrical males = 11.3 ± 2.8 bites; Mann-Whitney U: U = 63, P = 0.531, figure 2.1).

To determine if males had a response to symmetry, we compared the mean responses to symmetrical and asymmetrical image. When the two types of males were combined, males did not bite more at their symmetrical as compared to asymmetrical image (mean number of bites ± SE: towards symmetrical image = 11.4 ± 2.7 bites, towards asymmetrical image = 13.3 ± 2.8 bites; Wilcoxon signed-ranks test: Z = -

0.325, N = 25, P = 0.745). However, symmetrical males did respond to their symmetrical image with fewer bites than their asymmetrical image (Wilcoxon signed- ranks test: Z = -1.960, N = 10, P = 0.05; figure 2.1). There was no difference in the response of asymmetrical males (Wilcoxon signed-ranks test: Z = -1.225, N = 15, P =

0.220; figure 2.1).

Experiment 2: Male Preference for Female Size

Interaction test:

The strength of preference for the large female was calculated by subtracting the time the male spent within one body length of the small female from the time that the male spent within one body length of the large female. Thus positive scores indicate that the male spent more time with the large female and negative scores indicate that the male spent more time with the small female. The strength of preference for the large female did not differ significantly across years (mean strength

± SE: 2005 = 112.3 ± 32.7s, 2007 = 129.7 ± 27.8s; t test: t19 = -0.381, P = 0.71) so the data from both years was combined for the following analyses.

Both symmetrical and asymmetrical males spent significantly more time within one body length of the large female than the small female (Symmetrical males: paired t test: t7 = -5.161, P < 0.002; Asymmetrical males: paired t test: t12 = -3.921, P <

0.003; figure 2.2). However, symmetrical males had a significantly stronger preference for large females (mean strength ± SE: symmetrical males: 177.1 ± 34.3s; asymmetrical males: 91.2 ± 23.3s; paired t test: strength of association with small verses large females: t19 = 2.151, N = 21 males, two-tailed P < 0.05). There was also a significant correlation between strength of association and male size, where the strength of association for large females increased with male size (linear regression: r2

= 0.37, N = 18, F1,16 = 9.308; P< 0.01; figure 2.3). The mean size of symmetrical males is close to being significantly larger than the asymmetrical males (mean SL ±

SE: symmetrical males = 46.1 ± 1.5 mm, asymmetrical males = 41.9 ± 1.5 mm; two- 50

tailed t test: t16 = 1.908, P = 0.075). When male size is controlled for there is no significant difference between symmetrical and asymmetrical males preference for female size (Partial Correlation: correlation coefficient = 0.293, df = 15, P = 0.254).

The number of male displays, male approaches, and copulations towards the large female were not significantly different across years (Mann-Whitney U: male displays: U = 42.5, P = 0.621; male approaches: U = 35.5, P = 0.257; copulations: U =

41.5, P = 0.585), so the data from both years was combined for the following analyses.

Both the number of male displays and male approaches were greater toward the large females as compared to the small females (Wilcoxon signed-ranks test: male display:

Z = -2.58, N = 21 males, P < 0.01; male approaches: Z = -2.18, N = 21 males, P =

0.029; fig. 2.4). The number of copulations was not significantly greater towards the larger female, although there was a trend in this direction (Wilcoxon signed-ranks test:

Z = -1.389, N = 21 males, P = 0.165; figure 2.4).

Behaviors indicating female-female aggression (i.e. bites towards other female and display towards other female) were rare, occurring in only one test out of 21, so no analyses were conducted for these behaviors.

Standard Dichotomous Choice test:

Strength of preference for the large female in the dichotomous choice tests was calculated in the same manner as the strength of preference for the interaction test

(subtracting time with small female from the time spent with the large female).

Strength of preference differed significantly across years (mean strength of preference

± SE: 2005 = -464.4 ± 239.0s, 2007 = 263.8 ± 110.8s; two-tailed t test: t19 = -3.186, P 51

= 0.005). The number of days that the males were isolated also differed significantly across years (mean # isolation days ± SE: 2005 = 43.2 ± 7.7 days, 2007 = 22.1 ± 2.0 days; two-tailed t-test: t18 = 3.67, P < 0.003). Differences in the number of days isolated may explain differences in strength of preference across years as there was a significant positive correlation between male strength of preference for female size

2 and the length of the isolation period (linear regression: r = 0.37, N= 20, F1,18 = 10.54;

P < 0.005; figure 2.5). Because of the differences in isolation times, we analyzed the data comparing symmetrical and asymmetrical males preferences from the choice tests for each year separately.

For males collected in 2007, there was no difference between symmetrical and asymmetrical males in their strength of preference for the larger female (two-tailed t test: strength of association with small verses large females: t9.14 = 0.245, N = 21 males, P = 0.812). Combining symmetrical and asymmetrical males, males collected in 2007 spent significantly more time associating with the larger female than the smaller female (mean association time ± SE: smaller female = 344.4 ± 49.5s, larger female = 608.21 ± 65.26s; paired t test: t13 = -2.380, P < 0.04). For males that were collected in 2005 there was only one symmetrical male so no comparison between symmetrical and asymmetrical males was calculated. Combining the symmetrical and asymmetrical males, the males collected in 2005 spent more time associating with the smaller female as compared to the larger female, however this difference was not statistically significant (mean association time ± SE: small female = 727.4 ± 116.8s, large female = 263.0 ± 133.6s; paired t test: t6 = 1.944, P = 0.100). Males isolated for 52

30 days or less (cut off determined from the regression for the relationship of strength of male preference and isolation period and where the line crosses zero on the X-axis) exhibited a significant preference for large female size (mean association time ± SE: small female = 328.2 ± 54.0s, large female = 647.0 ± 74.3s; paired t test: t12 = -2.577,

P < 0.03), which corresponds to the preferences found with the other two indicators of male preference measured during the interaction test. Males isolated longer than 30 days had a close to significant preference for the smaller female in this second test

(mean association time ± SE: small female = 699.6 ± 113.3s, large female = 220.7 ±

95.7s; paired t test: t6 = -2.394, P = 0.054). Combing males from both years there is no significant difference between symmetrical and asymmetrical males in preference for female size when controlling for days isolated (Partial correlation: correlation coefficient = -0.131, df = 17, P = 0.593).

DISCUSSION

I predicted that symmetrical males would be less aggressive than asymmetrical males if male aggression could help explain the differences between large and small females in preference for bar number symmetry. I did not find a difference between symmetrical and asymmetrical males in overall aggression. However, the two types of males did differ in their response to their symmetrical image as compared to their asymmetrical image, as symmetrical males were less aggressive to their symmetrical image than asymmetrical males. The situation in which females could observe reduced aggression by symmetrical males however, should be rather uncommon as the 53 frequency of symmetrical males is much lower than asymmetrical males (25% symmetrical; Morris et al. 2006; Chapter 1). One might consider on the other hand, that in addition to the reduced aggression between two symmetrical males, the higher frequency of asymmetrical males would mean that females would be more likely to see asymmetrical males fighting than symmetrical males. Females may perceive asymmetrical males as more aggressive causing smaller females to avoid asymmetrical males.

During male-male interactions males often use phenotypic traits to relay information about certain behavioral aspects such as male aggression (Barlow 1983;

Pryke et al. 2001; Moretz 2005). Symmetry can be an effective signal in both inter- and intrasexual communication (for review see Swaddle 1999). The differential response to symmetry/asymmetry exhibited by symmetrical males suggests that at least symmetrical males use symmetry as a signal in male-male interactions and this trait could be a signal of some component of male aggression. Recent studies examining the correlation of behaviors across contexts (i.e. behavioral syndromes, for review see Sih et al. 2004), suggest that the aggressive behavior of a male in male- male interactions could spill over into other contexts such as mating. Therefore, females could eavesdrop on male-male interactions, using the same traits males use to assess an aggressive competitor to assess the aggressiveness of potential mates.

Swordtail females use information gained through eavesdropping on male-male interactions to make future mating decisions (personal communication Dubois).

Females exhibiting a lower tolerance for male aggression could then use information 54 gained through eavesdropping to avoid more aggressive mates. My results suggest that symmetry could be used by both males and females in some situations, to assess the aggressiveness of conspecifics.

Both symmetrical and asymmetrical males had a preference for large females when allowed to interact directly with two females. Population genetic models have shown that male preferences can spread in a population when males prefer female traits that are indicators of fitness, such as high fertility (Servedio & Lande 2006).

Large female size is correlated with increased fecundity in several Poeciliid fishes

(Kallman & Borkoski 1978; Thibault & Schultz 1978; Reznick 1993). Since both symmetrical and asymmetrical males exhibit a preference for large female size, male preference is not likely to be a factor that influences the shift in female preference exhibited by X. malinche.

While bar number symmetry did not influence male mating preference, male size did. The strength of preference for female size was correlated with male size such that larger males had a stronger preference for large females. These results are similar to those reported by Basolo (2004) for B. rhabdophora, in that there was a relationship between male size and strength of preference for female size. Larger males would benefit from mating with large more fecund females, while smaller males may not have the fighting ability to easily gain access to larger females. Smaller males with no preference for female size could have an advantage over small males exhibiting preference for large female size if the latter rarely gained access to these females due to male-male competition (Basolo 2004). 55

Future studies examining male mating preferences should consider several factors in their experimental design. First, allowing males to interact with females may be necessary to elicit male mate preferences. This may indicate that males use olfactory cues to assess females rather than body size per se. This type of test can only be used, however, if females do not exhibit female-female aggression. The lack of female aggressive behaviors in these tests indicated that female-female aggression is not likely to interfere with a female’s ability to gain access to the male or male preference in these fish. However, a standard dichotomous choice test could be used after an interaction period to examine male preferences in those cases where female- female aggression does exist. The other factor that influenced male preference for larger females was the time males were isolated. While all males appeared to prefer the larger female in the interaction test, males isolated for more than 30 days were more likely to spend time with the smaller female in the dichotomous choice test than those that had been isolated for less than 30 days. Switching their preference from larger to smaller females in the second test could indicate that the males that had been isolated longer were maximizing the number of females they mated with rather than continuing to try to mate with the larger female.

In summary, I suggest that differences in the aggressive behavior and frequency of symmetrical and asymmetrical males could play a role in producing the size differences in female mating preferences for symmetry in the swordtail

X. malinche. Future studies should determine if females of different sizes respond differently to male-male aggression, and if females use the same signals used by males 56 in intrasexual interactions to assess aggression levels of potential mates. Male mate preference, on the other hand, is not likely to produce the variation in female mating preferences as both symmetrical and asymmetrical males preferred larger females.

Detecting male mating preferences in swordtails may require that males can assess chemical cues, which leads to the questions of how and why large and small females might differ in this respect. 57

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Figure 2.1: Mean number of bites by symmetrical and asymmetrical males during a five-minute MIS trial. * P < 0.05

Figure 2.2: Comparison of the time spent with small vs. large females for both symmetrical and asymmetrical males. Bars represent mean value (± SE) of the association time. * P < 0.002.

Figure 2.3: The relationship between male size and strength of preference (difference in time spent with small and large females) during the interaction test. Negative scores indicate a preference for small females and positive scores indicate a preference for large females.

Figure 2.4: Comparison of the male behaviors directed towards small verse large females during the interaction test. Bars represent mean value (± SE) of the male behaviors. *P < 0.05.

Figure 2.5: The relationship between male isolation period and strength of male preference for female size during the choice tests. Negative scores indicate a preference for small females positive scores indicate a preference for large females.