Variation in Female Mate Preference for a Male Trait That Provides

Variation in female mate preference for a male trait that provides

information about growth rate in the swordtail Xiphophorus multilineatus

A thesis presented to

the Honors Tutorial College

Ohio University

In partial fulfillment

of the requirements for graduation

from the Honors Tutorial College

with the degree of

Bachelor of Science in Biological Sciences

Nicole L. Kleinas

August 2015

1 This thesis has been approved by

The Honors Tutorial College and the Department of Biological Sciences

______

Dr. Molly Morris

Professor, Department of Biological Sciences

Thesis Advisor

______

Dr. Soichi Tanda

Director of Studies, Honors Tutorial College

Biological Sciences

______

Dr. Jeremy Webster

Dean, Honors Tutorial College

2 Abstract

Sexual selection is comprised of intersexual mate choice and intrasexual competition. In order for female choice to be adaptive, the benefits of being choosey need to outweigh the costs. Females could benefit from preferences for male traits that relay information about male quality and/or that increase offspring fitness. Female preferences are affected by genotype, environment, or a combination of the two. In the study species, Xiphophorus multilineatus, males belong to one of four genetic size classes, and one of two genetic reproductive tactics. Between these alternative reproductive tactics, growth rate may be under disruptive selection. Since growth rate relates to fitness, it is possible that females assess a potential mate’s growth rate by evaluating variation in male vertical body bars. I identified two aspects of the vertical body bars that are correlated with male juvenile growth rate. In addition, I demonstrated that females from a population of exclusively sneaker males show a preference for the barring pattern that represents a slower growth rate, which supports the proposed tactical disruptive selection on growth rate. Females from the sneaker line were also choosier in their preferences, which could potentially indicate that the fitness advantage to growing slower as a sneaker male may be greater than the fitness advantage to growing faster as a courter male.

Table of Contents

Introduction

Sexual Selection: An Overview…………………………………………5

Xiphophorus as a Model System……………………………….……….5

Sexual Selection Theories……………………………………….………7

Male Signals and Female Choice…………………………………….….9

Growth Rate and Morphology………………………...………….….….10

Context Influences Female Preference…………………………...……..14

Materials & Methods

Breeding of males for assessing vertical bars…………………….……..18

Measurement of morphology……………………………………….…...18

Statistical analysis of growth rate and bar morphology…………………19

Breeding of females for preference tests…………………………...…...21

Creation of male animation for preference testing………………...……22

Female preference testing……………………………………….………26

Statistical analysis of female preference tests…………………………..28

Results

Male morphology and growth rate………………………………….…..30

Female preference tests………………………………………….…..….32

Discussion…………………………………………………………………...….38

Acknowledgements…………………………………………………………….44

References………………………………………………………………..….....45

4 Sexual Selection: An Overview

Darwin’s theory of natural selection was a turning point in the study of the natural sciences. Evolution by natural selection is driven by heritable traits that increase fitness, the ability of an individual to pass their genes onto the following generation. Fitness depends not only on the ability of an individual to survive, but upon the ability of an individual to mate, a system that is addressed by Darwin’s theory of sexual selection. Sexual selection consists of both intersexual selection (mate choice) and intrasexual selection (competition), typically by females and males, respectively (Darwin 1871). The details of this component of natural selection have been studied extensively, in particular, the morphological and behavioral cues used by each sex in assessing conspecifics in contests and as potential mates.

Xiphophorus as a Model System

The swordtail genus, Xiphophorus, is a system that has been studied in relation to sexual selection as early as Darwin’s study of the caudal fin extension (i.e. sword) in Xiphophorus helleri (Darwin 1871). Xiphophorus multilineatus was the species used in my study, but several other species have been studied in relation to sexual selection (X. nigrensis, Ryan et al. 1990; X. helleri, Basolo 1995; X. pygmaeus,

Hankinson & Morris 2003; X. cortezi, Morris et al. 2011).

Males of X. multilineatus belong to one of four genetically influenced (Y- linked gene) size classes: Y-L, Y-II, Y-I (courter males) and Y-s (sneaker males). The courter males comprise the three largest size classes and use courtship behaviors

5 exclusively, while Y-s males are smallest in size and perform both courting and sneak- chase mating behavior depending on social context. Courting behaviors are characterized by displays in which males erect their dorsal fin and darken their vertical body bars, while swimming back-and-forth in front of a female. Sneak-chase mating involves the male swimming rapidly after the female, and force copulating with the female, despite her preference against mating with the male. X. multilineatus females are known to prefer larger courter males (Morris et al. 2010) and males with more vertical bars (Morris et al. 2007). In Xiphophorus males, growth slows to a halt following sexual maturity, so the size of a male at sexual maturity is the size they will remain as an adult (Kallman 1989). Despite a disadvantage in the realm of female preference and male-male competition, the sneaker phenotype is thought to be maintained by an increased likelihood of ever reaching sexual maturity, as smaller sneaker males mature sooner than larger courter males (Bono et al. 2011). Although courter males reach sexual maturity later (and in doing so reduce their chances of reaching it at all), they do exhibit faster growth rates than sneaker males of the same age (Bono et al. 2011). However, recent data suggests that there may be a longevity trade-off with faster growth rate in courter males (Morris et al., in review, Figure 1).

Figure 1. Life histories of sneaker males (top) and courter males (bottom). The height of the bars denotes magnitude. Courters have faster feeding rates and faster growth rates, but reach sexual maturity later and die sooner. Courters also have increased mating success, but a shorter total reproductive lifespan. (Morris et al., in review)

Sexual Selection Theories

Intersexual mate choice can select for exaggerated traits, most of which are secondary sexual characteristics. Fisher (1930) put forth the idea that, if unchecked by other forces of natural selection, both the trait under selection by preference of the opposite sex and the preference itself will continue to become more exaggerated at an ever-increasing rate (termed a “runaway” process). When the reproductive advantage of the trait no longer outweighs the survival disadvantage, the “runaway” process will come to equilibrium.

Fisher’s theory of sexual selection through mate preference suggests that preferred traits become exaggerated due to an initial female sensory bias or preference for novelty and perhaps a slight genetic advantage, but after the trait falls victim to the

“runaway” process, the perceived benefit of mating with a male with a superior trait does not extend beyond producing offspring that will also be preferred by the females of their generation. In Xiphophorus, the phylogenetic evidence suggests that the

7 preference for the characteristic sword existed prior to the evolution of the trait itself, demonstrating a pre-existing sensory bias for large apparent male size (Basolo 1990;

Basolo 1995; Rosenthal & Evans 1998), implying that the exaggerated trait is preferred purely due to its attractiveness to females.

Alternatively, Zahavi (1975) described the “handicap principle,” in which the presence of a preferred trait indicates male genetic quality. He theorized that males of lower quality are unable to allot excess energy to the development of said traits. This hypothesis argues that females are selecting their mate not for the trait itself, but for the genotype of which the trait is indicative. Zahavi argues that the preference would be evolutionarily disadvantageous if the trait does not correlate directly with some aspect of the male’s genetic quality, indicated by the ability of the individual to allocate excess energy to develop the trait in question. He stated that the survival disadvantage of the exaggerated trait serves as a “test” that can only be passed by individuals of adequate quality, excluding individuals from the population which may have a high-quality trait but a low-quality genotype.

Genetic quality could also pertain to the growth strategy of an individual. For example, asymmetry (see “Growth Rate and Morphology”), could indicate that an individual optimizes growth over developmental stability (quality of development of physical characteristics in the face of environmental stress, Morris et al. 2012). It is important to note that genetic quality is contextual; some genetic traits may be favored in one context while others are favored in an alternative context (see “Context

Influences Female Preference”).

8 Male Signals and Female Choice

As outlined by van Doorn and Weissing (2004), a male courtship display may fall into one or more general classes: (1) obsolete signals, in which female preference for a trait has not been conserved, but the trait has been, due to it not incurring any significant cost to the males, (2) multiple receivers, in which the sexually selected traits exist in part for female preference and in part for male dyadic encounters, (3) unreliable signals, where the selected traits do not indicate male quality but are selected for by a runaway process, as described by Fisherian selection, (4) redundant signals, where multiple traits are all selected by female preference which has been shaped by the handicap principle, but that all indicate the same aspect of overall male condition, and (5) multiple messages, in which the multiple traits that are under selection pressure by females all reflect a different component of male quality.

For a female to evaluate two traits that indicate the same aspect of male quality, the cumulative benefit to fitness should outweigh the costs of demonstrating multiple preferences (Pomiankowski 1987). Even in systems in which different traits represent different properties of male condition, the benefits of selecting a mate by those parameters must outweigh the costs of evaluation. For example, swordtails use multiple cues when evaluating potential mates, such as body size and vertical body bar number and symmetry. In X. pygmaeus, females require information from chemical cues, vertical body bars and size in order to simply mate with the correct species

(Hankinson & Morris 2003). In this example, hybridization is not optimal, so the cost of evaluating multiple cues is outweighed by the benefit of mating with a conspecific.

9 The “multiple receiver hypothesis” can be tested in systems by evaluating both female preference for the trait and male behavioral responses to the trait in dyadic encounters. For example, the characteristic vertical body bars of X. multilineatus are evaluated by females in the context of mate selection and evaluated by males in the context of initiating contests (Morris et al. 1995). However, vertical body bars evolved in the context of female mate choice prior to the signal being co-opted by males in the context of male-male competition (Morris et al. 2007). Most sexually selected traits are assessed by both males and females (Andersson 1994), and therefore to determine if a trait is assessed for mate preference, it is important to examine preference in an environment where you can control for male-male competition.

Growth Rate and Morphology

A trait that is very important in relation fitness in swordtail fishes is growth rate. Growth rate varies across male genotypes, and as found in many other systems

(Arendt 1997) is influenced by both environment and genes (heritable). Therefore, it would make sense that females would be interested in assessing growth rate of potential mates. In many systems, body size would provide some information about growth rate when assessing males of the same age. In swordtails, body size does provide information about growth rate (larger males grow faster), however there is still variation in growth rate that body size alone does not reveal (Morris et al. 2012).

Symmetry in bar number (same number of bars on each side of the body) has recently become a trait of interest with reference to its representation of mate quality, and could

10 provide information about growth rate. The vertical bars of X. multilineatus can be laterally asymmetric in bar number, as demonstrated in Figure 2. The left side of the male (Figure 2, A) has ten vertical body bars while the right side (Figure 2, B) has eleven. Organisms seldom grow at a rate equal to their physiological maximum because it can be more advantageous to invest energy into the quality of growth rather than the quantity (Arendt 1997; Sibly & Calow 1984). There is often a trade-off between growth rate and developmental quality; often an individual growing at a fast rate will sacrifice some other characteristic, such as immunity (van der Most et al.

2011) or symmetry (Robinson & Wardrop 2002; Morris et al. 2012).

Interestingly, while X. multilineatus females have been shown to prefer symmetrical vertical bar patterns (Morris et al. 2012), variation in female mate preference for symmetry based on female size has been detected in two other species of swordtail fishes (X. malinche and X. cortezi, Morris et al. 2006). The variation in female mate preference for bar symmetry (larger females prefer asymmetric males) is a good example of how female mate preferences can be contextual (Jennions & Petrie

1997).

Figure 2. Photographs of a single asymmetric Xiphophorus multilineatus male. (A) The standard length (SL; red), body depth (blue) and the vertical body bar count (numbers) are shown (B) the body bar count is shown on the opposing side.

In X. multilineatus, symmetry of the vertical bar pigment pattern is under selection by potential mates and competitors (Morris et al. 2012). When faced with environmental stress during development, males may follow one of two growth strategies: they may develop slowly and symmetrically, or they may develop quickly

12 and asymmetrically. When individuals are raised in three intensifying levels of environmental stress, induced by food scarcity, their growth strategy is made evident.

With high resources, most individuals will develop symmetrically, as they have excess energy to allocate to developmental stability. Conversely, an environment with the highest level of stress (lowest food availability) will result in most individuals being asymmetric, as all of their energy will be invested in simple growth processes. It is the intermediate level of environmental stress that allows for a “choice” in growth strategy. (Figure 3)

Morris et al. (2012) demonstrated that X. multilineatus Y-II males sacrificed symmetry for increased growth rate, identifying an alternative growth tactic. In the same study, females showed a significant preference for symmetry in dichotomous choice tests when size was not varied, indicating a preference for a growth strategy in which symmetry is optimized over growth. Interestingly, males with unequal bar numbers are not significantly asymmetrical in the absolute amount of melanin

(Kleinas, unpublished data), bringing into question the mechanics of the development of the bars themselves.

Figure 3. A graphical depiction of fluctuating asymmetry in the face of environmental stress in two genotypes with opposing growth strategies, the first of which optimizes growth over symmetry (a), while the second optimizes symmetry over growth (b) (Morris et al. 2012).

Context Influences Female Preference

Female preferences can, in fact, be context-dependent. In order for female preferences to have evolved, they must have some heritable component, but female preference also varies with female condition (Cotton et al. 2006). For example, female preference for bar symmetry in X. cortezi and X. melinche is size-dependent, with larger females preferring asymmetric males (Morris et al. 2006). Possible explanations

14 for this variation in preference were evaluated in lab-raised X. multilineatus. Lyons et al. (2014) demonstrated that the strength of female preference for symmetry varied with female’s embryonic environment (brood size) and post-embryonic diet, which may indicate context-specificity based on either female condition or the unreliability of male traits in certain environments, as variation in the reliability of traits could lead to the existence of multiple, conflicting preferences. The same study also demonstrated that females raised on a low-quality diet had a stronger preference for larger male body size. These results suggest that females’ environment at the time of preference may influence which male growth strategy is ideal—optimizing growth in environments with low resource availability.

The context-dependence of this preference may indicate that females are actively choosing which male growth strategy (optimizing growth over development or development over growth) is most appropriate for the current environment. There is a mortality trade-off with growth rate in which the individuals growing most quickly are more likely to have decreased longevity (Morris et al., in review). Therefore, information gleaned by females from male sexual characteristics regarding growth rates could be pertinent to their mate selection if the resulting offspring will obtain a significant fitness advantage from either faster or slower growth. Female condition and life-history traits alter their mate preference, indicating that they may be using information about their environment to predict the most advantageous phenotype for their offspring, since the cost of choosiness must be outweighed by the fitness benefits

(the survival and successful reproduction of as many possible offspring).

15 Females may be able to assess male growth strategy by evaluating one or more traits that are correlated with growth rate and/or development. In X. multilineatus, the characteristic vertical bar pigment pattern has the potential to provide females with information regarding the growth rate of a potential mate. In addition to bar symmetry, which is hypothesized to represent a tradeoff between growth and developmental stability (Morris et al. 2012), other aspects of the vertical body bars may play a role in relaying information about male growth rate, such as the spacing between the bars or the span of the barred section of the body. Determining how male sexual characteristics vary with growth rate and how females interpret those changes would lend insight into the extent of selective pressures and evolutionary trade-offs within the species. Due to the extensive data collected on swordtails, we are able to delve deeper into the complex characteristics of their mating system, which serves as a platform for testing various sexual selection theories.

The purpose of this study was to (1) determine whether the spacing of vertical body bars (bar sprawl) or the amount of melanin within the bars in relation to body size (proportion melanin) provide information about a male’s growth rate and (2) whether females exhibit a preference for bar sprawl. Due to the context-specificity of female mate preference, predictions for the direction of preference for this trait are difficult. However, I hypothesized that females would prefer a spacing pattern that represents a faster male growth rate. This hypothesis was based on the results of a previous study, in which wild-caught females invested more energy into offspring after mating with a courter male, resulting in offspring reaching sexual maturity more-

16 quickly (Rios-Cardenas et al. 2013). Since this indicated that females’ investment in their offspring led to accelerated growth (at least when they developed and mated in the wild), it would be expected that they would also opt for the paternal phenotype that correlates with accelerated growth in the laboratory.

An alternative hypothesis, however, is based on recent work suggesting that there may be selection for sneaker males to grow slower than courter males (Morris et al., in review). Therefore, I also examined the possibility that the females from the two different genotypic lines (sneaker and courter) may have different preferences. Given that females from the sneaker line would have had a sneaker father, and would have grown up interacting with only sneaker males, these females may have a preference for males that grow slower than the male preferred by the females from the courter line.

17 Materials & Methods

Breeding of males for assessing vertical bars

The mothers of the males in this study were raised on high- or low-quality food, with 46% and 20% protein respectively (Murphy et al. 2014). Selected females were mated with courter males from the largest size class (Y-L) and allowed to drop fry. The fry from the second brood of the mothers were isolated after 15 days of age.

They were placed in individual 5-gallon aquaria with opaque dividers between tanks to prevent any visual social interactions. Fry were all raised on low-quality food (20% protein). Growth rate data about the males was measured in the aforementioned study and was made available for my use. Growth rate was measured as change in standard length (mm) per day.

Measurement of morphology

Males were photographed after sexual maturity using a Canon® Powershot SD

1200IS. The individuals were held flat against the glass of their aquarium, next to a ruler, and were photographed on each side. Using ImageJ (Rasband, W.S., NIH,

Maryland, USA), a series of measurements was made on each side of every individual: standard length (SL), body depth (BD), sword length (SW), bar span (BS), bar number, cumulative bar width (BW), body area (BA) and total melanin area (MA).

Standard length is the length of the body, from the snout tip to the caudal peduncle.

Body depth is the widest cross-section of the fish, measured perpendicularly to SL.

Sword length was measured as the length of the 3-4 fin rays protruding past the rest of

18 the caudal fin. I defined bar span as the distance from the beginning of the first vertical body bar to the end of the last vertical body bar. I did not include the vertical body bar through the eye, or any bars that intersected with the darkened operculum, as they are found on all individuals and difficult to measure accurately. Bar width was measured along the axis created by the SL measurement, to maintain consistency. Body area was measured circumnavigating the scaled body and excluding the dorsal, caudal, anal and pelvic fins (and portions of the pectoral fins that extended beyond the body itself). I defined melanin area as the area of the vertical body bars included in the bar number and bar width measurements. Three individuals did not have bars and were excluded from analysis.

Statistical analysis of growth rate and bar morphology

To examine the relationship between bar morphology and growth rate while directly controlling for body size, I used the following calculated variables in my analysis.

“Bar sprawl” was calculated as bar span (distance from first bar to last, Figure 4) by the product of standard length and bar number:

Bar Span (Standard Length x Bar Number)

This variable controls for body size and bar number, which would influence the bar span. Proportion melanin was calculated as total melanin area by body area:

Melanin Area Body Area

This variable was chosen as the amount of melanin is a trait females are known to prefer, and has been correlated with other behaviors, such as aggression (Kleinas,

19 unpublished data). I used linear mixed models (LMM) to determine the influence of growth rate on both bar sprawl and proportion of melanin, including mother as a random factor. Since bar measurements were obtained for both sides of each individual, I analyzed the left and right sides of all males in two separate models.

There was a marked difference between left and right (µ=0.00463 ± 0.00332, Figure

5), which decreased the precision of the analysis. As a result, I analyzed the largest and smallest sprawl measurements from each individual in relation to growth rate in separate analyses. I conducted all analyses in R (R Core Team 2013) and used the package car (Fox & Weisberg 2011). All assumptions of the models were met.

Figure 4. Morphological measurements used to calculate bar sprawl are shown. Red denotes standard length. Blue denotes bar span. Yellow denotes the width of each bar, while the black numbers denote the bar number.

Figure 5. The difference in sprawl values between the left and right side measurements. Negative values indicate that the left side measurement was larger, while positive values indicate that the right side measurement was larger.

Breeding of females for preference tests

Females were obtained from laboratory populations that have been maintained as either sneaker or courter lines for over 5 generations. Therefore, all of the females were descendants of the respective genotypic male and all courtship and mating experiences of the females had been with the same male genotype. Sexually mature females were selected from the populations and isolated in individual 5-gallon aquaria.

Individuals were isolated for a minimum of seven days prior to being tested.

Following completion of the preference test, the females were photographed using a

Canon® Powershot SD 1200IS.

21 Creation of male animation for preference testing

I used an animated model to test female mate preference for a bar variable that could provide females with information about growth rate. Both bar sprawl and proportion melanin were significantly correlated with growth rate (see results below).

I chose to test preference for bar sprawl rather than proportion melanin because my methods for creating the animation were such that manipulating bar sprawl could be done while keeping all other aspects of the bars (width, number, height) consistent between the models. A swordtail fish model was purchased from an animated template website (http://www.turbosquid.com/3d-models/maya-swordtail-male-rigged/755443).

The model was rigged using Autodesk Maya. The texture packages, containing the diffuse map, were edited in Adobe Photoshop. I selected a photograph of a male X. multilineatus and used the rubber stamp tool to remove the bars by copying the unbarred section cranial to the barred section, creating a barless body. This barless body section was used for both male models. In order to keep all other aspects of morphology consistent with the population, I used the mean bar width, bar number and body length values from the photograph analysis in the creation of the male models. A single vertical body bar of the mean width was created using the brush tool in

Photoshop. This bar was then copied eight times, creating a total of nine vertical body bars (the average from the photograph analysis). Being the variable of interest, the sprawl of the bars differed between the two models by four standard deviations, such that one model was two standard deviations above the mean, while the other was two below. The mean was calculated from the largest sprawl measurements from each

22 individual, as range of the smallest sprawl measurements was less than four standard deviations. The sprawl value was used to calculate the bar length from the standard length in pixels. The bar length was divided into ten sections, reflecting the ten spaces between the vertical body bars. The bars were spaced appropriately along the bar length axis. The caudal-most bar was shortened height-wise in an attempt to create a more natural effect. The middle bar was placed in the same location across both models, such that the wide (W) model had bars extending further caudally and cranially than the narrow (N) model. The eyes were reduced in size so that they appeared proportional in the final animation. After completing one side of the fish body, the image was reflected above the dorsal line axis to create the opposing side.

(Figure 6)

To create realistic fins, I replicated the fin-ray pattern from photographs and altered the colors, making the diffuse map background color most prominent so that the fins appeared transparent in the final animation. The transparency of the fins was further edited in Maya.

The fish was animated in Maya to mimic the typical courtship behavior of a male X. multilineatus, which includes swimming perpendicularly to the female while oscillating the erect dorsal fin.

For the background, a short video was made of the inside of an empty 5-gallon aquarium with a Top Fin® Aquarium Power Filter. This video was imported into

Adobe After Effects with the final animation from Maya, layering the fish on top of the background clip. The background clip looped for twelve minutes, while the male

23 animation looped for seven minutes, creating a final video containing five minutes of background alone followed by seven minutes of the background and animation running concurrently. Grey-scale was applied to the entire video in an attempt to control for differences in color perception by the fish from the LCD monitors upon which the animation would be displayed, as outlined by Rosenthal (1999). The frame of the animation was edited so that it was entirely contained by the sides of the aquarium containing the female [H x W]. The size of the fish was altered so that it appeared on the display 37mm in SL. The videos were imported into Adobe Media

Encoder to be exported as .mp4 files, which were each saved on individual 10 GB jump drives.

Figure 6. (A) The completed narrow model animation, with a bar sprawl of 0.04761. (B) The completed wide model animation with a bar sprawl of 0.06922. (C) The diffuse map used in the creation of the narrow male model displayed in A. (D) The diffuse map used in the creation of the wide male model displayed in B.

25 Female preference testing

The testing arena consisted of a 10-gallon aquarium placed perpendicularly between two VIZIO M322i-B1 32” 1080p 120Hz full-array LED HDTVs. The aquarium was elevated so that the bottom of the tank aligned with the bottom of the television screen. White Plexiglas was placed underneath and along the front and back of the aquarium to increase the contrast between the fish and the tank, as well as prevent the fish from seeing through the portions of the tank that did not display the animation. The tank was divided into three equal portions by string across the open top of the aquarium. A mirror was angled above the open tank in such a way that allowed the observer to view the fish’s movement without peering directly into the tank.

During the test, the observer was located behind a one-way mirror, as overhead movements tend to alter the fish’s behavior. When tests were not being conducted, a

Top Fin® Aquarium Power Filter was placed on the test aquarium to maintain water quality.

Preference tests were performed between 10:00 and 15:00. The overhead lights in the test room were turned off to prevent reflection from the aquarium onto the monitor screen. Prior to placing each female into the test tank, 2 mL of API STRESS

COAT ® was added to the water. The female was placed in a clear Plexiglas column in the center of the aquarium. Once the female was placed in the column, both animations were started. Each animation was played from a labeled 10GB flash-drive inserted into the television’s USB port. The side on which a specific animation played first was alternated between females of the same group, such that the first female from

26 the sneaker population was exposed to the W animation on the left screen first, while the second female from the sneaker population was exposed to the N animation on the same screen first. The first five minutes of the test, described as the acclimation period, consisted of the female contained by the column while both monitors displayed the identical background clips of an empty aquarium. Following the acclimation period, the animations of the courting males were displayed for one minute while the female was still contained; this period is described as the exposure period. After the exposure period, the test period began, during which the female was released from the

Plexiglas column and was free to move between the three sections of the aquarium while the male animations continued to play for five minutes.

During the test period, the observer recorded the movements of the female.

Behaviors were recorded using JWatcher 1.0, with a preexisting global definition file pairing every crossover between tank sections with a keystroke. This allowed for behaviors to be recorded in real time without requiring that the observer look away from the test set-up. The test period was included in the global definition file so that any keystrokes after the five-minute period were not recorded.

After the completion of one test, the female was captured and placed in an 8 oz. transport vessel while the observer switched the flash-drives containing the animations. Once the flash-drives were inserted into the appropriate monitors, the female was placed back in the Plexiglas column and the acclimation, exposure and test period were repeated as before. In the event that the female remained in the center section of the aquarium for the duration of both trials, or exhibited a side bias (no time

27 spent on one side of the aquarium for both trails), she remained isolated and was retested at least seven days later.

It should be noted the my experimental design avoided pseudoreplication because the male models were designed from population averages and the traits in question were created artificially, rather than from that of an exiting individual

(McGregor et al. 1992; Rosenthal 1999).

Statistical analysis of female preference tests

I made several measures of female responses during the preference tests to assess different aspects of female preference. Preference was measured as total time spent in the section adjacent to each model. The time a female spends with a male in laboratory preference tests has been shown to correlate with female mate choices made in the field (Morris et al. 2010). I used paired t-tests to compare the preference for W and the preference for N. Strength of preference was defined as the difference between the time spent with model W and time spent with model N. The sign of the strength of preference value relays the direction of the preference, such that a positive value represents a preference for W, while a negative value represents a preference for

N. To compare strength of preference between the females from the courter line and the sneaker lines, I used a one-way ANOVA. I also recorded which model the female approached first in each trial. To determine if first approach was a reliable measure of preference, a Mann-Whitney U test was used to compare strength of preference between females who first approached W and those that first approached N. A Fisher’s

28 Exact Test was used to compare the model females approached first between the two lines.

I also evaluated “choosiness” by quantifying the absolute strength of preference and number of cross-overs. A “cross-over” was defined as the female exiting the section of the aquarium adjacent to one model and entering the opposing section. A more choosy female would have a larger absolute difference in time spent with the two models, and would cross over between models less, as she would be more resolute in her mating choice. To evaluate if there was a difference in choosiness between the two lines, a Welch Two-Sample T-test was performed comparing the absolute value of strength of preference. A Welsh Two-Sample T-test was also used to compare choosiness between lines by number of cross-overs.

To further compare “choosiness” in females from the two genotypic lines, I determined if females exhibited the same preference across both trials or if they were switching their preference. If females switch preferences between trials, this would indicate that they are using bar sprawl as a means of identifying males. I used

Pearson’s correlation to quantify the relationship between strength of preference in the first trial and strength of preference in the second trial. I examined this correlation in both female lines. Statistical tests were performed using R (R Core Team 2013) and the package plyr (Wickham 2011).

29 Results

Male bar morphology and growth rate

Bar sprawl varied significantly with growth rate, for both the largest sprawl measurement (LMM: F1,52=10.6254, p=0.00197) and smallest sprawl measurement

(LMM: F1,52=7.1443, p=0.01002) of each individual. Individuals with wider bar sprawl (W) grew slower than individuals with narrower bar sprawl (N). Proportion melanin was also significantly affected by growth rate for both sides (Right, LMM:

F1,52=5.0888, p=0.02831; Left, LMM: F1,52=5.2224, p=0.026406). Individuals with faster growth had a greater proportion melanin.

30 A

Figure 7. The four measures of bar sprawl were plotted against growth rate, demonstrating the negative relationship between sprawl and growth rate across measurements. (A) The largest sprawl values from each male correlate with growth rate. (B) The smallest sprawl values from each male correlate with growth rate.

Female Preference Tests

There was no significant difference between time spent with the “narrow” (N) male model and time spent with the “wide” (W) male model across all females tested

(Paired T-test; t37=0.275, p>0.5). There was also no significant difference between preference for N and preference for W within females from the sneaker line (Paired T- test; t18=0.704, p>0.4) or within females from the courter line (Paired T-test; t18=-0.55, p>0.5, Figure 8). Furthermore, there was no difference in strength of preference between the two lines (One-Way ANOVA; F1,36=0.795, p>0.3).

Females overall spent more time with the model they approached first, although this result was not statistically significant (Mann-Whitney U: U=235, p=0.0636; Figure 9). When examining this relationship within the two lines of females, females from the courter line spent significantly more time with the model they first approached (Narrow model, Mann-Whitney U: U=70, p=0.033), while females from the sneaker line did not (Wide model, Mann-Whitney U: U=29, p>0.9).

In the first trial, there was a difference in the proportion of females from each line that approached each model first (Fisher’s Exact Test: p=0.048), such that sneakers more often approached the W model first (Figure 10). However, this was not true in the second trial (Fisher’s Exact Test: p>0.7).

Females from the sneaker line demonstrated a greater absolute strength of preference overall than females from the courter line (Welch Two-Sample

32 T-test: t34.4=-2.09, p=0.044). In addition, females from the sneaker line exhibited fewer cross-overs than females from the courter line, but only in the second trial

(Welch Two-Sample T-test; Trial 1: t32.8=0.674, p>0.5; Trial 2: t29.9=2.16, p=0.039).

Among the females from the courter line, there was a negative correlation between their preference in the first trial and that in the second trial (Figure 9; r=-0.506,

DF=17, p=0.027), indicating that their preference switched between trials; there was no such relationship among the females from the sneaker line (r=0.191, DF= 17, p>0.4), indicating that their preference remained stable across trials (Figure 11).

33 A

Figure 8. (A) The mean time females from the sneaker line spent with each model in each trial (B) Mean time females from the courter line spent with each model in each trial.

34 100

Wide

0 1" -20

-40

-60 Time Associating with (s) Time

Narrow

-80

-100 Approached Wide Approached Narrow Model First Model First

Figure 9. Mean Time spent with a model for females that approached the Wide (slow growing) model first as compared to females that approached the Narrow (fast growing) model first.

Figure 10. The number of females from each line that first approached each model. The females from the sneaker line were more likely than the courter line to approach the wide model first (Fisher’s Exact Test: p=0.048).

36 Courter Line Courter Line Sneaker Line

Sneaker Line Wide

Preference in Trial 1 (s) Narrow

Narrow Wide

Preference in Trial 2 (s)

Figure 11. Time spent with each model for each female across the two trials. The relationship across trials was negative for females from the courter line (r=-0.506, DF=17 p=0.027). There was no significant relationship between trials across females from the sneaker line (r=0.191, DF= 17, p=0.43).

37 Discussion

Females are known to have preference for mates based on particular male traits or combination of male traits that relay information about some aspect of male quality

(Andersson 1994). The consequences of these preferences are that the female has increased her fitness by increasing the fitness of her offspring. Female swordtail fishes may be interested in the growth rate of potential mates, as this trait has been shown to influence survival, both directly (Morris et al., in review) and indirectly via predation

(Biro et al. 2006). In fact, in X. multilineatus, there may be tactical disruptive selection on growth rate, such that sneaker males benefit from slower growth rates while courter males maximize fitness via faster growth rates (Morris et al., in review). Within a genetically influenced size class, age at sexual maturity is environmentally influenced

(Lyons et al. 2014), and so adult size does provide some information about growth rate within size classes. However, size does not represent all variation in growth rate, as two males may reach the same size through different growth rates. Fluctuating asymmetry (FA) allows for females to approximate male growth rate by evaluating symmetry and resource environment, however it is only a reliable signal in some environments. Here, I have identified two additional male traits that directly correlate with juvenile growth rate in X. multilineatus, at least one of which may be evaluated by females in mate selection.

I identified two male traits that are highly correlated with juvenile growth rate: proportion melanin and bar sprawl. The relationship between bar sprawl and growth rate was negative, such that increased growth leads to vertical body bars that are more

38 condensed. The relationship between these traits and juvenile growth rates in this species could lend insights into the formation of vertical body bars during development, such as the migration of melanophores into the barring pattern and the effect of growth rate on this process. In addition, more broad-scale measurement of these components of the vertical bar pigment pattern could have applications for approximating juvenile growth rate for males in the field.

In addition, I demonstrated that X. multilineatus females might be evaluating bar sprawl in mate selection. While there were no indications of an overall preference for the model indicating faster growth (narrow bar sprawl), I did detect evidence that females vary in their preference for this trait depending on their own genotype and environment. The females from the sneaker population showed a preference for the trait indicating slower growth rate (wide male model), in that they were more likely to first approach the wide model than the narrow model. First approach appears to be an additional indicator of mate preference as the females spent more time with the model they approached first. This measure of mate preference may be more useful in preference tests that use video animations, as it has been suggested that females lose interest in the video animations as the model male does not respond to the female the way a live male would in a preference test (Nicoletto & Kodric-Brown 1997).

Morris et al. (in review) suggests that there may be tactical disruptive selection acting on growth rate in males with different mating tactics in general, and the alternative reproductive tactics (i.e. sneakers and courters) in X. multilineatus specifically. They found that a faster growth rate in Y-L corresponded to decreased

39 longevity and proposed a model in which increased growth rate is favorable only when the fitness benefits of being a larger male as an adult outweigh the costs of increased mortality. According to this model, growth rate would only be under positive selection in courter males, as an increased growth rate would heighten an individual’s probability of ever reaching sexual maturity, which is already lower in courters than in sneakers. Sneakers, however, already reach sexual maturity earlier, and the maintenance of the “sneak-mate” reproductive tactic hinges on sneakers having a longer reproductive life span than courters, so a growth mortality trade-off would be detrimental. Therefore, this model would predict that females should prefer males that grow slower if they are mating with sneaker males, producing sneaker offspring, and males that grow faster if they are mating with courter males, producing courter offspring. To test this, we would need to demonstrate that offspring fitness is affected by female preference (Reynolds & Gross 1992; Hine et al. 2002; Sheldon et al. 1997), and that there is a heritable component of preference for this trait (Fisher 1930; reviewed in Bakker & Pomiankowski 1995).

My results can be compared to a previous study of mate preferences for vertical bars in the swordtail Xiphophorus cortezi. In this species, male mating tactic varies with male size, without the distinct genetically determined size classes found in

X. multilineatus. In X. cortezi, females were shown to prefer males with a smaller bar span (Morris et al. 2011). If my measure of bar sprawl is also correlated with growth rate in this species, female preference for bar span may indicate that females in X. cortezi prefer fast-growing males. An overall preference for faster growing males

40 would make sense in a system in which there is a fitness advantage to increased growth rate in all males. It would be interesting to further compare the fitness benefits of growth rate between these two species, as well as female mate preferences for bar sprawl in X. cortezi.

I did not detect a preference for either model by females from the courter line.

In fact, females from this genetic line demonstrated low levels of choosiness, with a tendency to switch preference between trials, such that if they associated with the wide model in the first trial, they would associate with the narrow model in the second.

There are two plausible explanations for these results: either the females recognized that the male was not responding or engaging in courtship behavior, causing them to redirect their efforts toward the other male, or they were actively switching their preference in an attempt to mate with a variety of males. Either of these explanation implies that the females were using the bar spacing as a method of identifying and distinguishing between different individual males. It is also possible that the females from the courter line did have a preference for the slow-growing male that was not shown statistically due to the small sample size of this study. Courter females spent more time with the model indicating a faster growing male (narrow model) in trial 2

(Figure 8), and they were more likely to approach the narrow model first (Figure 10), although neither of these differences were statistically significant. Given that these females had courter fathers, and had mated with courter males, preferring the faster growing male would have fit the hypothesis of choosing a mate that would provide their offspring with greater fitness (faster growth rates).

41 The sneaker population of females could be described as more choosy, as they exhibited a stronger absolute strength of preference than the courter line, and evaluated the different male models less thoroughly (quantified as number of cross- overs between stimuli). These data could suggest that being more choosey between these two types of males (slow growing over fast growing) is more important for the fitness advantage of the offspring of females from the sneaker genetic line than the courter genetic line. A population with only courter males (courter genetic line) for several generations could have shifted the cost/benefit relationship of faster growth, as offspring of the “choosing” female are no longer competing with sneaker males that reach sexual maturity sooner. Therefore, the cost to the females from the courter line of being choosy in relation to a trait indicating growth rate may not be outweighed by the benefits. Instead, females from this line could have been selected to attempt to mate with a several different males, which is why they were more likely to switch between models in the two trials. The benefits of multiple mating by females has been examined in other systems (insects, Arnqvist & Nilsson 2000; guppies, Evans &

Magurran 2000), and some of the benefits include increasing the opportunity for sperm competition and making sure that all eggs are fertilized (Jennions & Petrie

2000). Further study of the benefits of multiple mating in swordtails is warranted.

Females are often assessing multiple male traits, and therefore it is important to consider that X. multilineatus females evaluate potential mates via bar sprawl in conjunction with other traits, which could increase the ability of females to select optimal mates. For example, if a female was choosing between two symmetrical

42 males, and asymmetry is indicative of optimizing growth over developmental stability, she could use the bar sprawl measure to select the male that achieved said symmetry via faster growth, which would presumably indicate a genetic quality that allowed him to do so. In addition, bar sprawl could be evaluated in conjunction with body size to differentiate between male genotypes. The body size range of Y-II males overlaps with that of Y-I and Y-L males, but Y-II males exhibit faster growth rates than Y-I and

Y-L (Morris et al. 2012). This could allow for females to differentiate between genotypes, such as a smaller Y-II and a Y-I of the same size, or a larger Y-II and a Y-

L of the same size. This could also allow them to evaluate symmetry more honestly, since Y-II males exhibit fluctuating asymmetry (Morris et al. 2012). Further studies that examine how females assess these traits when more than one varies, and across different environmental conditions, will provide a better understanding of the selection on mate preferences.

In conclusion, this study has identified two new morphological measures that females could use to assess the juvenile growth rate of potential mates. In addition, my results suggest that females use this trait when evaluating potential mates. It has yet to be determined whether bar sprawl is assessed by males in male-male competition as well. By examining the role of this trait in intrasexual selection (competition) as well as intersexual selection (mate preference), we will gain a greater understanding of the importance of assessing growth rate in sexual selection.

43 Acknowledgements

First and foremost, I would like to thank my thesis advisor, Dr. Molly Morris.

Her advice and encouragement has been pivotal in the completion of this thesis. She has demonstrated great patience and enthusiasm as I have advanced in my education as a scientist. I attribute much of my professional and educational growth to my experiences in her laboratory. I would also like to acknowledge Dr. Soichi Tanda, who has offered me constant guidance throughout my four years at Ohio University.

Thirdly, I would like to thank the Honors Tutorial College for providing me with a unique and superior education in the field of biological sciences. In addition, the staff of the college has been an unprecedented support system through the trials and tribulations that often accompany the completion of an undergraduate thesis.

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