ABSTRACT FEMALE RESPONSES to MALE CHEMICAL CUES in PARDOSA MILVINA WOLF SPIDERS by Michael T. Stanley Females Often Use Male Si

ABSTRACT

FEMALE RESPONSES TO MALE CHEMICAL CUES IN PARDOSA MILVINA WOLF SPIDERS

by Michael T. Stanley

Females often use male signals and cues to locate potential mates and assess their quality. These male signals can be transmitted across one or multiple signaling modalities. In the wolf spider Pardosa milvina, males use a visual courtship display to attract female attention and encourage sexual receptivity. However, whether or not other signaling modalities influence female mate choice in this species is poorly understood. I hypothesized that male chemical cues may play a role in female mate choice in addition to visual cues. I tested females for their ability to detect and assess males based on their chemical cues, both isolated and when combined with a visual signal. Females did not change their activity in the presence of isolated male chemical cues. When presented with males either surrounded by or lacking their chemical cues, I found that while male courtship played a major role in female detection and attraction, females tended to spend less time near males when their chemical cues were present. This research suggests that while male courtship displays are necessary and sufficient for mate attraction, chemical signals may play a limited role by helping females more quickly assess a male’s visual display.

FEMALE RESPONSES TO MALE CHEMICAL CUES IN PARDOSA MILVINA WOLF SPIDERS

A Thesis

Submitted to the

Faculty of Miami University

in partial fulfillment of

the requirements for the degree of

Master of Science

Michael T. Stanley

Miami University

Oxford, Ohio

2018

Advisor: Ann L. Rypstra

Reader: Brian Keane

Reader: Nancy G. Solomon

Reader: Ann L. Rypstra

This Thesis titled

FEMALE RESPONSES TO MALE CHEMICAL CUES IN PARDOSA MILVINA WOLF SPIDERS

Michael T. Stanley

has been approved for publication by

The College of Arts and Science

and

Department of Biology

______Ann L. Rypstra

______Brian Keane

______Nancy G. Solomon

Table of Contents

List of Tables…………………….iv

List of Figures……………………v

Dedication………………………..vi

Acknowledgements………………vii

Introduction………………………1

Methods…………………………..5

Results……………………………10

Discussion………………………..11

References………………………..16

Tables & Figures…………………22

iii

List of Tables

Table 1………………….24

Table 2………………….25

Table 3………………….26

Table 4………………….27

Table 5………………….28

Appendix 1……………..34

Appendix 2……………..36

Appendix 3……………..43

List of Figures

Figure 1……………….22

Figure 2……………….23

Figure 3……………….29

Figure 4……………….30

Figure 5……………….31

Figure 6……………….32

Figure 7……………….33

Dedication

I would like to dedicate this thesis to my wife Gabby, who stuck by my side through thick and thin, always encouraged me to never give up, and continually inspires me to be a better person. I would also like to dedicate this to my parents (and former Miami alumni) Mary and Jim, as without their unconditional love and support I never would have gotten to where I am today.

Acknowledgements

I would first like to thank my advisor, Dr. Ann Rypstra, not only for providing excellent scientific insight when helping me with my experiments, but also for always being there to support and guide me through the crazy process that is graduate school. Thanks also to my excellent committee members Dr. Nancy Solomon and Dr. Brian Keane for sticking with me through a somewhat unorthodox scientific journey. I would also like to acknowledge Miami University for supporting my studies and providing funding for these experiments. Finally, I would like to thank all the members of the Rypstra lab, graduate and undergraduate alike, for ensuring that the Miami spider lab was always an interesting place to be.

vii

Introduction

Being able to find and choose a mate is crucial in organisms that reproduce sexually. In order to accomplish this task, many organisms rely on a variety of signals that must be received and interpreted against other stimuli in the environment (Guilford and Dawkins 1991). These signals can be visual, olfactory, tactile, auditory, or any combination of the above. This diverse range of signals allows them to be used in a wide variety of ways, such as to locate potential mates (Ryan 1991), to indicate that an organism is a conspecific (Byrne and Keogh 2007), to inform of mating status (Roberts and Uetz 2005), and to indicate to the opposite sex that a potential mate is of high quality (Knapp and Kovach 1991). Understanding the types of signals an organism uses in order to locate and choose a mate will therefore allow for a better understanding of what types of information an organism processes and pays attention to before engaging in reproduction.

In many species, signals sent by males are the primary way in which females choose a mate. These signals can be passive ornaments (Zahavi 1975, Husak and Swallow 2011) or active courtship displays (Girard et al. 2015, Zambre and Thaker 2017). Females then use these signals in order to judge male attractiveness and quality before committing to reproduction. This puts selective pressure on males to develop signals that will not only catch the female’s attention, but also allow them to indicate that they are of high enough quality that they will be worth the energy that females are about to invest in mating and reproduction (Zahavi 1975, Hamilton and Zuk 1982, Hill 2015). For example, in the scorpionfly Panorpa vulgaris (Mecoptera: Panorpidae), the number of nuptial salivary gifts a male can provide to a female accurately reflects his nutritional status (Engels and Sauer 2006). Additionally, in the eastern kingbird Tyrannus tyrannus (Passeriformes: Tyrannidae), early singing males and males with a high song rate also had long flight feathers, indicative of superior body condition (Murphy et al. 2008). By communicating their quality to females through the use of signals, males can increase their chances of successfully attracting a mate.

In many cases, females do not rely exclusively on single display modalities when evaluating males. Rather, males can signal their quality to females through the use of multiple signaling modalities (Partan and Marler 1999, Candolin 2003, Higham and Hebets 2013). By employing more than one mode of communication, males can more accurately inform females

1 about their quality and increase their probability of detection by females (Candolin 2003, Hebets and Papaj 2005). For example, in the squirrel treefrog Hyla squirella (Anura: Hylidae), Taylor et al. (2007) found that while vocalizations by males were sufficient for female attraction, females also used visual cues to discriminate between males with similar call qualities, preferring males with a higher quality visual cue (larger lateral stripe). Males can also benefit from multimodal displays by producing an enhanced response in female receivers compared to separate unimodal signals (Partan and Marler 2005). In the fruit fly Drosophila melanogaster (Diptera: Drosophilidae), males that produced a combined acoustic and chemical display had greater mating success (61%) than males which were limited to only acoustic (37%) or only chemical (10%) signals (Rybak et al. 2002). The ability of multimodal signals to help males signal their quality to females and enhance female responses highlights the potential for signaling in multiple modalities to be beneficial for organisms that rely on signaling for mate attraction.

Multimodal signals have actually been extensively studied in the context of arachnid sexual signaling. While many male web spiders rely on the plucking and tapping of female webs to initiate courtship and mating (Segoli et al. 2008, Wignall and Herberstein 2013), groups that are cursorial such as wolf spiders (Lycosidae) primarily rely on visual courtship displays in order to attract and secure a mate (Rovner 1968, Koomans et al. 1974). Many species, especially those in the genus Schizocosa, augment their visual leg waving displays with vibratory cues (Uetz and Roberts 2002, Hebets 2008). This multimodal display helps males communicate in a structurally complex environment with a variety of substrates, as males have been found to shift between primarily visual or seismic signals based on the surrounding substrate (Gordon and Uetz 2011). Females of multiple species detect multimodal cues faster than visual or seismic cues alone, suggesting that these multimodal displays enhance the detection of males (Uetz et al. 2009). Females can also use both modalities present in these displays to assess the condition of males. Males in better condition have significantly larger foreleg tufts (Uetz et al. 2002) and are able to produce louder and shorter seismic signals (Gibson and Uetz 2008, 2012), both of which are preferred by females. Visual and vibratory signals therefore appear to be important components of wolf spider courtship. However, this does not mean that these are the only signaling modalities which may play a role in female mate choice.

Spiders use chemical signals in many situations, such as microhabitat selection and trail following (Tietjen and Rovner 1980), foraging (Persons and Uetz 1996, Persons and Rypstra 2000), and predator detection (Barnes et al. 2002). However, while female-based chemical signals are used by males of many spider species in order to locate the opposite sex (Gaskett 2007, Foelix 2011), the extent to which females detect and assess males based on their chemical cues has received significantly less attention (Ross and Smith 1979, Ayyagari and Tietjen 1987, Huber 2005). There are many ways that females could benefit from using chemical information from males in addition to a more conspicuous visual display (Gomez-Diaz and Benton 2013). Chemical signals may allow a female to find a male in a visually complex environment. In the American lobster Homarus americanus (Decapoda: Nephropidae), for example, females were able to discriminate between occupied and unoccupied shelters from two meters away by following male chemical cues (Bushmann and Atema 1997). Male chemical signals could also be used by females to determine if a potential mate is worth her attention. Chemical cues carry information about a spider’s body condition (Hoefler et al. 2009b). A male’s body condition, or nutritional state, reflects his ability to successfully forage and cope with environmental pressures (Jakob et al. 1996). The ability of a female to accurately assess a male’s condition is important, because females that mate with males in good condition are able to increase their fitness by passing down these successful genes to their offspring (Kokko et al. 2003). However, the effort that females use to evaluate males display leaves them less focused on their surroundings, leaving them open to the possibility of attack or predation (Rypstra et al. 2017). Females could therefore be more successful if they had chemical information about males that allowed them to more quickly choose whether to mate or move on.

There have been a few studies suggesting that females from various spider taxa can attend to male chemical signals. In the neotropical nuptial gift-giving species Paratrechalea ornata (Araneae: Trechaleidae), chemical signals from the male silk wrapped around the gift induced female acceptance (Brum et al. 2012). Both males and females of the East African jumping spider Evarcha culicivora (Araneae: Salticidae) prefer the odor of an opposite-sex conspecific in olfactometer experiments (Cross and Jackson 2009). In the sex-role reversed wolf spiders Allocosa brasiliensis and Allocosa alticeps (Araneae: Lycosidae), male volatile pheromones are used to trigger female courtship (Aisenberg et al. 2010). Taken together, these

3 experiments indicate that females across spider taxa are able to detect male chemical cues and respond to them as part of their reproductive behavior.

The goal of this study was to better understand the role of male chemical signaling in female spider mate choice. Specifically, I wanted to explore the ability of females to use chemical information to assess male quality, as well as how chemical cues from males affected female responses to male courtship.

The wolf spider Pardosa milvina (Araneae, Lycosidae) provides a useful model system for studying visual and chemical communication and their relationship to sexual behavior. Males use air- and substrate-borne chemical cues in order to locate females (Searcy et al. 1999, Rypstra et al. 2009), determine their mating status (Rypstra et al. 2003), and determine female body condition (Hoefler et al. 2009b). Males then engage in a visually conspicuous courtship display meant to attract female attention and induce receptivity for mating (Koomans et al. 1974, Hoefler et al. 2008). Females are able to detect environmental chemical cues and can determine predation risk based on heterospecific chemotactile cues (substrate-borne chemical cues such as silk, excreta, etc.) from spider predators (Barnes et al. 2002, Lehmann et al. 2004). There is also evidence that females can detect male chemotactile cues, because they are able to distinguish between silk from courting and non-courting conspecific males (Khan and Persons 2015). However, the ability of females to gain information such as male body condition from chemical cues and the response of females to multimodal visual and chemical displays from males have yet to be formally investigated.

I hypothesized that P. milvina females would be able to detect male chemical cues and use them to distinguish males of different quality. Furthermore, I hypothesized that the addition of chemical cues to male visual signals would alter female behavior. In order to test these ideas, I used two different sets of experiments. In my first experiment, I compared the activity of females on isolated chemotactile cues of males that differed in their recent feeding history. If females are able to asses male quality based on these cues, I predicted that females would (1) spend more time with, (2) move less often on, and (3) move more slowly on chemotactile cue samples from higher quality males, in anticipation of an incoming courtship display. In my second experiment, I compared female responses to the presence of males with different recent feeding histories that either were surrounded by their chemotactile cues or were not. If females do use male

4 chemotactile cues to assist them in mate detection and assessment of the male’s visual courtship displays, I predicted that females would (1) more quickly approach and (2) spend more time near males that have their chemotactile cues present, similar to how males respond to female chemotactile cues (Rypstra et al. 2009).

Methods

I collected spiders in corn and soybean fields at Miami University’s Ecology Research Center, just north of Oxford, Ohio USA (39.532604N, 84.722998W). Females were caught as sub-adults in the field and allowed to reach maturity in the laboratory in order to ensure their virginity, because virgin female lycosids are more sexually receptive than mated females (Fernández-Montraveta and Ortega 1990, Rypstra et al. 2003). Males were caught as adults in the field (Hoefler et al. 2009b, Rypstra et al. 2009), because male lycosid mating status does not seem to affect male courtship effort or female receptivity (Norton and Uetz 2005, Jiao et al. 2011). I housed spiders individually in circular clear plastic containers (5cm diameter, 6cm height) with approximately 2cm of a damp soil and peat moss mixture covering the bottom in order to provide substrate and moisture. The spiders were maintained in an environmental chamber with a constant temperature (25 °C), constant relative humidity (50%) and a 13 h:11 h light:dark cycle. I kept spiders collected from the field in the laboratory for at least two weeks before I used them as experimental subjects. Mature females were used at least three days post- maturation. For normal laboratory maintenance I fed the spiders two 3mm long crickets (Gryllodes sigilattus) weekly.

Feeding regimes and body size

For both experiments, I randomly assigned male spiders to one of two feeding regimes: well-fed (WF) or food limited (FL). Well-fed males were fed two 3mm crickets three times over the course of one week, on days 1 (the start of the feeding regime), 3, and 5. The food limited males were fed two crickets only once, on day 1. I fed all females two crickets at the start of the experiment (day 1), and then once more the day before their behavioral trials (day 7) in order to control for hunger.

Before the start of their feeding regimes (day 0), I measured the carapace and abdomen widths of the males to the nearest 0.01mm using an optical micrometer (Wild Heerbrugg,

Switzerland). I measured the males again at the conclusion of their feeding regime on day 7. These measurements were used to verify that the different feeding regimes had an effect on male body condition, which was calculated as the ratio between the abdomen and carapace widths (Anderson 1974, Jakob 1991). This comparison controls for absolute body size, since the abdomen increases when the spider feeds while the carapace does not (Wilson 1970). The final body conditions of the well-fed and food limited males were compared using a Student’s t-test.

Isolated Chemotactile Cue Choice Trials

In my first experiment, I examined how females respond when presented with a choice between two different areas of isolated male chemotactile cues (Fig. 1; see also Persons et al. 2001). The activity of females on different male cues was tested by placing a female in an arena with one of three possible paired chemotactile cue combinations: WF-FL (n=16), WF-Blank (Blank= no chemotactile cue; n=17), and FL-Blank (n=17) (Fig. 1). This allowed me to determine if females react to male chemotactile cues and, specifically, if they differentiate the body condition of the male from the cues.

To collect male chemotactile cues, I placed individual males in cylindrical containers (15cm diameter) with filter paper (Grade 1, Whatman) lining the bottom at the conclusion of their feeding regime (day 7). At the center of the filter paper I cut out a 4cm hole and placed wet cotton on a plastic cap to provide the males with water. Males were then kept on the filter paper for 24 hours in the environmental chamber described previously. As the males move across the filter papers they deposit silk, feces and other excreta, which make up their chemotactile cue. I then removed the males from the containers and placed them back in their original homes. No males were reused for chemotactile cue deposition for the rest of the experiment. The filter paper circles from WF males and FL males, as well as blank filter paper circles (filter paper that a male had never been in contact with), were cut in half, and the halves from males on different regimes were placed side-by-side in a circular trial arena (15cm diameter, Fig. 1). Both halves created from a single WF or FL male were used in separate trials, therefore a total of 26 WF males and 21 FL males were used. The side that each cue was placed on was randomized with each trial to control for side bias. I then placed the arena under a camera (Panasonic PV-DV73, Japan) that was positioned to provide a top-down view in an isolated chamber.

At the beginning of the trial, the female was placed at the center of the arena inside an inverted plastic vial (4cm diameter, Fig. 1) and was allowed to acclimate for 3 minutes (Rypstra et al. 2003, Hoefler et al. 2008). I then removed the vial and video recorded the female’s movements for 15 minutes (similar to how long males were exposed to female cues in Rypstra et al. 2009). At the end of the trial, the female was removed and placed back into her container, the used filter paper halves were discarded, and the arena was washed with 70% ethanol to remove any traces of chemical cues. No females were used in more than one trial.

I quantified female movement using the EthoVision software (EthoVision® XT, Noldus, The Netherlands), which tracked the movement of the females at a rate of 10 frames per second. For each filter paper half, I measured four different activity metrics: (1) the duration of time females spent on it (s), (2) the distance females moved (cm), (3) the proportion of time they spent moving, and (4) the average velocity (cm/s). Any trials in which the females did not come into contact with both halves of the arena were excluded from analysis (WF-FL n=3, WF-Blank n=2, FL-Blank n=2).

Multimodal Trials

For my second experiment, I investigated how chemical signals alter the response of females to male courtship displays by manipulating the presence of male chemotactile cues. By also varying male feeding regimes as in experiment 1, I observed how male condition affects the quality of these signals. To do this, I exposed females to sequestered males of different conditions that were either surrounded by their chemotactile cue (WF=19, FL=20) or were not (WF=19, FL=20).

Trials were conducted in rectangular containers (31cm x 16.5cm x 7.8cm) lined with construction paper to provide an even and uniform surface across all containers (Fig. 2). The setup is derived from Rypstra et al. (2009) with the sexes reversed. I sequestered a male in an inverted clear glass vial (1.75cm diameter) at one end of the container (8cm from the back wall) on top of either filter paper that contains the cue he deposited (Cue males), or on top of a blank piece of filter paper (Blank males). In order to gather male cues, a new set of WF and FL males were split into Cue or Blank groups. For the Cue males, I placed them in cylindrical containers (9cm diameter) lined with 9cm filter paper (Grade 1, Whatman) with a small amount of wet

7 cotton in a plastic cap for 24 hours. The Blank males were kept in their homes until the trials began.

At the beginning of a trial, I placed a female at the opposite end of the container from the male (7cm from the front wall) underneath an inverted opaque plastic vial (2.2cm diameter), where she was allowed to acclimate for 3 minutes (Fig. 2). The female was then released and observed for 2 hours, in order to give her sufficient time to locate and respond to the male (Rypstra et al. 2003, 2009). I recorded how long it took the female to make contact with the filter paper surrounding the male (latency), the amount of time the female spent in contact with the filter paper surrounding the male (time on), the number of times the female contacted the male’s vial (approaches), and the number of times the male completed a set of courtship displays (courtship bouts), where separate sets of courtship displays were defined as periods of multiple leg raises and shakes that were separated by more than 10 seconds. In P. milvina, male courtship is characterized by a conspicuous visual display including leg raising and shaking behaviors (Koomans et al. 1974), which is easy to document.

Statistics

Since the data were non-normally distributed because females varied considerably in their responses to the male cues, non-parametric statistics were mainly used for analysis. For the isolated cue experiment, I determined if females reacted to male chemotactile cues in two ways. First, I compared all four female activity metrics (duration, distance, proportion of time moving, average velocity) on each pair of filter paper halves using a series of Wilcoxon signed-rank tests. I also compared female activity on all three different cue types across treatments. For each activity metric I combined all female activity data on WF cues, FL cues, and Blank paper (regardless of treatment) and performed a Kruskal-Wallis test. Since running tests on multiple activity metrics increases the likelihood of a Type 1 error, I used the Bonferroni correction to adjust the significance level to α=0.0125 (Bland and Altman 1995). In order to see if the size or condition of the males had an effect on female activity, I performed simple linear regression analyses on both the male’s final abdomen size (the width at the completion of the feeding regime) and final body condition against the four female activity metrics.

In the second experiment, I explored how the presence of different male signaling modalities affects female response by comparing the latency of females to contact the filter paper and the time females spent on the filter paper between Cue and Blank males using a Wilcoxon rank-sum test. In order to see if there was an interaction between cue presence and male feeding regime, I compared the latency and time on data across all four treatments with a Kruskal-Wallis test. I also compared the two metrics between WF and FL males using a Wilcoxon signed-rank test to determine if condition alone affects female behavior. Because two metrics were compared in each sample, I again adjusted the significance level with a Bonferroni correction, resulting in an α-value of α=0.025.

Since I hypothesized that females would make use of visual and chemical signals when evaluating a male, I used model selection to examine which traits of the male best explained a female’s response. Specifically, I ran three sets of model selection with female latency, time on and approaches as the separate response variables and male feeding regime, cue presence, courtship bouts, final abdomen size and final body condition ratio as the predictor variables. Model selection was determined by Akaike information criterion (AIC) and was run using a combined stepwise approach in which the starting point was the model including all the predictors, after which the predictors were selectively removed or re-added to improve AIC until the model could no longer be improved by changing the predictors. Top competing models were indicated by ΔAIC< 2 (Burnham and Anderson 2002).

To better understand how the male mating display itself was affecting females, I analyzed the strength and direction of the relationship between the number of male courtship bouts and female latency, time on and approaches to the male using Spearman’s rho. I was also interested in trying to determine if male courtship was affected by feeding regime or presence or absence of their chemical cue. To do this I compared the number of courtship bouts between WF and FL males using a Wilcoxon rank-sum test. I used this same comparison to examine differences between Cue and Blank males.

Results are presented as mean ± SE unless otherwise noted. All statistical analyses in both experiments were calculated using the R software (R Core Team 2016).

Results

Body Condition

Across all experiments, males maintained on the well-fed (WF) diet had a greater final body condition ratio (abdomen/carapace) than males on the food limited (FL) diet (t-test, 1.96 ± 0.03 vs. 1.72 ± 0.02, t=6.48, df=123, p<0.001).

Isolated Chemotactile Cue Choice

Overall, females did not alter their activity in response to isolated male chemotactile cues or differences in male body condition. Within treatments, I found that females did not show differences in activity when they were exposed to two different sets of cues. Females in the WF- FL treatment spent over 390 seconds on both the WF and FL sides of the arena (Wilcoxon signed-rank, V=84, p=0.433). Females did not spend less time with blank filter paper when it was paired with WF cues (V=67, p=0.678) or FL cues (V= 51, p=0.244) (Table 1). Females also did not differ in the distance they moved, the proportion of time they spent moving or their average velocity on either filter paper half in all three treatments (Table 1). A power test revealed that I would have needed a sample size of at least n=169 to find a significant difference using a Bonferroni corrected alpha-value of 0.0125 (Table 2). When the different activity metrics of all females on WF, FL and Blank cues were compared across all treatments, I found no statistically significant differences (Table 3).

I also explored the relationships between male size and female activity. Neither the male’s final abdomen size nor his body condition ratio were related to the activity of a female on his chemotactile cues (Table 4).

Multimodal Trials

Females tended to respond differently to males depending on whether or not chemical information was present. There was a non-significant tendency of females to spend more time on the filter paper around males without their chemotactile cue (Blank) compared to males with their cue (Cue) (Wilcoxon rank-sum test, W=567, p=0.054) (Fig. 3). Females did not differ in their latency to come into contact with the filter paper (W=787, p=0.795) (Fig. 4). When the four different treatments were compared, however, there was not a significant difference in terms of

10 time on the filter paper (Kruskal-Wallis, χ2=4.70, p=0.195) (Fig. 5) or latency to contact the filter paper (χ2=0.509, p=0.917) (Fig. 6). When only the feeding regime of the male was considered, I did not find differences in the latency of females to contact the filter paper around WF or FL males (WF=1414 ± 306.23 s, FL=1558.9 ± 318.04 s, Wilcoxon rank-sum test, W=573, p=0.541) or spend time on their filter paper (WF=1723.87 ± 309.55 s, FL=1480.63 ± 301.98 s, Wilcoxon rank-sum test, W=619, p=0.287).

According to model selection, male visual signals were more often included in the best models compared to the presence of a male’s chemical cue (Table 5). The best models that described female latency to contact a male’s filter paper included courtship and the male’s final body condition ratio. Courtship was also included in the best models for the amount of time that a female spent on a male’s filter paper, along with abdomen size and feeding regime. The only time that the presence of a chemical cue was an important predictor variable was in the best models for the number of times a female directly approached a male, along with courtship and feeding regime (Table 5).

When analyzed on its own, the amount of male courtship was significantly related to the amount of interest and attention that females paid to the males. Females approached males more often when males courted more (Spearman’s rho, ρ= 0.67, p<0.001, Fig. 7). Male courtship was also significantly positively correlated with the amount of time females spent on their filter paper (ρ=0.55, p<0.001). There was a significant negative correlation between courtship and latency, indicating that females moved faster towards males that courted more (ρ=-0.36, p=0.001). Male courtship was not affected by feeding status (WF 3.05 ± 0.80 bouts vs FL 1.65 ± 0.36 bouts, Wilcoxon rank-sum test, W=671.5, p=0.362) or the presence or absence of their chemical cues (Cue 1.67 ± 0.40 bouts vs Blank 3.00 ± 0.76 bouts, Wilcoxon rank-sum test, W=632, p=0.185).

Discussion

In this study, I examined the response of female Pardosa milvina wolf spiders to male chemotactile cues, both isolated and in the presence of a male. Overall I found no evidence that females altered their activity when presented with chemotactile cues from males that differed in recent feeding history. However, when presented with males displaying various signaling modalities, females tended to spend less time with chemical cue present males than males that

11 were limited to only displaying visually. This research suggests that females do not actively respond to isolated male chemotactile cues, but the multimodal combination of chemical and visual cues may interact to affect female preferences.

Females did not alter their movement when presented with isolated chemotactile cues from males of different quality, in contrast to my original hypothesis. This suggests that if females are able to detect these cues, their response is not exhibited through a change in their activity. Lack of detection seems unlikely, since females are able to respond to heterospecific chemical cues from predators (Lehmann et al. 2004) and alter their silk producing behavior when in the presence of different types of male silk (Khan and Persons 2015). Therefore, it is more probable that female P. milvina detect but do not actively respond to male isolated chemotactile cues. A similar lack of response to male chemical cues has been found with female Schizocosa ocreata wolf spiders (Plunkett 2010), supporting the idea that the presence of these cues without a visual component produces very little change in female wolf spider activity. These results indicate that chemical cues in isolation are not a strong enough signal to prompt an obvious female response, suggesting that the visual courtship display is the primary male signal that females attend to (Rypstra et al. 2003). There are multiple species in which a visual display is the primary method by which males signal to females (guppies, Houde 1997; peacocks, Loyau et al. 2005; fiddler crab, Callander et al. 2013). There is also much less selective pressure on P. milvina females to attend to male chemical cues, unlike the other way around. Males primarily use chemical detection to locate females and assess their quality, after which they begin their courtship display (Searcy et al. 1999, Rypstra et al. 2003). My results further support the idea of the male being primarily responsible for mate location, and only after the initiation of his display does the female attend to him. This strategy by the females would then not have produced strong selection on the ability to recognize and distinguish between male chemotactile cues.

When presented with males that were able to visually and either chemically signal females or not, females tended to spend less time near males with their chemotactile cue present regardless of feeding status (Fig. 3). This ran counter to my prediction of females spending more time with a male with his chemotactile cue present due to being better able to evaluate him for mating. A possible explanation of this is that females may have been able to evaluate these males more quickly. In this case, the multiple modalities that the male was presenting allowed the

12 female to quickly interpret both signals using parallel processing (Hebets and Papaj 2005), which is known to be used by female crickets to asses directionality and strength of a signal (Gabel et al. 2015). It is also possible that the chemical cue is acting as a secondary, redundant signal (Moller and Pomiankowski 1993), which allows females to more accurately assess male information that the female was receiving. In many birds, for example, multiple physical characteristics tend to display accurate data about a male’s aggression or attractiveness (Jones et al. 2016, Jones et al. 2017). This redundant signal could allow a P. milvina female to more quickly assess a male display. For males without the chemical information, females may be taking longer to monitor them visually before deciding whether or not to move on.

Unsurprisingly, male courtship was the best predictor of female response, and was highly correlated with female attention, because an increased amount of male courtship was positively related to the female choosing to approach and spend more time with the male (Fig. 7). These findings are consistent with the large body of research that has already explored the importance of the male visual courtship display to the female, (Rypstra et al. 2003, Hoefler et al. 2008, Rypstra et al. 2009, Havrilak et al. 2015). This display can be repeatedly measured across individuals (Hoefler et al. 2009a), and is an honest signal of male quality (Hoefler et al. 2008). Surprisingly, in this study I did not find that my feeding regimes affected the amount of male courtship. This could indicate that, while my feeding manipulations produced significant differences in male body condition, they were not extreme enough to truly alter male quality. This would explain why females did not show any preference for well-fed over food limited males, and instead were mainly influenced by the amount of male courtship. While the ability of males to court may not have been affected by their feeding regime, the strong effect that courtship has on female responses further supports the idea that male courtship is both necessary and sufficient for provoking a behavioral response in females.

One potential aspect of signaling that we did not consider in this study is the possibility of a third modality, seismic/vibrational cues, being transmitted by courting males in the second experiment. In a related genus of lycosid, Schizocosa, vibrational cues play a very important role in the male mating display (Scheffer et al. 1996, Uetz et al. 2009, Gibson and Uetz 2012). The use of vibration in P. milvina courtship has not been confirmed in experimental studies, but the leg-waving display of the males could potentially be transmitted through the substrate to females.

Therefore, in the multimodal cue experiment when males were present, the females may have been exposed to a more complex multimodal signal than just visual and olfactory. However, even if the seismic cues were present, I do not think that they would have significantly affected this experiment. For one, these cues would likely be coupled to the visual display of the males, which the females were able to clearly observe through the glass vial. Also, even if the glass vial dampened the seismic cues, they would have affected these cues equally across all the treatments. Since the male chemical cue was the only modality that was explicitly manipulated across treatments, the results of this experiment still provide information on the role of chemical signaling in female mate choice, even if vibratory signals may also have been present.

The extent to which different signaling modalities are utilized by male and female P. milvina to locate and assess one another has, until this point, not been fully explored, especially for females. Previous work suggests that for males, input from multiple modalities helps guide mating behavior. Males primarily use chemotactile cues to locate females and assess their mating status (Rypstra et al. 2003), but they can also visually assess females in order to localize their activity and modify their courtship effort (Rypstra et al. 2009). Females, on the other hand, have not been as thoroughly tested regarding their potential ability to receive inputs from multiple modalities, and have been thought to only assess males by visually observing their courtship displays (Rypstra et al. 2003). By observing female responses to male chemical stimuli, my experiments help solidify the dominance of the male courtship display while also acknowledging the minor role potentially played by chemical signaling.

In conclusion, my research suggests that chemotactile cues produced by males play at most a limited role in the mate choice of Pardosa milvina females, potentially as a secondary signal in conjunction with the male visual courtship display. Although this study did not fully support many of my original predictions, I feel that it highlights the importance of considering multiple modalities when investigating sexual signaling and mate choice. Additionally, more research needs to be done on the various factors that influence female mating decisions in addition to male visual displays. As mentioned earlier, the potential effects of seismic signals from P. milvina males have received essentially no attention (but see Sitvarin et al. 2016), and could potentially be an important factor in his courtship display. Additionally, females can be influenced not just by the male they are observing, but also by the environment in which they are

14 living and the other males with which they are interacting. While some wolf spider females change their mating preferences based on exposures they have as juveniles to certain phenotypes (Stoffer and Uetz 2016), further research on how experience with different types or numbers of males affects female mate choice is still needed.

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Tables and Figures

Filter paper half Filter paper half

♀ 4 cm 15 cm

Figure 1. Experimental arena design for the isolated male chemotactile cue choice experiment. The ♀ indicates where the virgin female spider was acclimated and released.

16.5 cm

8 cm

♂

Filter paper 31 cm

9 cm

♀

7 cm

Figure 2. Experimental arena design for the multimodal cue experiment. The ♂ indicates where the male was contained under a clear glass vial surrounded by filter paper which either contains his cue or is blank. The ♀ indicates where the female was released after being acclimated under an opaque vial.

Table 1. Summary of activity metrics (mean ± SE) for Pardosa milvina females when placed on three different sets of male cues: Well Fed-Food Limited, WF-Blank and FL-Blank. Duration (time spent on each cue), Distance (distance moved on each cue), Proportion Moving (proportion of time spent moving on each cue), and Average Velocity (average velocity on each cue) were not significantly different within each treatment. The data were compared with Wilcoxon signed-rank tests. Male cue Proportion Average Velocity treatment Duration (s) Distance (cm) Moving (cm/s) WF 492.21 ± 51.67 241.12 ± 28.49 0.12 ± 0.02 0.57 ± 0.08 FL 399.74 ± 52.05 242.50 ± 46.57 0.15 ± 0.03 0.67 ± 0.09 Wilcoxon V 84 71 52 48 p-value 0.433 0.900 0.433 0.323 N=16

WF 434.80 ± 43.54 267.26 ± 36.03 0.13 ± 0.02 0.65 ± 0.07 Blank 446.23 ± 46.83 280.82 ± 38.51 0.15 ± 0.03 0.75 ± 0.11 Wilcoxon V 67 64 80 74 p-value 0.678 0.579 0.890 0.927 N=17

FL 405.67 ± 40.50 322.22 ± 43.97 0.21 ± 0.04 0.90 ± 0.12 Blank 460.71 ± 43.44 347.23 ± 38.16 0.18 ± 0.03 0.86 ± 0.10 Wilcoxon V 51 59 106 107 p-value 0.244 0.431 0.174 0.159 N=17

Table 2. Sample sizes needed to obtain a power of 0.8 at a significance level of 0.0125 in the isolated chemotactile cue trials. WF= Well-Fed, FL= Food Limited Activity metric WF-FL WF-Blank FL-Blank Duration (s) 169 2967 3390 Distance (cm) 139,549 1359 516 Proportion Moving 198 489 252 Average Velocity (cm/s) 231 265 1271

Table 3. Summary of different activity metrics (mean ± SE) for Pardosa milvina females on Well- Fed (WF), Food Limited (FL) or Blank cues across all three treatments. Duration (time spent on each cue), Distance (distance moved on each cue), Proportion Moving (proportion of time spent moving on each cue), and Average Velocity (average velocity on each cue) were not significantly different across treatments. The data were compared with Kruskal-Wallis tests. Kruskal-Wallis Activity metric WF FL Blank χ2 p-value Duration (s) 454.22±33.48 418.24±32.22 437.99±31.48 1.339 0.512 Distance (cm) 244.91±22.89 265.17±32.26 296.51±26.64 1.820 0.403 Proportion Moving 0.125±0.016 0.180±0.025 0.164±0.019 3.453 0.178 Average Velocity (cm/s) 0.570±0.054 0.710±0.078 0.750±0.073 3.196 0.202

Table 4. A series of simple linear regressions revealed no significant correlations between either male final abdomen size or male final body ratio with any of the 4 female activity metrics. 2 r F1,64 p-value Final Abdomen Size Duration 0.0039 0.2475 0.621 Distance 0.0021 0.1353 0.714 Proportion moving 0.0318 2.1041 0.152 Average Velocity 0.0253 1.6582 0.203 Final Body Ratio Duration 0.0033 0.2125 0.646 Distance 0.0005 0.0331 0.856 Proportion moving 0.0262 1.7213 0.194 Average Velocity 0.0125 0.8091 0.372

Table 5. Results of AIC model selection for the female response variables Latency (time to contact male filter paper), Time On (amount of time spent on male filter paper) and Approach (number of times female attempted to contact the male). The male predictor variables included the number of courtship bouts (court), the final body condition ratio (ratio), the final abdomen size (ab), whether or not male was WF or FL (feed), and whether or not he had his chemotactile cue present (cue). Models with ΔAIC<2.00 were considered equally competitive models. Numbers in bold indicate the best models. The R2 represents the percent of variance accounted for. The w indicates relative model weights. Dependent variable Model AIC ΔAIC R2 w Latency court 1176.71 0.00 0.08 0.63 court+ratio 1178.68 1.97 0.07 0.23 feed+court+ratio 1180.59 3.88 0.05 0.09 feed+court+ab+ratio 1182.53 5.82 0.04 0.03 feed+cue+court+ab+ratio 1184.51 7.80 0.03 0.01

Time On court+ab 1161.73 0.00 0.22 0.44 feed+court+ab 1162.24 0.51 0.22 0.34 feed+court+ab+ratio 1163.75 2.02 0.22 0.16 feed+cue+court+ab+ratio 1165.57 3.84 0.21 0.06

Approach feed+court 167.48 0.00 0.36 0.42 feed+cue+court 167.78 0.30 0.36 0.37 feed+cue+court+ab 169.51 2.03 0.36 0.15 feed+cue+court+ab+ratio 171.51 4.03 0.35 0.06

Figure 3. Time that female P. milvina spent on filter paper near males that either contained his chemotactile cue (Cue, n=39) or were blank (Blank, n=39). Females tended to spend less time near males with their chemotactile cue present (Wilcoxon rank-sum W=567, p=0.054). The black line indicates the median, the box edges are at the 25th and 75th percentile, and the whiskers are either the minimum and maximum, or the interquartile range (IQR)*1.5 if points outside of this range are present.

Figure 4. Latency of females to contact male filter paper the either contained his chemotactile cue (Cue, n=39) or did not (Blank, n=39) was not significantly different (Wilcoxon rank-sum W=787, p=0.795). The black line indicates the median, the box edges are at the 25th and 75th percentile, and the whiskers are either the minimum and maximum, or the interquartile range (IQR)*1.5 if points outside of this range are present.

Figure 5. Time that females spent on filter paper near well-fed (WF) or food limited (FL) males that either contained his cue (Cue) or did not (Blank) did not differ between 2 treatments (Kruskal-Wallis χ =4.70, p=0.195) . The black line indicates the median, the box edges are at the 25th and 75th percentile, and the whiskers are either the minimum and maximum, or the interquartile range (IQR)*1.5 if points outside of this range are present.

Figure 6. Latency of females to contact male filter paper near well-fed (WF) or food limited (FL) males that either contained his cue (Cue) or did not (Blank) did not differ between treatments (Kruskal-Wallis χ2=0.519, p=0.917). The black line indicates the median, the box edges are at the 25th and 75th percentile, and the whiskers are either the minimum and maximum, or the interquartile range (IQR)*1.5 if points outside of this range are present.

Figure 7. The number of times a female approached a male was significantly positively correlated with the number of courtship bouts the male performed (Spearman’s ρ=0.67, p<0.001).

Appendix 1. Raw data of the body condition measurements of male spiders before and after their feeding regime. Car= carapace width (mm); Abd= abdomen width (mm); Pre= before feeding regime; Post= after feeding regime.

Food Well-Fed Limited Car- Abd- Car- Abd- Car- Abd- Car- Abd- Male ID Pre Pre Post Post Male ID Pre Pre Post Post WF3 0.79 1.43 0.91 1.5 FL1 0.78 1.57 0.81 1.60 WF4 0.88 1.48 0.87 1.71 FL4 0.85 1.92 0.88 1.46 WF5 0.97 1.47 0.89 1.65 FL6 0.83 1.43 0.77 1.35 WF6 0.92 1.45 0.91 1.59 FL7 0.86 1.49 0.85 1.74 WF7 0.91 1.51 0.87 1.69 FL9 0.90 1.53 0.84 1.28 WF8 0.94 1.52 0.80 1.55 FL10 0.74 1.40 0.76 1.48 WF10 0.89 1.34 0.86 1.56 FL11 0.85 1.42 1.02 1.38 WF11 0.80 1.57 0.87 1.43 FL12 0.82 1.48 0.82 1.47 WF12 0.76 1.47 0.78 1.58 FL13 0.82 1.46 0.82 1.59 WF13 0.95 1.55 0.86 1.74 FL14 0.84 1.67 0.82 1.59 WF14 0.75 1.43 0.70 1.55 FL16 0.85 1.59 0.89 1.52 WF15 0.74 1.30 0.80 1.57 FL17 0.92 1.85 0.75 1.44 WF16 0.61 1.25 0.86 1.46 FL18 0.84 1.33 0.90 1.50 WF17 0.83 1.64 0.81 1.63 FL2.1 0.84 1.38 0.83 1.23 WF18 0.93 1.33 0.79 1.77 FL2.2 0.65 1.24 0.83 1.25 WF19 0.84 1.29 0.90 1.4 FL2.3 0.68 1.22 0.69 1.27 WF2.1 0.78 1.48 0.90 1.71 FL2.5 0.84 1.30 0.78 1.26 WF2.2 0.57 1.17 0.72 1.4 FL2.6 0.59 1.51 0.65 1.37 WF2.3 0.73 1.18 0.80 1.28 FL2.7 0.81 1.38 0.79 1.27 WF2.5 0.74 1.18 0.71 1.41 FL2.8 0.81 1.37 0.78 1.25 WF2.6 0.89 1.22 0.89 1.54 FL2.9 0.76 1.76 0.76 1.50 WF2.9 0.70 1.66 0.73 1.91 FL2.11 0.90 1.93 0.87 1.54 WF2.10 0.80 1.33 0.82 1.57 FL2.12 0.95 1.25 0.64 1.35 WF2.11 0.71 1.52 0.83 1.73 FL2.13 0.77 1.46 0.82 1.55 WF2.13 0.73 1.44 0.75 1.52 FL21 0.78 1.53 0.89 1.55 WF2.14 0.69 1.37 0.66 1.67 FL2.16 0.79 1.41 0.89 1.56 WF2.15 0.77 1.26 0.71 1.44 FL2.17 0.88 1.51 0.82 1.47 WF20 0.67 1.42 0.68 1.67 FL22 0.76 1.53 0.91 1.39 WF21 0.81 1.32 0.76 1.37 FL23 0.75 1.27 0.87 1.59 WF2.16 0.79 1.79 0.73 1.57 FL24 0.82 1.52 0.93 1.49 WF2.17 0.75 1.51 0.78 1.8 FL26 0.88 2.21 0.81 1.78 WF22 0.93 1.51 0.85 1.57 FL27 0.81 1.67 0.76 1.20 WF23 0.77 1.49 0.81 1.71 FL28 0.90 1.72 0.88 1.68 WF24 0.81 1.42 0.86 1.57 FL29 0.86 1.25 0.85 1.34 WF25 0.78 1.53 0.89 1.61 FL2.20 0.72 1.56 0.80 1.32

WF26 0.87 1.49 0.84 1.66 FL2.22 0.96 1.86 0.77 1.33 WF27 0.83 1.56 0.92 1.7 FL2.23 0.91 1.83 0.84 1.34 WF28 0.83 1.44 0.80 1.53 FL2.24 0.99 1.81 1.01 1.55 WF29 0.85 1.50 0.87 1.79 FL2.25 0.95 1.62 0.89 1.55 WF2.20 0.84 1.53 0.84 1.54 FL2.26 0.81 1.56 0.91 1.35 WF2.21 0.97 1.42 0.86 1.53 FL2.27 0.66 1.46 0.65 1.17 WF2.22 0.85 1.40 0.81 1.44 FL2.28 0.81 1.71 0.76 1.36 WF2.23 0.95 1.64 0.90 1.5 FL2.28 0.84 1.56 1.10 1.49 WF2.25 0.74 1.74 0.71 1.57 FL2.30 0.91 1.32 0.81 1.44 WF2.26 0.89 1.54 0.72 1.53 FL2.31 0.94 1.78 0.87 1.79 WF2.27 0.92 1.68 0.64 1.71 FL2.32 1.05 1.47 0.88 1.52 WF2.28 0.86 1.30 0.79 1.43 FL2.33 0.84 1.49 0.82 1.37 WF2.29 0.76 1.84 0.98 1.68 FL2.34 0.78 1.38 0.82 1.48 WF2.29 0.94 1.39 0.99 1.49 FL2.37 0.79 1.50 0.93 1.46 WF2.32 0.74 1.42 0.70 1.36 FL2.38 0.85 1.45 1.02 1.46 WF2.33 0.84 1.42 0.84 1.4 FL2.40 0.86 1.27 0.71 1.26 WF2.34 0.81 1.51 0.85 1.64 FL2.41 0.90 1.36 0.86 1.43 WF2.35 0.82 1.40 0.97 1.85 FL2.42 0.74 1.28 0.93 1.55 WF2.36 0.82 1.44 0.83 2.18 FL2.43 0.64 1.09 0.94 1.38 WF2.37 0.85 1.34 0.75 1.73 FL2.44 0.94 1.44 0.88 1.52 WF2.38 0.95 1.40 0.94 1.56 FL2.45 0.72 1.40 0.80 1.34 WF2.40 0.81 1.56 0.81 1.55 FL2.46 0.95 1.59 0.93 1.61 WF2.41 0.92 1.51 0.83 1.67 FL2.47 0.91 1.58 0.91 1.55 WF2.44 0.83 1.42 0.83 1.60 FL2.48 0.86 1.32 0.79 1.20 WF2.45 0.83 1.56 0.92 1.79 FL2.49 0.94 1.34 0.83 1.42 WF2.46 0.88 1.37 0.76 1.67 FL2.51 0.96 1.36 0.88 1.42 WF2.47 0.91 1.45 0.89 1.73 FL2.52 0.86 1.32 0.80 1.25 WF2.49 0.98 1.63 0.92 1.89 FL2.53 0.88 1.38 0.87 1.73 WF2.50 0.98 1.41 0.85 1.57 FL2.54 0.88 1.41 0.82 1.33 WF2.51 0.84 1.44 0.85 1.70 FL2.55 0.86 1.44 0.79 1.55 WF2.52 0.89 1.40 0.82 1.56 FL2.56 0.92 1.46 0.92 1.57 WF2.53 0.95 1.42 0.87 1.66 FL2.57 0.81 1.39 0.96 1.32 FL2.58 0.99 1.47 0.96 1.50 FL2.59 0.85 1.29 0.82 1.27

Appendix 2. Raw data from isolated chemotactile cue experiment. Female ID= female participating in the trial; Male cue ID= set of male cues on which trial is occurring; Distance moved= distance covered by female (cm); In zone= amount of time a female spent in a defined zone (s); Moving= amount of time female spent moving over 1.1 cm/s (s); Velocity= average velocity of the female (cm/s).

Female ID Male cue ID Distance moved In zone In zone Moving Velocity WF3 FL1 1 Total 226.7030401 868.468468 6.506506 10.610616 0.253442633 FL1 9.746166676 2.702705 1.497911 WF3 212.2741129 7.60761 0.246011417

FL1 Blank 3 Total 702.6345869 237.537536 659.659662 125.425436 0.786566407 FL1 252.9892314 53.553564 1.065947775 Blank 443.0896259 70.070071 0.678593395

WF4 FL4 6 Total 1271.975509 349.449448 542.742743 310.810815 1.422800917 WF4 502.7011892 122.82282 1.440202177 FL4 754.5607792 184.68469 1.405306083

WF4 Blank 7 Total 306.4829782 501.701701 394.394393 47.447448 0.342824413 WF4 170.5989305 25.725727 0.344021628 Blank 126.5031313 19.319316 0.320752876

FL4 Blank 8 Total 969.7043937 353.453453 542.842842 214.114098 1.08432359 FL4 410.5594819 86.086082 1.180964465 Blank 548.9516443 125.725713 1.011439801

WF5 Blank 9 Total 714.0095948 419.819821 467.867866 207.307302 0.797691366 WF5 337.0521551 100.500498 0.80284956 Blank 345.8888813 100.400398 0.74744328

FL6 WL6 11 Total 357.6404125 112.812812 781.48148 61.56156 0.398931148 FL6 102.5602472 29.329328 0.909925926 WF6 247.2608825 29.929929 0.317865922

Blank FL6

12 Total 988.2665441 443.043044 417.917917 209.409406 1.103597511 Blank 453.6250618 99.799801 1.032281247 FL6 461.2298066 96.396395 1.106287133

FL7 WF7 13 Total 389.5141649 387.087086 511.11111 71.771778 0.434824735 FL7 186.7627674 36.236239 0.48260735 WF7 195.2789374 34.434437 0.385312434

WF7 Blank 14 Total 180.9004351 698.798798 201.301301 10.71071 0.202260255 WF7 138.3161472 2.102103 0.19959241 Blank 42.13130961 8.508507 0.209294811

Blank FL7 15 Total 786.1841567 427.827826 469.46947 179.679674 0.878816146 Blank 369.4412543 81.881875 0.866162394 FL7 401.8826354 95.195196 0.863956923

WF8 FL9 16 Total 433.182085 557.957957 339.239238 78.578578 0.483626453 WF8 249.6698444 44.844844 0.451031111 FL9 174.9607221 32.032033 0.515896623

Blank WF8 17 Total 945.313544 420.72072 467.06707 226.726721 1.056576675 Blank 456.7667249 108.508508 1.088006625 W8 464.7988396 113.013007 1.004834526

FL9 Blank 18 Total 302.4257566 256.056054 626.926929 46.146149 0.338323998 FL9 95.76178929 14.714717 0.374133862 Blank 199.2793881 30.330332 0.320013025

WF10 FL10 19 Total 156.5755319 503.903903 396.096096 36.836835 0.1749652 85 WF10 86.1281942 20.92092 0.17095583 FL10 70.10684578 15.815815 0.179351456

WF10 Blank 20 Total 1083.387471 469.469471 382.982983 224.02402 1.212258162 WF10 530.9665389 113.813812 1.142933772 Blank 504.1130562 104.004005 1.321114784

FL10 Blank 21 Total 228.7635853 875.275274 24.324324 25.925929 0.255975362 FL10 196.8343551 16.816819 0.226567008 Blank 27.81515814 8.508509 1.143511853

FL11 WF11 22 Total 555.0365737 606.006003 282.182182 11 1.611614 0.620780942 FL11 348.6778879 65.165165 0.581226768 WF11 194.6558176 44.544545 0.689823226

Blank WF11 24 Total 850.5823166 414.114115 474.474474 176.176174 0.953148251 Blank 424.7146242 88.088091 1.025597938 WF11 405.8523132 84.58458 0.869683468

Blank FL11 25 Total 236.6602797 706.206205 191.691691 47.547552 0.262926625 Blank 136.0162919 20.02002 0.192628691 FL11 97.85046547 26.42643 0.510457512

WF12 FL12 26 Total 168.9073051 0 900.100099 15.115111 0.188576674 WF12 0 0 0 FL12 168.5236913 15.015011 0.188169417

WF12 Blank 27 Total 228.0343808 863.863863 34.634635 21.021027 0.254759938 WF12 178.9653437 7.507509 0.208375718 Blank 46.97916706 12.912918 1.360353374

WF13 FL13 29 Total 228.6272643 756.656656 139.53954 21.421416 0.25582286 WF13 178.1704503 10.010007 0.235845089 FL13 47.1394672 11.011009 0.350128838

WF13 Blank 30 Total 299.6944451 312.912913 582.082081 28.228232 0.334818574 WF13 158.737737 19.819828 0.507290528 Blank 129.523389 7.107103 0.224486215

FL13 Blank 31 Total 949.3766263 456.156156 434.434433 236.236231 1.061949696

FL13 477.5554369 115.415411 1.053384632 Blank 460.9386777 118.018017 1.069138012

WF14 FL14 32 Total 384.0014915 670.47047 225.025025 54.354357 0.428862505 WF14 264.3149927 30.230235 0.394282052 FL14 107.4703157 21.021022 0.487791222

WF14 Blank 33 Total 704.9162972 530.630628 338.338339 116.916918 0.786652613 WF14 331.6014873 44.544542 0.625155499 Blank 345.9354146 67.46747 1.034079907

WF15 FL14 34 Total 719.4205609 345.945946 547.647646 177.577567 0.799267296 WF15 253.155656 60.760754 0.73198989 FL14 442.8912164 112.212208 0.80871564

WF15 Blank 35 Total 770.5704782 602.202205 137.937938 131.931933 0.863585282 WF15 459.2799394 79.879881 0.769833375 Blank 220.241943 38.438436 1.596674306

WF16 Blank 38 Total 497.1166024 359.959961 538.438438 99.499502 0.552290386 WF16 219.8451376 48.248258 0.610748889 Blank 262.7957232 49.449444 0.488160928

FL16 Blank 39 Total 379.0036033 338.238237 561.661661 66.86686 0.422666448 FL16 147.9182719 29.929927 0.437708355 Blank 229.6406081 36.636632 0.41120449

FL17 WF17 40 Total 642.6754947 458.558556 430.830832 155.755756 0.718559398 FL17 307.9507947 80.180182 0.678524189 WF17 314.0348111 72.57257 0.72958321

Blank WF 17 41 Total 914.2097814 549.249248 345.545548 211.611621 1.025598635 Blank 515.9576984 122.022027 0.947851675 WF17 371.9962852 85.485493 1.088849424

Blank FL17 42 Total 390.9613319 181.581581 626.626626 68.668666 0.437466836 Blank 142.1510217 30.030028 0.782849436 FL17 226.8953794 36.136136 0.365889392

WF20 FL21 46 Total 309.545837 899.499498 0.100101 17.017016 0.345979285 WF20 308.5407816 16.816816 0.345126228 FL21 0.060799703 - 0.607389644

FL21 WF21 49 Total 192.9379221 863.863863 33.933933 8.308309 0.215646661 FL21 169.6614554 3.603603 0.197635025 WF21 21.88618395 4.404405 0.646872682

WF23 FL23 50 Total 434.8428148 454.854854 443.443443 51.551555 0.487168305 WF23 219.8311006 20.42042 0.489329908 FL23 210.0593508 29.929934 0.475740873

WF22 Blank 52 Total 162.8944858 0 359.459458 0.900901 0.182434523 WF22 0 0 0 Blank 61.6361815 0.600601 0.175027132

Blank FL23 54 Total 312.8004315 565.265264 334.334335 30.230227 0.350636929 Blank 196.0036722 15.115112 0.346807788 FL23 114.5828166 14.714715 0.351129519

WF24 FL24 56 Total 482.7612379 458.358357 439.239237 135.13514 0.586285536 WF24 225.8527352 86.78679 0.559729289 FL24 245.1535059 46.246246 0.58731022

WF25 Blank 57 Total 596.9872659 338.038037 537.137159 159.55956 0.74362877 WF25 198.2721863 97.097098 0.821201928 Blank 373.7486962 60.460459 0.696595065

Blank F L24 58 Total 925.233461 541.341341 352.852853 221.321329 1.034596185 Blank 504.8552225 121.221232 0.939549835

FL24 402.286733 96.596595 1.146276276

FL26 Blank 59 Total 684.9321116 198.398395 597.897901 130.830843 0.765120437 FL26 153.7926717 28.12813 0.775170935 Blank 440.4930164 85.885898 0.742955501

Blank FL26 60 Total 1330.915039 306.706703 526.326326 298.498494 1.48523695 Blank 495.1434219 116.316306 1.620734796 FL26 741.0345294 168.668673 1.41520455

FL27 Blank 61 Total 1025.282734 415.615614 470.670671 167.667653 1.147756009 FL27 538.627271 99.599587 1.308581403 Blank 448.3705718 63.163163 0.958122361

Blank FL27 62 Total 170.9805906 0 900.200199 5.805805 0.190 614458 Blank 0 0 0 Fl27 170.9805906 5.805805 0.190614458

WF26 Blank 63 Total 309.1680537 132.332333 765.165165 44.644644 0.345170869 WF26 59.44218106 11.61161 0.449188649 Blank 246.1258459 32.332334 0.323568529

Bl ank WF26 64 Total 204.2305198 714.714714 185.385385 5.405407 0.228140765 Blank 154.5321495 1.101102 0.21621515 WF26 49.48269084 4.204205 0.274324112

FL28 WF28 65 Total 875.3739107 446.746745 426.226225 188.588587 0.985572508 FL28 401.9903106 79.179177 0.899816963 WF28 436.4384624 100.300302 1.03809994

Blank WF28 66 Total 726.6934528 486.586585 409.90991 164.06405 0.812861675 Blank 356.6800497 71.371365 0.742185738 WF28 341.5456255 89.089081 0.833221247

Blank FL28

67 Total 776.2493571 476.276278 415.415415 196.696707 0.870144864 Blank 371.177002 86.386392 0.781960801 FL28 376.8775781 104.804811 0.921666348

FL29 WF29 68 Total 569.3543511 441.341339 443.543544 116.316 316 0.651977309 FL29 300.341997 57.857858 0.697122783 WF29 256.2979077 48.048048 0.584436496

Blank WF29 69 Total 348.2086632 620.320321 279.479478 18.218222 0.388670899 Blank 221.3044787 9.709713 0.359192812 WF29 126.5946732 8.408409 0.453128187

Blank FL29 71 Total 881.1840456 265.465466 431.13113 406.506506 1.678045886 Blank 435.9742178 71.07107 1.642920597 FL29 381.0766736 327.32733 2.161814714

Appendix 3. Raw data from multimodal cue experiment. Female ID= female used in trial; Male ID= male contained under glass vial, either Well-Fed (WF) or Food Limited (FL). Chem Cue?= whether or not chemotactile cue of male present (Yes or No); Latency= amount of time it took female to first contact male filter paper (s); Time On= amount of time female spent on male filter paper (s); Male court= number of male courtship bouts; Female approach= number of times female contacted male glass vial.

Female ID Male ID Chem Cue? Latency (s) Time On (s) Male court Female approach 2.1 WF2.6 Y 50 327 1 7 2.2 WF2.2 N 5250 71 0 2 2.3 FL2.1 Y 4378 429 0 0 2.4 FL2.2 N 140 2179 5 13 2.5 WF2.3 Y 3 19 2 0 2.6 WF2.29 Y 179 304 0 0 2.7 WF2.38 Y 24 3478 11 6 2.8 FL2.3 Y 7 131 5 4 2.9 WF2.5 N 3252 147 1 3 2.10 WF2.1 Y 994 693 1 4 2.11 FL2.5 N 23 115 1 0 2.12 FL2.6 Y 246 47 1 1 2.13 FL2.7 N 1921 298 1 3 2.15 WF2.10 N 94 81 1 0 2.16 WF2.11 N 289 3230 10 4 2.17 FL2.11 N 440 6518 9 16 2.18 WF2.9 Y 252 3146 9 9 2.19 FL2.8 Y 670 4916 5 11 2.20 FL2.9 Y 618 1279 2 1 2.21 WF2.13 N 1545 2 0 0 2.22 WF2.14 Y 1309 298 1 1 2.23 WF2.15 N 523 1560 7 1 2.24 FL2.12 Y 2695 2 0 0 2.26 FL2.13 N 0 0 0 0 2.29 WF2.16 N 1035 1051 6 7 2.30 WF2.17 Y 0 0 0 0 2.31 FL2.16 N 0 0 0 0 2.33 FL2.17 Y 0 0 0 0 2.35 FL2.20 Y 187 26 0 0 WF 2.36 2.21 Y 611 1437 2 2 2.37 WF2.22 N 320 3757 26 9 2.38 WF2.23 Y 75 5389 6 11 2.39 WF2.25 N 606 6137 5 1 2.40 FL2.22 Y 327 132 1 1

2.41 FL2.24 N 2393 2141 1 1 2.42 FL2.23 Y 162 7038 1 1 2.44 WF2.26 Y 2478 33 0 1 2.47 WF2.27 Y 1114 603 0 1 2.48 FL2.25 N 2904 1024 1 4 2.49 FL2.26 N 41 62 1 1 2.50 WF2.28 Y 243 3 0 0 2.51 WF2.29 N 163 90 0 1 2.52 FL2.28 Y 594 234 0 0 2.53 FL2.30 N 300 260 0 2 2.54 FL2.32 N 566 5003 7 13 2.55 FL2.34 N 345 1200 1 3 2.56 WF2.32 Y 5466 70 0 1 2.57 WF2.33 Y 7178 1 0 0 2.58 FL2.37 Y 5475 995 1 0 2.59 FL2.38 Y 272 14 1 0 2.61 FL2.31 Y 5540 12 0 0 2.62 FL2.33 N 323 408 7 1 2.63 WF2.34 N 4860 2340 0 0 2.64 WF2.35 N 5976 55 0 0 2.65 WF2.36 N 221 3913 7 5 2.66 FL2.40 N 598 5174 4 10 2.67 WF2.37 Y 374 1103 3 1 2.71 FL2.41 Y 4130 2 1 0 2.72 FL2.42 Y 187 3693 0 3 2.73 WF2.40 N 600 1460 0 1 2.74 FL2.43 N 1670 1030 2 1 2.75 FL2.44 N 272 821 3 5 2.76 WF2.44 Y 1553 1686 3 6 2.77 WF2.45 Y 377 4462 4 4 2.78 WF2.41 Y 2609 4591 2 2 2.80 WF2.47 Y 1250 793 0 1 2.81 FL2.46 Y 386 5 0 0 2.82 FL2.47 Y 625 2634 0 1 2.84 FL2.49 Y 6358 842 1 1 2.85 FL2.51 Y 431 366 1 2 2.86 FL2.52 Y 877 586 0 0 2.87 WF2.49 N 634 4539 3 3 2.88 WF2.50 N 809 1227 0 0 2.89 WF2.51 N 1210 5990 1 3 2.90 WF2.52 N 49 586 3 3 2.91 WF2.53 N 157 835 1 6 2.92 FL2.53 N 562 1184 2 5

2.93 FL2.54 N 3885 2922 0 0 2.94 FL2.55 N 5 3257 1 5 2.95 FL2.56 N 5168 697 0 0 2.96 FL2.57 N 50 1538 0 1 2.99 FL2.58 N 6585 11 0 0