Personality in the Brush-Legged Wolf Spider: Behavioral Syndromes and Their Effects on Mating Success in Schizocosa Ocreata

Personality in the Brush-legged Wolf Spider: Behavioral Syndromes and their Effects on Mating Success in Schizocosa ocreata

A thesis submitted to the

Graduate School

of the University of Cincinnati

in partial fulfillment of the

requirements for the degree of

Master of Science

in the Department of Biological Sciences

in the College of Arts and Sciences

Trinity D. Walls

B.A. Washington University in Saint Louis

July 2018

Committee Chair: George W. Uetz, Ph.D.

Cincinnati, OH

Abstract

Recent studies have shown that animal “personality” demonstrates consistent behavioral variation at the individual level that persists across lifestages and contexts. The most commonly measured behavioral syndrome involves a “bold” to “shy” continuum, in which individuals are evaluated based on their willingness or latency to engage in risk-taking behaviors. I examined bold-shy behavioral syndromes in the brush-legged wolf spider, Schizocosa ocreata. Spiders were repeatedly given open field tests and later exposed to simulated predator stimuli. All spiders were tested as juveniles and adults. Results of open field tests showed individual S. ocreata exhibit consistent behavioral patterns associated with either end of the continuum of bold (exploratory) to shy (freeze) behavioral syndromes. These differences persisted across contexts, as well as lifestages (juvenile, adult). Bold spiders exhibited shorter latency to explore in an open field and to resume exploration after a simulated predator than did their shy counterparts, but also showed more variation in latency to resume exploration after a simulated predator. After reaching maturity, females were given a two-choice test using video playback of male courtship to analyze differences in mate choice, while males were exposed to female cues to assess courtship vigor. While open field behaviors and responses to simulated predators were correlated, personality type did not show significant effects on male courtship in the presence of female cues or female mate preference in the context of video playback. Males and females of differing personality types were also paired in a two-by-two factorial design to assess the effect of personality on overall mating success. No differences in mating success were found, suggesting that personality type measured using bold-shy attributes may affect somatic traits but not reproductive traits in this study.

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Acknowledgements

I would like to thank the University of Cincinnati and the Yates Fellowship program for providing financial support and space to conduct this research. I thank my advisor, Dr. George

Uetz, for providing indispensable guidance and direction for the last two years, as well as for his encouragement and confidence throughout this process. I thank the other members of my research advisory committee, Dr. Elke Buschbeck and Dr. Nathan Morehouse, who were also fundamental in helping me revise and improve my ideas and project designs. I thank the post- doctoral researchers Dr. Brent Stoffer and Dr. Alex Sweger for their patience and constructive feedback during my time here. I also thank my lab members, Emily Pickett, Madeline Lallo, and

Tim Meyer for their encouragement and advice, and the numerous undergraduates who performed animal husbandry. Lastly, I thank the spiders for being complex enough to display distinct personalities and making this research possible.

Table of Contents Abstract…………………………………………………………………………………………. ii Acknowledgments …………………………………………………………………....……...... iv List of Figures……………………………………………….………………………………….. vii Introduction………………………………………………………………………………………..1 Methods…………………………………………………………………………………………....6 Collection and Care.………..………………………………………………….………...... 7 Establishing Personality………………………………………………………….……...... 8 Open Field Test……………………...... 8 Correlating Personalities with Behaviors in Other Contexts …………………………...... 9 Simulated Predator Stimulus Test……..…………………………………………..9 Female Mate Choice………………………………………………………………………9 Male Courtship……………………………...……………………………………………10 Live Mating Trials of Different Personalities…………………………………………10 Differences Between Research Seasons………………………………………………………...11 Statistical Analyses…………………………………….…………………………………11 Results……………………………………………………………………………………………13 Part I: Lab-reared spiders in an open field...... 13 Part II: Lab-reared spiders’ responses to a simulated predator stimulus………………..14 Part III: Field-exposed spiders in an open field.…………..…………………………….15 Part IV: Field-exposed spiders’ responses to a simulated predator stimulus………..…..15 Part V: Lab-reared vs field-exposed ……………………………………………………..15 Part VI: Female responses to a two-choice test.………………………………...... 16 Part VII: Male courtship on female silk.…………………………………………………17 Part VIII: Mating personalities together..………………………………………………..17 Part IX: Lab-reared spiders in extended open field test.………………………………...18 Part X: Lab-reared spiders’ responses to a simulated predator stimulus…………………18 Discussion……………………….……………………………………………………………….19 Future Directions.………………………………………………………………………………..25 References……………………………………………………………………………………….29

Figures…………………………………………………………………………………………...35

List of Tables and Figures

Figure 1: Lab-reared Penultimate Latency to Explore in Open Field across Trials

Figure 2: Lab-reared Mature Latency to Explore in Open Field across Trials

Figure 3: Distribution of Initial Open Field Responses in Lab-reared Penultimate Females

Figure 4: Distribution of Initial Open Field Responses in Lab-reared Penultimate Males

Figure 5: Distribution of Initial Open Field Responses in Lab-reared Mature Females

Figure 6: Distribution of Initial Open Field Responses in Lab-reared Mature Males

Figure 7: Lab-reared Penultimate Female Latency to Recover from Predator Stimulus

Figure 8: Lab-reared Penultimate Male Latency to Recover from Predator Stimulus

Figure 9: Correlation between Open Field Latency and Predator Stimulus Latency in Lab-reared Penultimates

Figure 10: Lab-reared Mature Female Latency to Recover from Predator Stimulus

Figure 11: Lab-reared Mature Male Latency to Recover from Predator Stimulus

Figure 12: Correlation between Open Field Latency and Predator Stimulus Latency in Lab-reared Matures

Figure 13: Field-exposed Penultimate Latency to Explore in Open Field across Trials

Figure 14: Field-exposed Mature Latency to Explore in Open Field across Trials

Figure 15: Distribution of Initial Open Field Responses in Field-exposed Penultimate Females

Figure 16: Distribution of Initial Open Field Responses in Field-exposed Penultimate Males

Figure 17: Distribution of Initial Open Field Responses in Field-exposed Mature Females

Figure 18: Distribution of Initial Open Field Responses in Field-exposed Mature Males

Figure 19: Field-exposed Penultimate Female Latency to Recover from Predator Stimulus

Figure 20: Field-exposed Penultimate Male Latency to Recover from Predator Stimulus

Figure 21: Correlation between Open Field Latency and Predator Stimulus Latency in Field- exposed Penultimates

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Figure 22: Field-exposed Mature Female Latency to Recover from Predator Stimulus

Figure 23: Field-exposed Mature Male Latency to Recover from Predator Stimulus

Figure 24: Correlation between Open Field Latency and Predator Stimulus Latency in Field- exposed Matures Figure 25: Lab-reared Mature Female Latency to Recover from a Predator Stimulus using Boldness Scores Figure 26: Lab-reared Mature Male Latency to Recover from a Predator Stimulus using Boldness Scores

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Introduction

Behavioral variation and its drivers have been the subject of considerable research, particularly within lower animal taxonomic levels. Changes in animal behavior have been attributed to weather, age, habitat, food abundance, predator proximity, resource abundance, breeding season, etc. (Relyea 2001), although responses may vary among taxa. Ecotypic behavioral variation between populations of the same species is also found, e.g., coastal garter snakes accepting slugs as prey while inland garter snakes reject them (Arnold 1981), and differences in attack latency values for the same prey between grassland and riparian spider populations (Hedrick and Riechert 1989). However, even more specifically and in some instances surprisingly, variation between individuals in the same population has also been recently shown to have a sizable influence on animal responses.

Over the last few decades, ethologists and behavioral ecologists have begun addressing a previously understudied facet of animal behavior that may have substantial impact on animal responses in different contexts: behavioral syndromes and “personality” (Gosling 2001; Réale et. al. 2007). In the past, personality has primarily been considered in humans and other complex animals with large mental capacities (McGuire et. al 1994; Capitanio 1999) because such traits are generally thought of as arising from characteristic patterns of thinking, behaving, and feeling

(Pervin and John 1997) that have been shaped through individual experience. While this may be true, animal personality has been defined more simply as consistent behavioral differences between individuals that persist across time and contexts (Dall, Houston, and McNamara, 2004;

Kralj-Fišer and Schuett, 2014). This simplified definition allows for personalities to be quantified in less complex animals and also allows for consistent individual variation to be examined rather than average population behaviors.

The idea of animal personality is somewhat contrary to the well-documented and adaptive idea of behavioral plasticity, wherein responses of animals are hypothesized to change based on external or internal stimuli (Stamps and Biro 2016). Many studies have demonstrated behavioral plasticity both in vertebrates and in invertebrates (Crosland, 1997; Hobert, 2003;

Scheiner, Page, and Erber, 2004), and this plasticity can be exemplified in many forms.

Developmental plasticity involves changes in behavior based on rearing or events which occurred in the past, while temporal plasticity involves changes in behavior associated with age, and contextual plasticity involves changing behavior based on the circumstances present when that behavior is expressed (Stamps and Biro 2016). Though this is conceptually different from the implications of animal personality, it does not mean personality and plasticity are mutually exclusive. Personality does not necessarily imply lack of plasticity, but rather that plasticity may be more limited than previously assumed. Animals may well be able to modify their behaviors based on changes in their environments, but individuals may still be pre-disposed to exhibiting some behaviors over others.

One of the core tenets of animal personality is the correlation of an individual’s behaviors across multiple situations. In essence, suites of measurable behaviors that are correlated across different contexts are associated with personality types, and these suites are termed behavioral syndromes (Sih et. al 2004a;b). Several continua have been defined to characterize the behaviors of various species. Dingemanse (2002) distinguished proactive and reactive individuals in a flock of great tits (Parus major) and determined that proactive individuals flew along the periphery while reactive individuals kept close to the interior. The proactive vs reactive scale is the most all-encompassing continuum, in that it incorporates bold and shy behaviors as well as aggression levels, which may or may not be correlated with each other. Alison Bell and Andrew Sih have

2 conducted extensive work on sticklebacks and found a correlation between boldness under the risk of predation and aggression towards conspecifics in a high predation pressure environment, but this correlation was not detected when predation pressure is low (2007). The aggression syndrome is another popular continuum where some individuals consistently exhibit a greater likelihood of attacking conspecifics than other individuals. Reichert and Hedrick (1993) found that more aggressive Agelenopsis aperta individuals (funnel web spiders) attack both prey and conspecifics more quickly. Maupin and Reichert (2001) later discovered that aggressive individuals were also more likely to engage in superfluous killing of potential prey without consumption, and that more aggressive females are also more likely to cannibalize males.

The most commonly measured descriptor of animal personality is the “bold” to “shy” continuum, where individuals are often assessed according to their responses to open arenas where they are allowed to explore or otherwise react to a new environment. Bold pumpkinseed sunfish (Lepomis gibbosus) have been shown to adjust more quickly to a new environment (in this case the laboratory), be more willing to feed on more exposed prey, and be more willing to examine potential predators than shy sunfish (Wilson et. al. 1994; Coleman and Wilson 1998;

Sih et al. 2004a). Boldness has also been positively correlated with greater dispersal in killifish

(Rivulus hartii) and great tits (Fraser et. al. 2001; Dingemanse et. al. 2003), and with greater parasite infection in pumpkinseed sunfish (Wilson 1998). The bold or shy continuum has been used in numerous spider studies in the past but primarily with social spiders (Pruitt et. al 2013;

Grinsted et. al. 2013). Social spiders showed task differentiation within colonies that were correlated with personality type (Grinsted et. al 2013). This study will utilize the bold or shy continuum to assess whether personalities exist within (solitary) S. ocreata populations which could help explain significant variation observed between individuals.

Another key aspect of personality in animals is that the measured behaviors are repeatable. Repeatability measures the proportion of variation that results from variation between individuals or groups. Repeatability is also referred to as the intra-class correlation coefficient

(ICC) and is measured as the between-group (or between-individual) variance divided by the sum of the between-group variance plus the within-group variance (Nakagawa and Schielzeth

2010). This ratio is important because repeatability could be low due to high within-individual variation or low between-individual variation, so accounting for both factors allows the observer to determine the behavioral consistency of individuals. If the behaviors measured are not consistent, then the animals may not be displaying personalities, or the tests being used may not be applicable to the animals being examined.

While animal personalities have been studied more extensively in the last few years, research has primarily involved understanding how behavioral syndromes of individuals influence social structure and behavior. Regarding invertebrates, much has been done using

Stegodyphus and Anelosimus social spiders. It was found that Stegodyphus colonies with more bold individuals attacked prey with more force (more spiders attacking) than colonies with less bold spiders (Wright, Keiser, and Pruitt 2015). Likewise, studies involving Anelosimus studiosus showed that colonies with a greater proportion of aggressive females (who were more likely to capture prey) had higher overall colony mass than colonies with lower frequencies of this behavioral phenotype (Pruitt and Reichert 2011). However, a colony with too many bold individuals displayed an increase in intraspecific conflicts, and shyer spiders were less likely engage predators and risk mortality which also increases overall colony survival (Pruitt and

Reichert 2011; Jandt et. al. 2014). Both asocial and social phenotypes benefited from the presence of unlike phenotypes. The aforementioned studies have provided insight regarding the

4 costs and benefits of having multiple personality types in groups of social animals. However, few studies have been conducted involving the individual effects behavioral syndromes have on solitary species. Chad Johnson and Andrew Sih did find a correlation between voracity

(aggression shown in the context of prey capture) and precopulatory sexual cannibalism in the six-spotted fishing spider (Dolomedes triton) (2005), suggesting that behavioral syndromes do exist within solitary spiders. Moreover, behavioral syndromes may have consequences for overall mating success. Sexual selection and mating success have been studied in many invertebrates, but few studies have examined the influence of personality in this context.

The behavior of the brush-legged wolf spider Schizocosa ocreata (named so for the large tufts that appear on the front forelegs of reproductively mature males) has been extensively studied and characterized over the last 40 years. Studying these spiders has increased understanding of how vibratory and visual signals are produced and received, how mate quality information contained in signals is assessed in mate choice, and how these behaviors contribute to reproductive isolation between species (Uetz et. al 2016). While much is known about these spiders, no studies have attempted so far to determine the presence or absence of personality in this species. Recent studies have found that spiders show behavioral variation based on prior experience or exposure to conspecifics (Rutledge et. al 2010; Rutledge and Uetz 2014; Stoffer and Uetz 2015, 2016, 2017). This study sought to quantify behaviors exhibited by individuals when presented with a standardized open field test to determine whether Schizocosa ocreata demonstrate repeatable behaviors and personality types or simply demonstrate behavioral plasticity in each situation. Spiders were then retested to determine if personality type influenced antipredator behaviors, sexual selection, and mating success.

Methods

Study Species

The Brush-legged wolf spider Schizocosa ocreata (Hentz) is primarily found in the leaf litter in deciduous forests throughout eastern North America (Dondale and Redner 1978; 1990).

These ground-dwelling spiders are mobile hunters who feed on assorted insect prey, and are sexually dimorphic, with females being larger than males (Dondale and Redner 1978), and mature males having tufts of bristles on their forelegs (Dondale and Redner 1978; Uetz and

Dondale 1979). These spiders are well-studied due to their multi-modal signals. They communicate using visual, vibratory, and chemical cues (Scheffer et. al 1996; Uetz 2000), with males in particular exhibiting complex, ritualistic courtship behaviors (Stratton and Uetz 1981,

1983, 1986; Scheffer et. al 1996; Uetz et al 2009). Courtship involves both a visual signal, where males raise and wave their tufted forelegs, as well as multiple vibratory signals, including a percussive element (where males strike their chelicerae on the ground and tap their legs, also known as “jerky tap”) and a stridulatory element (using a file and scraper mechanism in their pedipalps to produce sound) (Rovner 1975; Stratton and Uetz 1981, 1983, and 1986; Uetz and

Stratton 1982).

Females have been shown to prefer males with larger tufts in free-mating trials (Persons and Uetz 2005) or as isolated visual courtship signals (McClintock and Uetz 1996; Scheffer et. al

1996; Uetz and Roberts 1992; Uetz and Norton 2007). Male tuft size and symmetry show dependence on development and successful foraging history, as well as infection (Gilbert & Uetz

2016), indicating that tufts are an honest indicator of fitness (Scheffer et. al 1996; Uetz et. al

2002). While studies have shown that females are able to evaluate males using vibratory or visual signals in isolation (Gibson and Uetz 2008; McClintock and Uetz 1996; Scheffer et. al

1996; Uetz and Norton 2007), the combined signals elicit greater receptivity and convey redundant information regarding male quality and feeding history (Uetz et. al 2002; Uetz et al.

2016).

Female S. ocreata are generally monandrous, mating more than once only infrequently.

Males exhibit more of a scramble competition polygyny mating system where they mate with multiple females but must indirectly compete with other males for females’ attention (Roberts and Uetz 2005; Norton and Uetz 2005). Female Schizocosa ocreata would therefore seem to demonstrate Bateman’s principle (Bateman 1948), in that they do not benefit from subsequent matings and consequently try to choose the best male, while males can increase reproductive success by mating as many times as possible. These spiders are also occasionally cannibalistic, with females eating the males around 10-15% percent of the time (Persons and Uetz 2005). All of these elements of sexual selection and mating have been studied in this species, but not in the context of personality.

Collection and Maintenance

Lab-reared” spiders were captured in the field during the fall and raised in the lab for approximately 12 to 14 weeks before trials began. “Field-exposed” spiders were caught during the spring and raised in the lab for approximately 2 to 4 weeks before trials began. Juvenile S. ocreata were caught in September and October (for winter research season) and March to April

(for spring research season) from 2016 to 2018 at the Cincinnati Nature Center Rowe Woods in

Clermont County, Ohio (N 39° 7' 32.894'' W 84° 14' 57.692”). Spiders were kept in individual opaque deli containers (9 cm diameter) with constant access to water through a cotton wick which connected the enclosure to a reservoir of water below. A cylindrical sponge forced through a hole at the top of the container allowed for constant airflow. Spiders were fed 2-3 1/8

7 inch crickets (Acheta domesticus) twice a week using an aspirator. Spiders were also kept on a

13L:11D cycle at 21°Celsius. All trials were performed during either the winter or spring season after spiders reached their penultimate lifestage (at which point genitalia begin to become visible and spiders may be sexed).

Establishing personality

Open Field Test

One to two weeks after juveniles molted to the penultimate lifestage (last lifestage before reaching maturity), each spider was placed in a 15 cm clear plastic arena on top of filter paper.

Arena walls were coated with Vaseline to prevent escape. Each spider was allowed to react to the open field and behavior was recorded for 60 seconds. Trials were recorded with a SONY

Handycam HDR-XR260. The spider was then removed from the arena, returned to its home container, and fed. All spiders were fed 24 hours before every trial, and trials were conducted on three consecutive days for each spider. Procedures (including weighing and photographs) were repeated 7-14 days after spiders molted to adulthood.

Based on an initial assessment of the distribution of latency values for exploratory behavior, spiders were assigned to personality types. Spiders that consistently began exploration

(walking around the arena and probing with their legs) within 10 seconds for each trial were classified as “bold” spiders. Spiders that froze (did not move or immediately pressed their bodies against the wall) for more than 10 seconds were classified as “shy.” Spiders that frenetically ran around the arena for more than 10 seconds were initially classified as “run” but were left out of future analysis due to small sample size (less than 2% of spiders in some trials).

Correlating Personalities with Behaviors in Other Contexts

Simulated Predator Stimulus Test

Individual spiders were placed in 15 cm clear plastic arenas on filter paper. Spiders were then allowed to acclimate for 60 seconds. Then a prod test was administered (Pruitt et. al 2013), wherein spiders were touched with forceps on their right anterior forelegs. The same person conducted all trials to standardize the intensity of prodding, and trials were then run for 5 minutes and recorded with a SONY Handycam HDR-XR260. Initial reactions and latency to return to normal behavior were recorded. Forceps were sterilized with 70% ethanol between each trial. Trials were repeated for 3 consecutive days, and spiders were fed 24 hours before every trial.

Female Mate Choice

Two to three weeks after reaching maturity, females were placed in the center of a 20 cm clear plastic arena. Each arena contained two Apple iPod® Touch portable media players for video playback of courting male spiders. To avoid pseudo-replication, three different male exemplars were used to represent the average and extreme body conditions and courtship rates most likely to be encountered in a normal population. Each female was exposed to playback from a single exemplar, digitally modified to show increased tuft size in one monitor and decreased tuft size in the other. Each female was only used once. All trials were conducted in the absence of male vibratory signals. Females were exposed to the videos for 5 minutes, and left- right order of videos of males with small or large tufts was varied to avoid side bias. All trials were recorded with a SONY Handycam HDR-XR260. Receptivity was indicated when females settled (dropped their bodies to the ground and extended their legs), pivoted (slowly turned their bodies towards the males) or performed a tandem leg extend (turned towards the males and

9 arched their abdomens upwards while extending their front legs) (McClintock and Uetz 1996;

Uetz et. al 2009). The sum of receptivity displays, number of visits to each screen and total time spent at each screen (quantified as when the female was within a half inch distance from the screen) were also recorded. Females were classified as bold or shy prior to exposure to the two- choice test (see Establishing Personality-Open Field Test).

Male Courtship

Previous studies have shown that males can determine a female’s reproductive state, hunger level, and mating status (mated or unmated) from chemotactile cues in female silk

(Roberts and Uetz 2005). The goal of this experiment was to determine if bold and shy males show differences in courtship rates based on their own personalities. To test this, males aged two to three weeks post maturity were exposed to female chemical signals left on silk. Females of unknown personality types were placed in 15 cm plastic arenas on filter paper and allowed to lay down silk for approximately 12 hours. The following day, females were removed and males were placed in the arenas and allowed to court for 5 minutes. All trials were recorded using a SONY

Handycam HDR-XR260. Latency to move, number of cheliceral strikes, jerky taps, bouts, average taps per bout, and average strikes per bout were all recorded (Delaney et. al 2007). Each male was exposed to female cues only once.

Live Mating Trials with Different Personalities

Males and females were paired in a 2 x 2 factorial design, with bold and shy males and bold and shy females offering a total of four possible personality matches to analyze overall mating success. Females were placed in 15 cm clear plastic arenas and allowed to lay down silk on filter paper for 15 minutes. Males were then dropped into the arena and allowed to interact with the female for 5 minutes. All trials were recorded using a SONY Handycam HDR-XR260.

Latency to court, time between start of courtship and first receptivity display, total courtship time, number of receptivity displays, total number of bouts, and total bouts divided by the courtship duration were recorded. Overall mating success (mated, not-mated, or cannibalized before mating) was also recorded.

Differences Between Research Seasons

The original design for this experiment did not include an open field test. However, after noting differences in latency values to begin exploring during the acclimation period for the predator stimulus assay, an open field assay was included. However, for the first two research seasons (conducted in 2016 and 2017), the open field assay was the acclimation period before the predator stimulus test, and consequently was only 60 seconds. For the final research season

(2018), open field and predator stimulus assays were conducted separately to extend the open field trials. Spiders were subjected to one trial per day (alternating between assay types - open field and predator stimulus - to prevent habituation) over a six day period until three replicates of each trial were conducted. Open field trials were changed to 5 minutes each instead of 60 seconds, and predator stimulus trials remained unchanged. Spiders were subjected to the same trials as penultimate instars and as adults, and all other subsequent tests (involving mating success and mate preference) remained the same.

Statistical Analyses

For the open field assay, all spider behaviors were labeled as explore, freeze, or run for each trial. Trial number, sex, and lifestage were analyzed as fixed effects in a nonparametric survival analysis. Latency to begin exploring in the open field was the dependent variable. Each spider’s identification number was nested within trial number as a random effect, and trial number was nested within lifestage. Spiders that explored during all three trials were relabeled as

“bold” personalities, while spiders that froze during all trials were relabeled as “shy” personalities. Running spiders were analyzed for open field behavioral consistencies only and were excluded from further analyses because of their low numbers. Spiders that demonstrated both exploratory and freezing behaviors were labeled as “unclear” personalities, and were initially withheld from further analyses. An ICC (intraclass correlation coefficient) was calculated by dividing the between-individual variance over the total variance to determine repeatability of individual behaviors across trials.

Simulated predator stimulus trials were analyzed similarly to open field tests with trial number, sex, lifestage, and initial response to open field (explore, freeze) as fixed effects.

Latency to resume normal behavior after the introduction of a predator stimulus was the dependent variable. To determine if behaviors were correlated across behavioral assays, during the 2018 research season (and the female mate choice and male courtship trials of the spring/summer 2017 research season), spiders were also given a boldness score associated with their open field test results in addition to a personality category. Boldness scores (ranging from 3 to 0) represented the number of times a spider explored during its open field tests. Spiders who began exploring within 10 seconds during every trial were given a score of 3. Those who froze during every trial were given a score of 0. Boldness scores and personalities were used to determine if latency to begin exploration in an open field test was correlated with latency to resume exploration after a simulated predator stimulus.

Female mate choice tests were analyzed using a nonparametric survival analysis for the latency to move after being introduced to the arena between bold and shy females. An ANOVA

(when data were normally distributed) and a Kruskal Wallis analysis (when data were not normally distributed) were used to determine the differences in overall display rates between

12 females and to determine differences in responses to a large-tufted male minus the responses to a small-tufted male in terms of visits to each screen, time spent at each screen, types of displays

(settle, pivot, or tandem leg extend) exhibited to each screen, and total displays to each screen for bold and shy females. A nonparametric survival analysis was also used to analyze differences in latency to court on female silk between males. Male courtship on female cues was analyzed using univariate ANOVAs and Kruskal Wallis tests to determine differences in total cheliceral strikes, taps, and bouts of courtship as well as mean strikes and mean taps per bout due to personality type. Variables that were not normally distributed were square-root transformed to improve normality. If normality was not significantly improved, a Kruskal Wallis test was used.

Live mating trials were analyzed using a nonparametric analysis, an ANOVA, and a

Kruskal Wallis test with male and female personalities as fixed effects. Response variables for males included latency to court, courtship duration, total bouts of courtship, and bouts of courtship divided by the total courtship duration for males, while time from start of courtship until first receptivity display and total receptivity displays were measured for females. Rates of successful mating, non-successful mating, and cannibalism were also analyzed.

RESULTS

Results from season 1 (Fall 2016-Winter 2017)

Part I: Lab-reared spiders in an open field

A Shapiro-Wilk test showed that the latency values to begin exploring in the open field test were not normally distributed (N=773, W=0.830009, p<0.0001). A nonparametric proportional hazards survival analysis showed that sex had an effect on latency to begin exploring for penultimate individuals (X2=5.7937, df=1, p=0.0161), but trial number did not

(X2=0.7713, df=2, p=0.6800; Figure 1). There was no difference in latency values to begin

13 exploring for penultimate individuals, suggesting responses are repeatable (ICC=0.4598). Mature spiders also showed an effect of sex (X2=7.8960, df=1, p=0.0050) but not trial number

(X2=1.0969, df=2, p=0.5778, Figure 2). Latency values showed repeatability across trials

(ICC=0.4561). Freezing was the most common behavior in all trials except for mature females

(Figures 3-6). When penultimate and mature spiders were combined into a full model, lifestage

(penultimate, mature) was not significant (X2=0.7487, df=1, p=0.3869). Sex was significant

(X2=13.3655, df=1, p=0.0003). Trial number (nested within lifestage) was not significant

(X2=1.8222, df=4, p=0.7684, ICC=0.3905).

Part II: Lab-reared spiders’ responses to a simulated predator stimulus

A Shapiro-Wilk test showed that the latency values to return to normal behavior after the introduction of a predator stimulus were not normally distributed (N=773, W=0.7361, p<0.0001). A nonparametric proportional hazards survival analysis showed that sex, lifestage, and trial number (nested within lifestage) had no effect (sex: X2=1.7666, df=1, p=0.1838; lifestage: X2=0.5190, df=1, p=0.4713; trial number: X2=3.2660, df=4, p=0.5143). Initial reaction to a new environment had an effect (X2=73.3322, df=1, p<0.0001). Wald test results were reported. A Pearson’s correlation with an ANOVA showed a significant correlation between the latency to begin exploring in an open field and latency to begin exploring after a simulated predator stimulus for penultimate females and penultimate males (females: R2=0.2516,

F=9.5277, df=1, p=0.0024, slope=0.6978; males: R2=0.2209, F=5.8472, df=1, p=0.0172, slope=0.5603, Figures 7-9). A significant correlation was also found for mature females but not for mature males (females: R2=0.1978, F=6.4714, df=1, p=0.0119, slope = 0.7155; males: R2=

0.1053, F=1.5478, df=1, p=0.2156, slope = 0.2422, Figures 10-12).

Results from season 2 (Spring-Summer 2017)

Part III: Field-exposed spiders in an open field

A Shapiro-Wilk test showed that the latency values to begin exploring in an open field for field-exposed spiders were also not normally distributed (N=611, W=0.8202, p<0.0001). A nonparametric proportional hazards survival analysis was used. Sex, lifestage, and trial number were not significant (sex: X2=1.8869, df=1, p=0.1696; lifestage: X2=0.9455, df=1, p=0.3309; trial number: X2=0.2530, df=4, p=0.9926). There were no differences in individual latency values across trials (ICC=0.2446).

Part IV: Field-exposed spiders’ responses to a simulated predator stimulus

A Shapiro-Wilk test showed that the latency values to return to normal behavior after the introduction of a predator stimulus for field-exposed spiders were also not normally distributed

(N=611, W=0.6223, p<0.0001). A nonparametric proportional hazards survival analysis showed no differences in lifestage or trial number (lifestage: X2=1.3030, df=1, p=0.2537; trial number:

X2=1.5986, df=4, p=0.8090). Initial reaction and sex were significant (initial reaction:

X2=460.5223, df=2, p<0.0001; sex: X2=965.6318, df=1, p<0.0001). A Pearson’s correlation with an ANOVA showed a significant correlation between the latency to explore in an open field and the latency to resume normal behavior after a simulated predator stimulus for penultimate females but not for penultimate males (females: R2=0.2211, F=8.0217, df=1, p=0.0052, slope =

0.6193; males: R2=0.1751, F=3.2271, df=1, p=0.0754, slope=0.7483, Figures 19-21). A significant correlation was also found for mature males but not for mature females (males: R2=

0.3238, F=13.0050, df=1, p=0.0005, slope = 0.6891; females: R2=0.0919, F=1.3897, df=1, p=0.2402, slope = 0.1605, Figures 22-24).

Part V: Lab-reared vs field-exposed

A nonparametric proportional hazards survival analysis found significant differences between the latency to start exploring in an open field between lab-reared and field-exposed spiders for both matures (X2=8.4424, df=1, p=0.0037) and penultimate individuals (X2=55.0721, df=1, p=<0.0001). A nonparametric proportional hazards survival analysis also found significant differences between latency to resume exploration after a simulated predator stimulus between lab-reared and field-exposed spiders for both matures (X2=35.9614, df=1, p=<0.0001) and penultimate individuals (X2=42.8943, df=1, p=<0.0001). A two-way chi-squared contingency analysis revealed that lab-reared and field-exposed spiders also had significantly different proportions of spiders exploring, freezing, and running in their respective populations, with more exploring spiders present in the field-exposed population and more freezing and running spiders present in the lab-reared population (X2=73.59, p<0.0001).

Part VI: Female mate choice

The latency to move values in the female mate choice test were not normally distributed

(N=59, W=0.8745, p<0.0001). A nonparametric proportional hazards survival analysis revealed a significant difference between the latency to move values for females with different boldness scores (X2=8.1110, df=3, p=0.0438). The amount of time spent in front of large-tufted male vs small-tufted males was not significantly different between female personality scores

(F3,54=0.3250, p=0.8073). All other variables were not-normally distributed, and only the normality of the number of visits to large-tufted male vs. small-tufted male was significantly improved by square-root transformation (N=59, W=0.9623, p=0.0651). An ANOVA showed the number of visits to large-tufted males vs. small-tufted males was also not significantly different between females (F3,54=0.4059, p=0.7494). A Kruskal Wallis analysis showed that there were no differences between females for overall total displays (X2=2.2450, df=3, p=0.5231), total

16 displays to a large-tufted male vs displays to a small-tufted male (X2=1.4389, df=3, p=0.6964),and the number of tandem leg extends (TLEs), settles, and pivots to large or small- tufted males (TLEs: X2=2.0748, df=3, p=0.5570; ;settles: X2=0.4372, df=3, p=0.9324; pivots:

X2=1.5570, df=3, p=0.6692).

Part VII: Male courtship on female silk

Male latency values to move were not normally distributed (N=39, W=0.5841, p<0.0001). A nonparametric proportional hazards survival analysis revealed no significant difference between males’ latency to move based on boldness score when placed on female silk

2 (X =3.8921, df=3, p=0.2733). An ANOVA showed no differences between males for total taps during courtship and total bouts of courtship (taps: F3,35=0.2413, p=0.8669; bouts: F3.35=0.7003, p=0.5584). After being square-root transformed, normality for total cheliceral strikes and average strikes per bout were improved (total strikes: W=0.9786, p=0.6681; average strikes per bout: W= 0.9744, p=0.5221), and an ANOVA showed there were no differences between males for total cheliceral strikes and average strikes per bout of courtship (total strikes: F3,35=0.9659, p=0.4201; average strikes per bout: F3,35=0.5469, p=0.6536). A Kruskal Wallis analysis showed no differences between average taps per bout of courtship between males (X2=1.2216, df=3, p=0.7478).

Part VIII: Mating personalities together

Using data combined from both 2017 and 2018 (the second and third research seasons), an ANOVA showed that neither male personality nor female personality had an effect on the likelihood of overall mating success (male personality: X2=0.2406, df=1, p=0.6238; female personality: X2=0.0534, df=1, p=0.8172). Latency to court was not normally distributed (N=39,

W=0.6367, p<0.0001). A nonparametric proportional hazards survival analysis revealed that

17 male personality had no effect on male latency to court (X2=2.1521, df=1, p=0.1424), but female personality was borderline (X2=3.7350, df=1, p=0.0533), and the interaction between male and female personality was significant (X2=8.0633, df=1, p=0.0045). An ANOVA showed there were no effects of female or male personality on total receptivity displays exhibited by females

(male personality: F=0.5028, df=1, p=0.4843, female personality: F=0.3264, df=1, p=0.5725).

Square-root transforming total bouts of courtship for males improved normality (N=39,

W=0.9492, p=0.0773), and neither male nor female personality had an effect on total bouts of courtship (male personality: F=0.9605, df=1, p=0.5565; female personality: F=0.6163, df=1, p=0.6372). A Kruskal Wallis showed neither male nor female personality had a significant effect on male courtship duration (male personality: X2=0.0019, df=1, p=0.9649; female personality:

X2=1.9407, df=1, p=0.1636), bouts/courtship duration (male personality: X2=0.3528, df=1, p=0.5525; female personality: X2=0.0622, df=1, p=0.8030), or time lapsed until first receptivity display is exhibited by the female (male personality: X2=1.8323, df=1, p0.1759; female personality: X2=3.5325, df=1, p=0.0602).

Results from season 3 (Fall 2017-Winter 2018)

Part IX: Lab-reared spiders in extended open field test

The latency values to begin exploring in an open field were not normally distributed

(N=851, W=0.4123, p<0.0001). A nonparametric proportional hazards survival analysis showed that sex, lifestage, and trial number were not significant for latency values to begin exploring in extended open field tests (sex: X2=1.1435, df=1, p=0.2849; lifestage: X2=1.3758, df=1, p=0.2408; trial number: X2=1..6715, df=4, p=0.7959, ICC=0.14).

Part X: Lab-reared spiders’ responses to a simulated predator stimulus

The latency values to return to normal behavior after the introduction of a simulated predator stimulus were not normally distributed (N=821, W=0.5465, p<0.0001). A nonparametric proportional hazards survival analysis showed that sex, lifestage, and trial number were not significant for latency to recover from a simulated predator stimulus (sex: X2=1.1695, df=1, p=0.2795; lifestage: X2=0.0902, df=1, p=0.7639; trial number: X2=1.8880, df=4, p=0.7563). Boldness score was not significant for penultimate individuals or mature individuals

(penultimate: X2=4.1437, df=3, p=0.2464; mature: X2=4.3923, df=3, p=0.2221 Figures 25-26).

DISCUSSION

This research has revealed that individual S. ocreata exhibit behavioral differences that persist across time and context, consistent with current definitions of animal “personality” (Dall,

Houston, and McNamara, 2004; Kralj-Fišer and Schuett, 2014). Penultimate spiders demonstrated repeatable reactions to the open field test in that some consistently explored the arena while others consistently froze. After being classified as “bold” (for those who explored) or shy (for those that froze), penultimate spiders also showed repeatable latency to resume normal behavior after being prodded by a simulated predator stimulus. Shorter latency to begin exploration in an open field was significantly correlated with shorter latency to resume exploration after the introduction of a predator stimulus (and vice versa), and these patterns persisted across trials. This correlation across trials suggests that S. ocreata likely exhibit

“behavioral syndromes” as well as personality.

These spiders were also subjected to the same trials after reaching maturity, and the same patterns were found. All latency values were consistent across trials within the same assay.

However, during the first research season, as penultimate individuals and as mature individuals, female spiders showed consistent latency values both in the open field and with the simulated

19 predator, and these values were correlated between assays. In contrast, while penultimate males’ latency to return to exploratory behavior after a predator stimulus was correlated with the latency to begin exploration in an open field, this correlation disappeared after reaching maturity. Mature males’ latency to begin exploring an open field was not correlated with their latency to resume exploration after being prodded. In the second and third research season, this correlation was flipped, in that mature female latency values were not correlated between assays, but male latency values were. However, within the same assays, individual latency values were still consistent between trials. Similar results and age effects (differences between lifestages) have been found in other recent arthropod studies which support these findings (Stanley et. al 2017).

These distinct behavioral syndromes suggest that while average population behaviors are important, it is also necessary to account for the behaviors of individual animals.

While spiders showed consistency in behavior across trials for both open field and simulated predator stimulus trials, lab-reared and field-exposed spiders showed some notable differences. Unlike field-exposed spiders, the lab-reared spider population had an overall greater proportion of shy spiders than bold spiders. This could suggest that personality may also be influenced by the environment in which an individual is raised. Field-exposed spiders would likely have encountered predators and other aversive stimuli prior to the simulated predator stimulus trials, whereas lab-reared spiders were housed in individual opaque containers from early instar stages. Previous exposure to threat stimuli could allow field-exposed spiders to learn from previous predator-prey scenarios and habituate to simulated threats more quickly. On the other hand, bold spiders could be better adapted to their complex environments, and their quick recovery rates may be better suited for avoiding predation. Shy individuals may be selected against in the field and therefore not be as prevalent. Mature lab-reared spiders also showed a

(nonsignificant) trend of decreasing latency to begin exploration from trial 1 to trial 3, implying that mature lab-reared spiders might have a component of learning or habituation influencing responses to the open field test. In addition, while “running” spiders made up roughly 5 to 10% of the population of lab-reared spiders, they were virtually non-existent in the field-exposed population. Again, this could suggest that lab-reared spiders may experience conditions during development which may change their behaviors from what is seen in field-exposed spiders. On the other hand, field-exposed spiders might not exhibit running behavior because of the increased risk that continuous movement poses in their natural environments. Constant rapid movement could potentially attract the attention of visual predators like birds, so frenetic movement could be disadvantageous.

In the 2018 research season, open field tests were lengthened to five minutes and separated from predator stimulus trials instead of the original 60 second acclimation period conjoined with the predator stimulus trial. The results of trials conducted in 2018 still showed repeatability across trials for both penultimate and mature spiders. The distribution of data was not as strongly bimodal as it had been with the 60 second trials, but the pattern remained the same.

Contrary to expectations, few correlations were found between open field results and mate preference or mating success. Mature bold and shy males showed no differences in latency to court or any elements of courtship when only exposed to female cues. Simultaneously, while mature bold and shy females differed in their latency to move when given a choice between large and small-tufted males, they displayed no differences in preference or receptivity for males when exposed to video playback. In addition and irrespective of personality type, females also showed no preference for large-tufted or small-tufted males, which diverges from previous results where

21 females showed more receptivity to large-tufted males (McClintock and Uetz 1996; Scheffer et. al 1996; Uetz 2000; Uetz and Roberts 2002; Uetz and Norton 2007; Uetz et al. 2017). Both studies could be investigated further to see if personality type affects different elements than were tested at this time. In this study, bold and shy males were exposed to chemical cues (in silk) from females of undetermined personality types. In a follow-up study, bold and shy males could be exposed to silk from females with predetermined personality types to determine if (1) the personality type of the female has any effect on the properties of her silk, (2) if that effect is detectable by courting males, and (3) if bold and shy males show a preference for bold or shy female silk. Females could also be exposed to both visual and vibratory courtship elements during video playback since this study only exposed them to visual signals. However, live mating trials showed no differences between any paired personalities. Overall courtship duration, male latency to court, latency for females to be receptive, and all other variables showed no differences. There was a significant interaction between male personality and female personality for male latency to court, and further analysis showed that bold males seemed to take longer to court bold females, as did shy males with shy females. It is possible that a larger sample size could strengthen these trends. However, overall mating success was not significantly affected by male or female personality type. Therefore, it seems unlikely that further two-choice tests or male courtship trials will illuminate any differences, and further live mating trials are unlikely to produce notably different conclusions. Based on these results, bold-shy personality type as assessed in this study appears to directly influence somatic traits but not traits involving reproduction. Personality type may regulate more voluntary and flexible behavior such as reacting to a predator, but it does not appear to impact courtship or mate choice.

While differing personalities have been observed, repeating test results and also modifying experimental procedures could also help expand the dimensions of the possible personalities of these spiders. This experiment used forceps as a prod stimulus to simulate a predator’s approach, and the forceps were used to touch the right anterior foreleg of each animal.

Other studies assessing animal personalities have used different methods. Multiple studies have used a paintbrush to touch the abdomen or back of an animal and produced significant repeatable results showing differences in behavior between individuals (Pruitt et. al 2013; Mather and

Anderson 1993). Forceps were used in this study because a previous study (unpublished) used the same method and produced notable reactions from the spiders. However, either tool could be used, and touching the back of an animal could produce different results. This study could be performed again but using a paintbrush to touch the abdomen of the spiders to see if the results are corroborated or if different results are produced. Secondly, critics have argued that two different types of open field tests exist and they measure different personality traits (Carter et. al

2013 state; Misslin and Cigrang 1986; Walsh and Cummins 1976). In a forced open field test, subjects are placed in an open field and observed without access to a hide or any form of retreat.

In a free open field test, entry into the open field is voluntary. Misslin and Cigrang (1986) argue that free open field tests measure curiosity and exploratory levels, while forced open field tests measure either fear or anxiety. However, boldness is defined as willingness to engage in risk- taking behavior. Moving around in a new environment is risky in that the movement might make the subject more susceptible to detection by predators. Therefore, I believe both free and forced open field tests measure some aspect of boldness. This study could and should be conducted again using a free open field test to compare results, but using a forced open field test does not invalidate the results or negate the use of boldness as a personality trait.

Why should individual variance in behaviors and personalities exist in a population of S. ocreata wolf spiders? One reason is that different personalities may be more or less beneficial at different times. As stated previously, being a bolder individual may increase the likelihood of prey capture. However, it could simultaneously increase the risk of being eaten by a predator due to higher levels of motion. Many spiders are preyed on by birds and other animals that track their prey through movement. Shy spiders may decrease their risk of predation due to their propensity to “freeze” in uncertain or potentially dangerous contexts. At the same time, this behavior may make them more unlikely to go after potential prey. In addition, while different personalities showed no significant differences in mating success or mate choice, previous studies have shown that while females prefer males with larger tufts, females are also more likely to mate with the first male that moves (Clark and Uetz 1992; Scheffer et al. 1996). If bold spiders are more likely to move and explore first, their chances of successful mating may increase simply due to greater likelihood of encountering a female. Females are also monandrous, so males that move the most could have an advantage. Lastly, given that S. ocreata live in a seasonally changing environment, changes in selection pressures could make one personality more fit than another depending on the current state of the habitat.

Another concern often raised in studies of animal personality is the “jingle fallacy”

(Gosling 2001; Bell 2007; Yuen et. al 2017). The jingle fallacy ensues when multiple assays are used to measure the same trait when they should be used to measure different traits. This study used both open field tests and simulated predator stimulus tests to measure boldness levels, and correlations were found between these two tests. However, other studies have found contrasting results (Yuen et. al. 2017), and critics have argued that prod tests and open field tests measure different personality traits. Prod tests are often considered startle tests and may be considered

24 measurements of anxiety rather than boldness. Likewise, open field tests have been used to measure levels of exploration as a separate trait from boldness. Studies of personality should standardize test results and try to use the same terms for simplicity. However, animals display varying levels of complexity and behavioral variation, so it is entirely possible that prod tests measure one trait in some animals and another trait in others. However, correlations were found between the two assays for this study, and these tests may measure different traits in different animals, so an open field test and a prod test appear to be applicable for boldness tests for S. ocreata.

This study provides further evidence of the complexity of arthropods and other presumed

“less complex” animals as a whole, and it also highlights the importance of individuality.

Although personality is primarily applied to humans and other animals of “higher taxa,” it can also be applied to lower taxa, and thereby provide insight regarding why animals behave differently in the same environment. The personality types and behavioral syndromes exhibited by S. ocreata in this study can potentially aid future research seeking to determine the impact personality type has on individuals, and advance understanding of how individual variation influences animals’ behavior.

FUTURE DIRECTIONS

While this study has demonstrated the existence of different personality types in S. ocreata, evidence for behavioral syndromes is incomplete. The conclusions of this study could be further supported by determining the influence of personality type on other behaviors, e.g., foraging. Jumping spiders and orb-weavers are well-known for their abilities to distinguish between different types of prey as well as to assess the overall risk a potential prey presents.

Previous studies involving Portia (araneophagus jumping spiders) have shown that while personality type may not influence the size of prey captured, it can influence the time required to choose between prey sizes when given two choices (Chang, Ng, and Li 2016). Wolf spiders are also good candidates for such tests as they are not only adept at prey capture but also exhibit specific prey capture tactics, e.g., a “shoulder roll” in which cases they flip on their backs when tackling large prey in order to increase their abilities to manipulate their targets (Rovner 1980).

Such complex prey capture tactics present possibilities regarding different methods of prey capture as well as differing degrees of likelihood to take on larger prey targets.

Personality type could also be assessed and compared across species. Gosling (2001) explains the need to compare personalities across various animal groups to get a more accurate and objective perspective of what constitutes “aggressiveness.” Gosling gives the example of how a black mamba (Dendroaspis polylepis) would generally be considered a very aggressive species, but individual black mambas may be considered less aggressive than others when compared within a population. In the case of S. ocreata, there is a sister species (S. rovneri) that has identical morphological characteristics except that males do not develop tufts after reaching maturity (Stratton & Uetz 1981, 1983, 1986; Stratton 2005), even though females are indistinguishable. However, these species are reproductively isolated by courtship behavior.

While they are physically able to interbreed, females only respond to the courtship signals of conspecifics, and S. ocreata males use multi-modal courtship signals while S. rovneri use unimodal signals (McClintock and Uetz 1996). Comparing the personalities of these sister species could yield interesting results regarding the relative bold and shy tendencies between and within the groups.

One aspect of personality that has not been highly explored, particularly in spiders, is its potential correlation with an individual’s ability to learn. Spiders appear to possess the ability to learn based on previous experience. Male S. ocreata learn to eavesdrop on other courting males and begin courting even without female cues (Clark, Roberts, and Uetz 2012). This study also revealed a trend (though not significant) of females exploring more quickly in the third open field trial than the first open field trial, implying that females may have begun habituating to the trials. Potential differences in the capacity to learn could add another layer to the complexity that personality types represent, and differences in these capacities could be a product of previous experience.

The aforementioned correlations between different behaviors based on personality could also potentially explain why maladaptive behaviors exist in populations (Sih et. al. 2003,

Johnson and Sih 2005; Bell 2007). While many studies in the past have focused on one behavior at a time, the presence of personalities suggests that behaviors may be linked together into identifiable behavioral syndromes (Bell 2007). These links could explain why some individuals exhibit sub-optimal behaviors in one context but optimal behaviors in another. Studying every behavior separately might overlook effects of other correlated behaviors. This could also help explain the loss or gain of behavioral traits across phylogenies.

Another aspect of measuring personality that has been brought up in previous studies is the need for observer agreement. Personality studies clearly need to measure the same animal multiple times as the definition of personality postulates consistent behavioral differences between individuals. Repeating tests for each animal satisfies this requirement, but Gosling and others argue that more than one researcher should observe each specimen through the trials to prevent bias from occurring from using only a single scientist. One scientist may acknowledge

27 more subtle behaviors in the study while another may not. This can often depend on the experimenters’ relative familiarity with the animals in question. Having multiple people test the same individual and observe each test can hopefully lead to a consensus about each behavior and consequently make results more reliable (Gosling 2001; Block 1961). As a result, this study could be repeated but with multiple observers to further increase the accuracy and account for any potential bias presented by only having one observer. However, only having one observer may also increase the consistency with which each animal was assessed and consequently does not pose an issue in this study. Overall this study met the requirements for measuring personality in individuals in a population and seems to support the idea that distinct bold and shy personalities exist within S. ocreata.

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FIGURES

Females Males

Figure 1. Latency to begin exploring in an open field arena across trials (trial 1-blue, trial 2- orange, trial 3-gray) for penultimate lab-reared females and males

Females Males

Figure 2. Latency to begin exploring in an open field arena across trials (trial 1-blue, trial 2- orange, trial 3-gray) for mature lab-reared females and males.

100% 90% 80% 34.33% 34.85% 39.93% 70% 60% 50% 40% 47.76% 53.03% 51.52%

Proportion 30% 20% 10% 17.91% 12.12% 9.09% 0% TRIAL 1 TRIAL 2 TRIAL 3

Figure 3. Overall distribution of behaviors (explore-red, freeze-blue, tan-run) exhibited by lab- reared penultimate females during open field trials.

100% 90% 26.60% 27% 26.20% 80% 70% 60% 50% 40% 62.50% 65.10% 63.90%

Proportion 30% 20% 10% 10.90% 9.80% 0% 7.90% TRIAL 1 TRIAL 2 TRIAL 3

Figure 4. Overall distribution of behaviors (explore-red, freeze-blue, tan-run) exhibited by lab- reared penultimate males during open field trials.

100% 90% 80% 41.79% 48.44% 70% 54.55% 60% 50% 40%

Proportion 30% 53.73% 50.00% 39.39% 20% 10% 0% 4.48% 1.56% 6.06% TRIAL 1 TRIAL 2 TRIAL 3

Figure 5. Overall distribution of behaviors (explore-red, freeze-blue, tan-run) exhibited by lab- reared mature females during open field trials.

100% 90% 23.80% 31.30% 80% 36.50% 70% 60% 50% 65.10% 40% 65.60% 55.60% Proportion 30% 20% 10% 11.10% 7.90% 0% 3.10% TRIAL 1 TRIAL 2 TRIAL 3

Figure 6. Overall distribution of behaviors (explore-red, freeze-blue, tan-run) exhibited by lab- reared mature males during open field trials.

Figure 7. Nonparametric survival analysis depicting proportion of bold (red) and shy (blue) lab- reared penultimate females recovering from a simulated predator stimulus.

Figure 8. Nonparametric survival analysis depicting proportion of lab-reared bold (red) and shy (blue) penultimate males recovering from a simulated predator stimulus.

300

250

200

Latency to Resume150 Normal Behavior (s) 100

0 0 10 20 30 40 50 60 Latency to Begin Exploring (s) Figure 9. Pearson’s correlation showing correlation between latency to begin exploring in an open field arena and latency to resume normal behavior after a predator stimulus for penultimate females (red) and males (blue).

Figure 10. Nonparametric survival analysis depicting proportion of lab-reared bold (red) and shy (blue) mature females recovering from a simulated predator stimulus.

Figure 11. Nonparametric survival analysis depicting proportion of lab-reared bold (red) and shy (blue) mature males recovering from a simulated predator stimulus.

300

250

200

150

100

Latency to Resume Resume to Latency Behavior(s) Normal 0 0 10 20 30 40 50 60

Latency to Begin Exploring (s)

Figure 12. Pearson’s correlation showing correlation between latency to begin exploring in an open field arena and latency to resume normal behavior after a predator stimulus for mature females (red) and males (blue).

Females Males

Figure 13. Latency to begin exploring in an open field arena across trials (trial 1-blue, trial 2- orange, trial 3-gray) for penultimate field-exposed females and males

Females Males

14. Latency to begin exploring in an open field arena across trials (trial 1-blue, trial 2-orange, trial 3-gray) for mature field-exposed females and males

100% 90% 80% 70% 56.67% 55.74% 57.63% 60% 50% 40%

Proportion 30% 20% 43.33% 44.26% 42.37% 10% 0% TRIAL 1 TRIAL 2 TRIAL 3

Figure 15. Overall distribution of behaviors (explore-red, freeze-blue) exhibited by field- exposed penultimate females during open field trials.

100% 90% 80% 70% 53.66% 67.50% 63.41% 60% 50% 40%

Proportion 30% 46.34% 20% 30.00% 36.59% 10% 0% 2.50% 0.00% 0.00% TRIAL1 TRIAL 2 TRIAL 3

Figure 16. Overall distribution of behaviors (explore-red, freeze-blue, tan-run) exhibited by field-exposed penultimate males during open field trials.

100% 90% 31.67% 31.15% 28.33% 80% 70% 60% 50% 40% 68.33% 68.85% 71.67% Proportion 30% 20% 10% 0% TRIAL 1 TRIAL 2 TRIAL 3

Figure 17. Overall distribution of behaviors (explore-red, freeze-blue) exhibited by field- exposed mature females during open field trials.

100% 90% 80% 41.46% 46.34% 48.78% 70% 60% 50% 40%

Proportion 30% 56.10% 51.22% 51.22% 20% 10% 0% 2.44% 0.00% 2.44% TRIAL1 TRIAL 2 TRIAL 3

Figure 18. Overall distribution of behaviors (explore-red, freeze-blue, tan-run) exhibited by field-exposed mature males during open field trials.

Figure 19. Nonparametric survival analysis depicting proportion of field-exposed bold (red) and shy (blue) penultimate females recovering from a simulated predator stimulus.

Figure 20. Nonparametric survival analysis depicting proportion of field-exposed bold (red) and shy (blue) penultimate males recovering from a simulated predator stimulus

Figure 21. Nonparametric survival analysis depicting proportion of field-exposed bold (red) and shy (blue) mature females recovering from a simulated predator stimulus

100% 90% 80% 70% 60% 50% 40%

30% Percent Exploring Percent 20% 10% 0% 0 50 100 150 200 250 300 Latency to Resume Exploration (s)

Figure 22. Nonparametric survival analysis depicting proportion of field-exposed bold (red) and shy (blue) mature males recovering from a simulated predator stimulus

300

250

200

150

100

Latency to Resume Resume to Latency 50 Normal Behavior (s) Behavior Normal

0 0 10 20 30 40 50 60 Latency to Begin Exploring (s)

Figure 23. Pearson’s correlation showing correlation between latency to begin exploring in an open field arena and latency to resume normal behavior after a predator stimulus for field- exposed penultimate females (red) and males (blue).

300

250

200

150

100 Latency to Resume Resume to Latency Normal Behavior (s) Behavior Normal 50

0 0 10 20 30 40 50 60 Latency to Begin Exploring (s)

Figure 24. Pearson’s correlation showing correlation between latency to begin exploring in an open field arena and latency to resume normal behavior after a predator stimulus for field- exposed mature females (red) and males (blue).

Figure 25. Nonparametric survival analysis depicting proportion of lab-reared mature females recovering from a simulated predator stimulus using boldness scores (blue-score of 0 (least bold), yellow-score of 1, green-score of 2, red-score of 3 (most bold)).

Figure 26. Nonparametric survival analysis depicting proportion of lab-reared mature males recovering from a simulated predator stimulus using boldness scores (blue-score of 0 (least bold), yellow-score of 1, green-score of 2, red-score of 3 (most bold)).