Courtship signaling, sexual selection, and the potential for acoustic

communication in the “purring” wolf spider Gladicosa gulosa

A DISSERTATION

Submitted in partial fulfillment of

the requirements for the degree of

Doctorate of Philosophy

Department of Biological Sciences

University of Cincinnati

By

Alexander Lee Sweger

University of Cincinnati

Cincinnati, Ohio

Dissertation Advisor: Dr. George W. Uetz

ABSTRACT

Animals communicate across any number of diverse modalities, including multiple modalities simultaneously. Investigating the evolution of differing modalities often requires unique models and a thorough dissection of the pressures that shape the emergence and continued changing of conspecific signals. Vibration, as a modality for communication, is both incredibly widespread (particularly in insects and spiders) and poorly investigated relative to its most closely associated modality, airborne sound. Spiders, in particular, are an excellent model for studying vibration, and they uniquely do not seem to directly perceive airborne sound. This is especially intriguing when considering certain species, such as the ‘purring’ wolf spider

Gladicosa gulosa, that produce an audible sound during courtship interactions, raising questions about both how and why this sound exists. This thesis sets out to provide quantitative demonstrations of the nature and structure of the courtship signal, investigate the possible mechanisms and value of producing and receiving this signal, and suggest a potential explanation for the evolution of this signaling phenomenon. I recorded and characterized the male courtship signal in this species in both vibratory and acoustic modalities, and identified behaviors that were significantly predictive of mating success for both males and females, as well as their relative correlations to one another. I also measured the role of the substrate in both signal production and reception of the acoustic signal, and provided evidence of behavioral shifts in response to isolated conspecific acoustic signals. Finally, I set these data within a broader context of North

American wolf spiders and suggest an evolutionary framework for the evolution of the G. gulosa signal. I found that this species is unique in the strength of its signal relative to other species, but not in the mechanisms it uses for production, and the airborne signal appears to be largely a byproduct of an abnormally strong vibration. However, this byproduct is still detectable and

produces a marked response in females, which in turn may drive signal exaggeration through copulatory decisions. Ultimately, this all fits into a framework through which a truly adaptive acoustic signal might emerge, and I lay out both the potential process for this emergence as well as the future studies needed to fully demonstrate this phenomenon.

Table of Contents

General Introduction ...... 1

References ...... 7

Chapter 1 - Characterizing the vibratory and acoustic signals of the "purring" wolf spider,

Gladicosa gulosa (Araneae: Lycosidae)

Abstract ...... 14

Introduction ...... 15

Methods...... 19

Results ...... 21

Discussion ...... 24

References ...... 28

Tables & Figures ...... 36

Chapter 2 - Behaviors associated with mating success in the "purring" wolf spider

Gladicosa gulosa

Abstract ...... 40

Introduction ...... 41

Methods...... 44

Results ...... 47

Discussion ...... 49

References ...... 51

Tables & Figures ...... 56

i

Chapter 3 - The role of the substrate in the production of acoustic and vibratory signals in

the wolf spider Gladicosa gulosa

Abstract ...... 59

Introduction ...... 60

Methods...... 63

Results ...... 65

Discussion ...... 66

References ...... 68

Tables & Figures ...... 70

Chapter 4 - Behavioral responses to isolated acoustic signals in the purring wolf spider

Gladicosa gulosa

Abstract ...... 73

Introduction ...... 74

Methods...... 77

Results ...... 80

Discussion ...... 81

References ...... 83

Tables & Figures ...... 88

Chapter 5 – A comparison of vibratory and acoustic signal production in co-occurring

lyscosid species

ii Abstract ...... 93

Introduction ...... 94

Methods...... 97

Results ...... 99

Discussion ...... 100

References ...... 102

Tables & Figures ...... 104

Chapter 6 – Conclusion: An evolutionary framework for the emergence of an adaptive

acoustic signal in Gladicosa gulosa

General Conclusions ...... 109

Evolutionary Framework ...... 114

Future Directions ...... 116

References ...... 119

Tables & Figures ...... 120

iii DEDICATION

This dissertation is dedicated to Hildred Dean Sweger, who taught me to pursue every endeavor

with authenticity, determination, and unwavering tenacity.

ACKNOWLEDGEMENTS

There are a great number of people to whom I owe a considerable debt for their help and support in the completion of this dissertation. Above all else, I would like to thank my doctoral advisor, Dr. George Uetz, for providing unflinching support during every high and low throughout my graduate education, and never interfering with even my most outlandish ideas.

My committee members (Drs. Elke Buschbeck, John Layne, Michal Polak, and Peter Scheifele) provided countless insights and constructive edits to my work, as well as intellectual guidance and technical expertise. I would further like to acknowledge my former undergraduate mentor,

Dr. Matthew Persons, for not only guiding me towards my graduate career, but maintaining support and friendship as I progressed beyond his teaching. Former and current Uetz Lab members, including but not limited to- Dr. Brent Stoffer, Dr. Rachel Gilbert, Dr. Brian Moskalik,

Mark Tiemeier, Dr. Shira Gordon, Emily Pickett, Brittany Hutton, Tim Meyer, Elizabeth Kozak,

Madeline Lallo, and Trinity Walls- were always a sounding board for my best and worst ideas, and the final product would not be complete without their help.

iv Special thanks go out to additional individuals who provided both intellectual and technical advice and support throughout this process. I would like to thank John Pattee from Pioneer Hill

Software for technical support in troubleshooting my bioacoustics program, Dr. Randall

Allemang for help in instrument calibration, Corey Vaughn for investing his personal time in aiding with several projects, William Lewis for agreeing to translate at least one of my references from the original German, and every organization that took a chance on one of my proposals and provided the funds I needed to complete this work.

Finally, I owe everything to the friends and family that never once questioned my goals or my pursuit of this degree. Every stress and pressure of my graduate education was made all the more bearable by the support system that they provided.

v General Introduction

1 Communication plays an important role in the evolution of animal behavior. While sensory systems and pre-existing sensitivities may shape the modalities and signals used in both intra- and interspecific communication (Endler 1992; Endler & Basolo 1993; Bradbury &

Vehrencamp 2011), the signals produced and perceived by individual species have vital and often complex interactions with the forces of natural and sexual selection. Effective communication can be used to avoid dangerous agonistic interactions, attract and/or persuade potential mates, ward of predators, or facilitate social interactions. These signals can exist across a variety of sensory modalities; many species utilize visual (Endler et al. 2005; Osorio &

Vorobyev 2008), tactile (Geldard 1977), chemical (Bossert & Wilson 1963; Wilson & Bossert

1963; Schultz 2004), acoustic (Wiley & Richards 1978; Ewing 1989; Gerhardt & Huber 2002;

Brumm & Slabbekoorn 2005), vibratory (Cocroft & Rodriguez 2005; Hill 2008; Cocroft et al.

2014), and even electrical signals (Hagedorn & Heiligenberg 1985). Moreover, many species will use more than one modality either sequentially or simultaneously, resulting in complex multicomponent or multimodal signals (Partan & Marler 1999; Rowe 1999; Uetz et al. 2016).

The plasticity of signaling behavior makes it a facet of animal behavior that is ideal for studying the ecology and evolution of an animal species, and the selective forces that may be acting on it.

Within the context of communication and animal behavior, spiders have been increasingly popular study systems. Spiders have been recognized for their diversity, worldwide distribution, and critical role as both predators and prey in food webs across a surprisingly diverse range of ecosystems (Foelix 2011). However, they are gaining traction as important study systems within behavioral phenomena (Herberstein 2004)- including predator-prey interactions, sociality, cognition, complex sexual competition and selection, and even behavioral syndromes and animal personality. Many of these complex behaviors are mediated by similarly

2 complex communication signals, as they are in other animal species, making them suitable models for the study of communication (Witt & Rovner 1982). Courtship and mating interactions within spiders, in particular, are intriguing systems within which to study signaling behavior, primarily because of the potential for sexual cannibalism in most species. Female spiders in many species engage in either pre-copulatory or post-copulatory sexual cannibalism (Buskirk &

Frohlich 1984), forcing males to face (and communicate with) a potential predator in the pursuit of increasing fitness. This direct conflict between natural and sexual selective forces creates an ideal situation in which to study effective communication, and previous studies have found complex signals in many species across a variety of modalities (Kronestedt 1996; Elias et al.

2006; Gaskett 2007; Elias et al. 2010; Clark et al. 2011), and in some cases, truly multimodal signals (Uetz & Roberts 2002; Rypstra et al. 2009; Uetz et al. 2016). Still, spiders provide a unique opportunity for one specific modality, because of both their morphology and their well- developed sense for it: vibration.

Vibration is both an understudied and overwhelmingly abundant modality for communication. Despite estimates of hundreds of thousands of arthropod species alone that utilize vibration either exclusively or in conjunction with another signaling modality, the number of studies in vibratory communication in animal species has only recently begun to grow

(Cocroft & Rodriguez 2005; Hill 2008). Nonetheless, there are a number of vertebrate and invertebrate species that use vibration in conspecific communication, and there is increasing evidence that in many insect species acoustic communication may have evolved from the vibratory modality (Cocroft & Rodriguez 2005). Vibration is also unique in that it is inextricably linked to airborne sound (Caldwell 2014). The two are more closely linked than any other two modalities, yet because many animals use different sensory organs for their detection, they

3 remain somewhat biologically separate communication pathways. It is within this particular framework that spider morphology and behavior is unique among other terrestrial arthropods.

Both web-building and wandering spider species have extremely sensitive and well- developed sensory systems for the detection of vibrations. There is some evidence that the mechanoreceptors in some spider species may count among the most sensitive mechanical organs in the animal kingdom (Barth 2004). With few exceptions, vibration is arguably the primary means by which most spiders sense their world, and in many cases it has also been co- opted as the primary means of conspecific communication. However, unlike most arthropod species that communicate via some form of vibration, spiders do not possess any structures for the direct perception of airborne sound. Many insect species have a series of mechanoreceptors for detection of both vibration (in the form of subgenual organs or campaniform sensilla) and airborne sound (in the form of tympana and chordotonal organs), but spiders have only a series of vibratory receptors (in the form of slit sensillae and lyriform organs) positioned all over the body (Barth 2004; Hill 2008; Foelix 2011). While these receptors are remarkably sensitive, and there is increasing evidence that they are sensitive enough to provide some detection of airborne environmental sound (Lohrey et al. 2009; Shamble et al. 2016), the role of acoustic signals in relation to vibratory communication is still poorly understood in spiders. Still there are a few species in which airborne sound has been observed in relation to conspecific communication

(Kronestedt 1996; Kotiaho et al. 1996; Sweger & Uetz 2016), though it is still not clear if the airborne sounds are artefactual or somehow detected as part of the communication system. These spiders could possibly serve as atypical models for potential acoustic communication, and may even shed some light on the environmental circumstances through which an acoustic modality evolves from a vibratory precursor.

4 One such species that has garnered attention for its production of an airborne sound is the

“purring” wolf spider Gladicosa gulosa. A series of short, anecdotal papers observe that courtship of males is audible at some distance (Davis 1904, Lahee 1904, Allard 1936, Kaston

1936), though little to no quantitative measures are provided, and the only recordings date back to 1969 (Harrison). While its taxonomic position and identification have been discussed (Brady

1986), and the primary mechanism through which it produces its courtship signal has been identified (Rovner 1975), next to nothing else has been recorded with regards to its behavioral ecology or communication. Despite its general absence in the literature, the few papers that do focus on this species emphasize the audible nature of its courtship. Given the previously discussed morphology of sensory organs and the seeming lack of any relevance for acoustic signals in spiders, this species raises interesting questions regarding the audible nature of its courtship, and might serve as a model for studying the relationship between vibratory and acoustic communication in animals.

This dissertation is constructed around a series of projects designed to 1) build a foundation of basic knowledge about the production, reception and general structure of the courtship signal in G. gulosa, 2) quantify behaviors in males and females during courtship and identify possible mechanisms of sexual selection, and 3) provide a context for airborne signaling in this species and a possible evolutionary framework for its potential adaptive use of acoustic communication. In chapter 1, I provide a qualitative and quantitative characterization of the vibratory and acoustic signal using laser Doppler vibrometry. Chapter 2 evaluates male and female behaviors in a courtship and mating context, providing statistical analysis of which behaviors are most predictive of mating success and correlations between male and female behaviors. In chapter 3, I focus on the role of the environment in signal production, assessing the

5 relative production of vibration and airborne sound on a series of vibrating and non-vibrating substrates, making the case that the habitat of G. gulosa may provide natural “sounding boards” for efficient sound production. Chapter 4 tests the potential for behavioral responses in male and female conspecifics to isolate acoustic cues, demonstrating that the acoustic signal alone is sufficient to elicit changes in the general behavior of females and possibly influence the courtship interaction. Chapter 5 provides a survey of several co-occurring lycosid species, finding that the same vibratory/acoustic relationship exists across species, but G. gulosa presents a considerably stronger signal than both larger and smaller species. Finally, in chapter 6 I present a summary of the main points of each project as they fit within a broader proposed evolutionary framework, through which the potential adaptive value of the acoustic signal might be assessed.

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10 Rypstra, A. L., Schlosser, A. M., Sutton, P. L., & Persons, M. H. 2009. “Multimodal signalling: the relative importance of chemical and visual cues from females to the behaviour of male spiders (Lycosidae).” Animal Behaviour 77(4): 937-947.

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'purring' wolf spider, Gladicosa gulosa (Araneae: Lycosidae).” Bioacoustics 25(3): 293-303.

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(Lycosidae): an emerging model for study.” In: Naguib, M., Mitani, J. C., Simmons, L. W.,

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11 Wilson, E. O., & Bossert, W. H. 1963. “Chemical communication among animals.” Recent

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12 Chapter 1- Characterizing the vibratory and acoustic signals of the "purring" wolf spider, Gladicosa gulosa (Araneae: Lycosidae)

This chapter has been published:

Sweger, A.L. and G.W. Uetz. 2016. Characterizing the vibratory and acoustic signals of the

"purring" wolf spider, Gladicosa gulosa (Araneae: Lycosidae). Bioacoustics 25: 293-303

13 ABSTRACT

Both air-borne acoustic signals and substrate-borne vibrations are prevalent modes of animal communication, particularly in arthropods. While a wide variety of animals utilize one or both of these modalities, the connection between them is still ambiguous in many species. Spiders as a group are not known for using, or even perceiving, acoustic signals, despite being well-adapted for vibratory communication. Males of the "purring" wolf spider Gladicosa gulosa are reported to produce audible signals during courtship, although the literature on this species is largely anecdotal. Using a laser Doppler vibrometer and an omnidirectional microphone in controlled conditions, I recorded and characterized the visual and mechanical (both substrate-borne and air-borne) signals of this species in an attempt to provide a qualitative and quantitative overview of its signal properties. I found that the vibratory signal is composed of two primary repeating and alternating elements, consisting of pulses of stridulation and percussive strikes, as well as a less common, but repeatable, third element which mechanism and function remains unclear. I also characterized a measurable air-borne component to the signal that is significantly correlated with the amplitude of the vibratory signal, which I suggest is a byproduct of the strong vibration. Neither modality correlated significantly with male body size or condition. Although the exact role of the acoustic component is unclear, I speculate that the unique properties of signaling in this species may have value in answering new questions about animal communication, specifically regarding the emergence of acoustic signals from a vibratory precursor.

14 INTRODUCTION

While acoustic communication is widely recognized as a standard mode of communication for a variety of animal species (Bradbury & Vehrencamp 2011), it is also intrinsically connected with vibration as a means of communication (Hill 2008, Cocroft et al.

2014). "Sound" (air-borne) and "vibration" (substrate-borne) constitute different facets of a coupled mechanical signal and are differentiated primarily by the medium through which they flow. Increasingly, vibration is gaining recognition as a prominent modality, with hundreds of thousands of species potentially utilizing substrate-borne vibratory signals for conspecific communication (Cocroft & Rodriguez 2005, Hill 2008, Cocroft et al. 2014). Studies on animal vibration elucidate a previously understudied but highly prevalent mode of communication, and are beginning to shed light on the evolutionary origins of air-borne acoustic communication

(Cocroft & Rodriguez 2005, Hill 2008, Cocroft et al. 2014). While there are numerous examples of animals that utilize both sound and vibration, particularly in arthropods (Cocroft & Rodriguez

2005), the degree of connection between these two modalities is still largely unknown for many species, as true "bimodal" acoustic signaling can be difficult to demonstrate (Caldwell 2014), and is largely dependent on the physiological independence of the receptor organs in the target receiver. Thus, the connection may be even more ambiguous for species that do not possess structures adapted to sense both air-borne sound and substrate-borne vibration.

Wolf spiders (Araneae: Lycosidae) are known to communicate using multiple modalities, including chemical (Searcy et al. 1999, Persons & Rypstra 2000, Persons et al. 2001, Barnes et al. 2002, Roberts & Uetz 2005, Gaskett 2007, Sweger et al. 2010), visual (McClintock & Uetz

1996, Clark et al. 2011, Uetz et al. 2011, Clark et al. 2012), and vibratory (Rovner 1967, Rovner

1975, Elias et al. 2006, Gibson & Uetz 2008, Gordon & Uetz 2012, Hebets et al. 2013) signals,

15 and in some cases, species will use multimodal communication (Hebets & Uetz 1999, Uetz &

Roberts 2002, Hebets 2005, Rypstra et al. 2009, Uetz et al. 2009, Gordon & Uetz 2011,

Stafstrom & Hebets 2013, Uetz et al. 2013). However, male courtship in many species involves complex vibrations with structural components of the courtship often serving as one of the defining behavioral differences between closely related species (Uetz & Denterlein 1979,

Stratton & Uetz 1981).

With several thousand species worldwide, the prevalence of substrate-borne vibratory signaling across lycosid spiders is not fully understood, and is further complicated by the convolution of terminology related to air-borne and substrate-borne signals. However, several key genera have been studied extensively in relation to mechanical signaling during courtship.

Rovner's work in the genus Rabidosa documented the production of and response to male signals

(1967), and while his initial work identified them as "acoustic" in nature, his follow-up work identified a substrate-coupled mechanism not only in Rabidosa, but in several North American genera (1975), suggesting that the primary mechanical signaling modality was substrate-borne.

Since that time, the genus Schizocosa has been perhaps the most well-studied in relation to substrate-borne vibration. Several members of this genus utilize multi-component signals, consisting of stridulation, body tremulation, and active percussion with the whole or part of the body (Stratton & Uetz 1981, Elias et al. 2006, Gibson & Uetz 2008), and substrate-borne vibration is believed to be ancestral to the more derived visual and multimodal signals that are present in several species (Hebets et al. 2013). Despite a mixture of terminology for the mechanical signals, and a few references to "audible" signals (Rundus et al. 2010), the literature largely suggests that mechanical communication in wolf spiders is substrate-bound. While research increasingly suggests that spiders are capable of responding to air-borne acoustic stimuli

16 (Gordon & Uetz 2008, Lohrey et al. 2009, Caldwell 2014), there is no evidence of tympanal organs capable of direct pressure reception, there is little to no concrete evidence that they utilize airborne acoustic signals in intraspecific communication, and there are few species that seem capable of producing a sufficient air-borne signal to effectively communicate.

To my knowledge, only two species have been described as "audible" at a substantial distance from the focal spider. The European drumming spider Hygrolycosa rubrofasciata has been documented as having a substantial air-borne sound associated with the drumming behavior inherent to its substrate-borne communication (Kronestedt 1996). Females of this species appear to respond to both forms of the signal (Parri et al. 2002) and the percussive signal has been extensively studied in connection with female preference (Parri et al. 1997, Parri et al. 2002), male quality and viability (Kotiaho et al. 1996, Mappes et al. 1996, Kotiaho et al. 1998, Kotiaho et al. 1999, Rivero et al. 2000), and the evolution of chorusing behavior (Kotiaho et al. 2004).

However, this European species bears a more distant lineage to North American lycosid species, and does not appear to utilize multi-component mechanical signals.

The "purring" wolf spider, Gladicosa gulosa, a ground-dwelling wolf spider that occupies deciduous forest habitats throughout the mid-western and eastern United States (Brady

1986, Dondale & Redner 1990), has also been described as "audible" to observers at considerable distances. However, unlike Hygrolycosa, this species is not well-studied, and what little literature exists is largely anecdotal. The focal point of nearly every published anecdote is the audible nature of male courtship in this species (Davis 1904, Lahee 1904, Allard 1936,

Kaston 1936, Harrison 1969), with some authors suggesting that males can be heard anywhere from 10 feet (Allard 1936) to upwards of 6 meters away (Harrison 1969). While the majority of the claims are nearly a century old and empirically unfounded, Harrison (1969) does provide the

17 only published recordings of the species, noting its audible volume in the study. It remains unclear if the initial suggestion about air-borne acoustic communication in this species is an artifact, given that we lack updated and properly controlled recordings of male courtship.

However, if this species does have the potential to produce a sufficient air-borne component, it could serve as useful system to evaluate the evolution of new signal modalities.

My objective was to record and characterize the courtship signals of this species, and provide an assessment of any visual or mechanical (both substrate-borne and air-borne) signals that males may produce when courting. Here, I characterize the presence of various signals and their constituent components, provide qualitative and quantitative descriptions of spectral and temporal aspects of the male courtship signal, and realistically evaluate historic claims regarding this species. I also discuss its potential as a model for the future study of acoustic and vibratory communication in animals.

18 METHODS

Spider Collection and Maintenance

All male and female spiders used in this study were collected post-maturity in leaf litter from a deciduous forest location in New Richmond, OH in August-September of 2013. Spiders were brought into the laboratory at the University of Cincinnati and maintained on a Spring photoperiod (13h:11h light:dark) in stable environmental conditions (23-25°C, 65-75% humidity). Each individual was housed in a small deli dish (approx. 9.5 cm dia. at base) and were fed 2-3 house crickets (Acheta domesticus) twice per week and given water ad libitum. Prior to trials, each individual was photographed using a stereo dissecting scope (OMAX CS-W43C2-

D3-L144L-C90) using ScopeImage 9.0 imaging software, and weighed using a microbalance

(RadWag AS220/C/2) to determine size, mass and body condition. Condition indices (a measure of the relationship between mass and size) were determined from residuals of the regression of mass x cephalothorax width using the method described in Jakob et al. (1996). Post-trial, male spiders were euthanized with CO2 and stored in 75% ethanol for cataloguing purposes.

Recording and Assessing Male Behaviors

I recorded 14 individual male G. gulosa in this study. To stimulate courtship, a female conspecific spider was placed on a clean Fisherbrand P8 filter paper substrate (09-795F) for approximately 2 hours prior to recording. In many wolf spider species, males will respond to chemotactile cues from females in isolation. Prior to recording, I removed the female, and placed a male on the filter paper substrate inside a hollow rectangular arena (13 x 7 cm). I then simultaneously recorded any vibratory, acoustic, or visual signal produced by males during courtship. All signals were recorded inside an anechoic and vibration-isolated booth. Video was

19 recorded using a Sony Handycam (HDR-XR260), and male visual behaviors (if present) were evaluated from video recordings. Substrate-borne vibrations were recorded using a PDV-100 laser Doppler vibrometer (125 mm/s/V sensitivity, 500 mm/s max, 96 mm standoff distance), and air-borne acoustic signals were recorded using an iSEMcon omni-directional microphone

(1/4" front end, 125 dB max, 20 cm standoff distance). Both instruments were connected to an external sound card (Roland QuadCapture) and calibrated with a 1 kHz tone (LDV at 50% FS,

Microphone at 94 dB). Digital signal processing of male acoustic and vibratory signals was conducted in SpectraPLUS-SC (24 kHz sampling rate, 2048 FFT, Hanning window), with independent scaling and calibration for each signal. After initial qualitative evaluation of the air- borne acoustic signal, a 100 Hz high-pass filter was applied in order to facilitate cleaner quantification of signal levels, as most of the acoustic signal was completely masked below this frequency.

20 RESULTS

Elements of the Substrate-borne Signal

The substrate-borne vibratory signal in this species is composed of three discrete and simple elements (Fig.1), two of which were prominent in every recording. The first is a stridulatory element generated through a pulse of the pedipalps, striking the plectrum across the pars stridens in a stridulatory organ first described by Rovner (1975). Each stridulatory pulse is short (370.3 ± 24.0 ms) and contains anywhere from 6-25 separate rapid (31.95 ± 0.90/s) impulses (with each impulse containing a series of strikes across the teeth of each pars stridens), that are generally connected in a train of 2-6 pulses. Pulse amplitude can vary (6.58 ± 0.56 mm/s

RMS), and each pulse occupies a fairly broad frequency range, with most of the signal ranging from 100-4500 Hz. Males generally do not move around while producing this stridulatory element, as the pedipalps need to be in contact with the substrate in order to properly conduct the vibration (Rovner 1975).

The second element consists of a sharp percussive strike using the abdomen. These strikes are short (6.67 ± 0.56 ms) and high amplitude (68.47 ± 6.08 mm/s RMS). Males frequently move around the arena while generating percussive strikes in a series of about 5-10 over the course of several seconds. Like most percussive signals, each strike occupies a broad range of frequencies, with signal energy extending from low frequencies up through 12000 Hz

(the upper limit of instrument recording capacity).

In four of the total recordings, a third discrete element was present (Fig. 1). While the element was not present in every male recorded, it was repeatable and distinct in the four males that produced it. It consists of a low amplitude (0.578 ± 0.02 mm/s RMS) signal resembling 3-4

21 "ticks" that are produced with considerable regularity once every 1-2 seconds over the course of several minutes. The ticks occupy relatively low frequencies compared with the other elements

(100-2000 Hz) and they do not occur alongside the other two elements. During production of this third element, the spider remains completely still with only subtle movement from the pedipalps, not beginning movement again until returning to full courtship with the first two elements, at which point the third element is no longer present. It remains unclear what mechanism is used to produce this element, possibly the same organs used in stridulation.

Temporal structure of the signal, while varied, has a qualitative pattern, as shown in

Figure 1. Elements 1 and 2 are almost always produced repeatedly in a series, with each series consisting of only one of the elements and alternating the elements between separate series. The visual behavior of males follows a similar pattern, with each male pausing to generate a series of stridulatory pulses, followed by movement around the arena while creating a series of percussive strikes. This courtship occurs in discrete bouts of 1-2 minutes, followed by a pause of several minutes before the next bout. It is during this intermediate time interval that the third element, if present, is produced.

Presence of the Airborne Signal

I found that an acoustic component to this signal is qualitatively present, as each male spider was audible to the human ear at up to a 1 meter away (Sweger & Uetz, personal observation). The acoustic component is also quantifiable, though at low levels (37.3 ± 1.0 dBSPL) above background noise (32.5 ± 1.0 dBSPL). The quiet acoustic "purring" that occurs is temporally associated with the stridulatory component of the vibratory signal and is largely masked on a waveform by low-frequency background noise. However, when looking at the

22 spectrogram of both signals (Fig. 2), the air-borne signal is visible for both elements, and at higher frequencies than most of its vibratory component. The third element, when present, is sufficiently masked by background noise such that it is not quantitatively or qualitatively identifiable in the acoustic recording.

Gross Morphology and Signal Production

I sampled multiple segments of courtship containing both primary elements of the signal and compared them to natural variations in size and body condition among males. I found no significant association between size and amplitude of either signal (Vibratory: N=14, R2=0.0010, p=0.9123; Acoustic: N=14, R2=0.0017, p=0.8868 ), nor did I find an association between body condition and either signal (Vibratory: N=14, R2=0.0503, p=0.4406; Acoustic: N=14,

R2=0.08846, p=0.3017). However, I found a significant positive correlation between the relative amplitude of the vibratory and acoustic signals (N=14, R2=0.3120, p=0.0379) (Fig.3).

23 DISCUSSION

This species utilizes two different mechanisms in producing vibration- stridulation and percussion- both of which are prominent mechanisms in both substrate-borne and airborne mechanical communication. Each element is produced with an independent mechanism, and these elements are discrete and repeated, influencing both the temporal structure of the vibratory signal and the visual behavior of the male during courtship. Given the previous evidence for both visual and vibratory signals influencing female mate choice in wolf spiders (McClintock & Uetz

1996, Hebets & Uetz 1999, Hebets & Uetz 2000, Hebets 2005, Gibson & Uetz 2008) with particular emphasis on the substrate-borne vibratory signal (Kotiaho et al. 1996, Hebets et al.

2013), the discrete and repeatable structure of this courtship signal may make this species useful dissecting signal complexity within a specific modality. Future studies can easily manipulate signal elements in relation to varying courtship rate, element ratios, and overall pattern. While my current data do not suggest a direct connection between signal amplitude and body size, additional studies could explore connections between individual signal parameters and aspects of male quality or female preference, and given the role of these signal facets in female preference in Hygrolycosa (Parri et al. 1997, Parri et al. 2002), I hypothesize that G. gulosa may follow similar trends.

The third element of the vibratory signal raises additional questions about signals, their production, and their function, both within this species and in arthropod communication networks as a whole. Its mechanism for production is unknown, though visually there appears to be subtle movement of the male pedipalps during its production, suggesting a similar mechanism to the stridulatory element. Its role within the broader courtship signal is also unknown, and could possibly be an artifact of the spider "warming up" for the substantially stronger elements

24 of the substrate-borne signal, similar to a behavior described in Hygrolycosa (Kohler &

Tembrock 1987). However, given its dramatically lower amplitude relative to other elements, its distinct regularity, and its presence exclusively between bouts of more pronounced courtship, I speculate that it could also have a role as some sort of timing mechanism, though the theoretical intended receiver for this timing is not immediately clear. Regardless, its production and function may raise questions about conspecific communication that translate to other species utilizing similar mechanisms.

In characterizing the various components and possible modalities inherent to this signal, I might speculate about its value in the context of a variety of unanswered questions in arthropod communication. Based exclusively on my recordings, it remains difficult to determine the relevance of any air-borne acoustic signal. Its temporal and spectral correlation with the substrate-borne signal suggests that it is a physical by-product of the strong vibratory signal.

However, in flat frequency weighting, the air-borne signal is masked almost entirely at lower frequencies, and is difficult (if possible) to visualize on a waveform. The signal is clearly visible above the background much more readily at mid- to high frequencies, though still quantitatively low-amplitude, especially in comparison to species that traditionally utilize air-borne sound.

Despite this, the qualitative presence of the air-borne signal is clear. In both lab and field settings, a quiet environment allows males of this species to be heard easily at upwards of 1 meter (Sweger & Uetz, personal observation). While not traditionally "loud", it does represent a considerable signal for a species that is not known to use acoustic communication, and given that this species seems to breed in very early spring (Brady 1986, Dondale & Redner 1990), the acoustic environment may be more accommodating to low-amplitude acoustic signals. At the

25 very least, anecdotal reports of air-borne acoustic signals in this species are not far off, although controlled measures of the signal create a much more realistic portrait of the acoustic component.

Though studies are needed to confirm the function (if any) of the air-borne signal, the lack of tympanal organs in lycosids suggests that bimodal acoustic signaling is unlikely in this species. I hypothesize that a more likely function for this signal is in the extension of the active space of the signal. Substrate-borne signals are highly attenuated by breaks in the solid communication medium, and an air-borne signal capable of inducing vibration in nearby substrates could potentially extend the active range of a signaling male (Mazzoni et al. 2014). It is also unclear to what degree individual male behavior influences the production of the air-borne component. Previous studies in Schizocosa suggest a flexibility in microhabitat choice and relative production of different signal components based on available habitat (Hebets et al. 2008,

Gordon & Uetz 2011), and varying shapes, sizes, and species of deciduous litter are likely to have equally varying acoustic properties. Additional recordings and behavioral studies in this species could determine which substrates contribute most to the production of the air-borne signal, and if they are utilized as such.

This species and its courtship signals may present opportunities to investigate the unique conditions under which new modes of communication might have evolved. Independent reception and usage of substrate-borne and air-borne signals is rare, but not unprecedented, even in arthropods (Latimer & Schatral 1983, Hill & Shadley 2001, Caldwell 2014). Certain environmental conditions, combined with current variations in behavior, signal production, and mate choice pressures may create opportunity for the adaptation of a signal that would otherwise be a simple redundancy or biophysical artifact. Combined with the complex patterning and

26 discrete signal elements of courtship in this species, communication in this wolf spider could serve as an atypical, but elegant, model for the evolution of signals and their functions.

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35 TABLES & FIGURES

Figure 1. A waveform of an example of the substrate-borne vibratory courtship signal in male G. gulosa, with the three characterized elements of the signal- (1) stridulation; (2) percussion; (3) third "tick" element, which occurs between larger bouts of the other two elements. The hatched line indicates a large temporal gap between segments, though both segments are scaled to the same relative amplitude.

36

Figure 2. Two spectrograms showing the relative amplitude for both primary elements of the substrate-borne and air-borne signal. Both spectrograms represent separate modality recordings of the same 1.5-s interval.

37

Figure 3. Regressions of the vibratory signal against a scaling metric (left), and the substrate-borne signal against the air-borne signal (right). While gross morphology did not appear to correlate with signal strength, amplitudes of the separate modalities were tightly correlated.

38 Chapter 2- Behaviors associated with mating success in the "purring" wolf spider Gladicosa gulosa

Submitted to Ethology (in revision)

Sweger, A.L., Vaughn, C.J., and G.W. Uetz 2017

Behaviors associated with mating success in the "purring" wolf spider Gladicosa gulosa

39 ABSTRACT

Studies of sexual selection in animal systems frequently use courtship and receptivity behaviors as metrics for mating success, and this is generally built upon data associating such behaviors with mating. Spider systems are becoming increasingly popular in sexual selection research, and the wolf spider Gladicosa gulosa has the potential to help answer important questions in animal communication. However, this species is understudied, and no quantitative data exists that demonstrates which male and female behaviors may be indicators of mating success. We paired male and female G. gulosa and recorded the behaviors involved in their courtship and mating. We found a number of behaviors in both males and females that were significant predictors of mating success, and we suggest which features of the courtship interaction are perhaps most predictive of mating. Beyond building a foundation for future work, we also suggest that the correlation between certain male and female variables may shed light on some of the more unusual features of this species.

40 INTRODUCTION

Animal mating systems vary considerably, and sexual selection pressures will often shape conspecific communication during courtship and mating, resulting in a diversity of signals across multiple modalities (Bradbury & Vehrencamp 2011). Some species will engage in either sequential or simultaneous multimodal signaling, and often the function of these multimodal signals is not always clear. Still, the behaviors associated with courtship and mating- both for courting males and choosy females- can provide insight into the selection pressures that shape conspecific communication and can ultimately serve as useful metrics in predicting mating success during experimental manipulations. Establishing quantitative associations between certain behaviors and mating success can elucidate which features or modalities may be most effective at communicating male quality or female receptivity.

Spider mating systems are increasingly popular models for sexual selection studies, primarily because of the risk of sexual cannibalism that exists in every species at some level

(Eberhard 2004, Huber 2005). The possibility of cannibalism in spiders places males at an intersection of natural and sexual selection, placing a high stress on effective communication, often resulting in complex courtship behaviors (Witt & Rovner 1982, Herberstein 2010). This communication also frequently occurs across multiple modalities, as numerous spider species have been shown to actively utilize chemical cues (Schultz 2004, 2013, Gaskett 2007), vibratory cues (Rovner 1975, Masters & Markl 1981, Kronestedt 1996, Barth 2004, Cocroft & Rodriguez

2005, Elias et al. 2005, 2010, Gibson & Uetz 2008) and visual cues (McClintock & Uetz 1996,

Elias et al. 2006, Clark et al. 2011, Uetz et al. 2011). Moreover, several species use several of these modalities concurrently, with males producing multimodal signals that are evaluated by females (Hebets & Uetz 1999, Uetz & Roberts 2002, Uetz et al. 2009, 2013, 2016). There is a

41 high potential for raising and answering unique and critical questions about effective communication using spider systems, but dissection of complex behaviors requires an understanding of which behaviors have direct consequences to fitness, and in many systems that data has yet to be established.

The "purring" wolf spider Gladicosa gulosa has potential to answer relevant questions about communication in arthropods, though the species itself is understudied. Only a handful of previous studies have been conducted in this species, and most are largely anecdotal (Davis

1904, Lahee 1904, Allard 1936, Kaston 1936, Harrison 1969). However, nearly every study on this species references an audible "purring" sound produced by males. While we have previously discussed the character of the signal itself and the typical stridulatory and percussive mechanisms for producing it (Sweger & Uetz 2016), the production of airborne sound has special relevance to evolutionary questions about communication. Moreover, as this appears to be a courtship signal, it has additional relevance to questions of sexual selection in this species.

To date no study has evaluated the role of various signal components and behaviors in predicting mating success, data which is invaluable understanding sexual selection and communication in this species.

Our objective in this study was to both qualitatively and quantitatively assess the behaviors relevant to mating success in this system. We hypothesize that, similar to other lycosid species as well as many animal species, males and females should both engage in a variety of behaviors that could serve as significant predictors of mating success and are likely complexly interconnected. Additionally, given the repeated anecdotal references to airborne sound in the male courtship of this species, we hypothesize that the strength of the male signal may play a role in successful copulation. Our goal is that this data may illuminate some aspects of sexual

42 selection in this species, and possibly provide some insight into hypotheses regarding the potential for its use of acoustic signals in intersexual communication.

43 METHODS

Spider Collection and Maintenance

Individuals were collected from a deciduous forest habitat in New Richmond, OH and were reared with controlled lab conditions (13:11 light:dark, ~23-25 degrees, relative humidity

65-75%.) and provided food and water ad libitum. All male and female spiders used in this experiment were mature at the time of collection, so prior mating experience could not be determined. However, all individuals were collected in September and October of 2014, prior to the breeding season established by Kaston (1948). Individuals were fed two to three appropriately sized crickets the day before trials to control for hunger.

Prior to trials, each spider was weighed to the nearest milligram using a microbalance

(RadWag AS220/C/2) and photographed with a stereo dissecting scope (OMAX CS-W43C2-D3-

L144L-C90) and imaging software (ScopeImage 9.0) to evaluate size and body condition. We calculated body condition by taking the residual values for each spider from a regression of mass against a standard scalar for body size (cephalothorax width). This calculation has been previously shown to be the most reliable metric for body condition, particularly with regard to controlling for variation in body size (Jakob et al. 1996). Following the experiment, males and unmated females were euthanized with CO2 and stored in 75% ethanol for cataloguing. Mated females were monitored for egg sac production for several weeks before being catalogued in a similar manner.

Trial Pairing and Recording

Trial pairings were conducted in a clear, circular plastic arena (~20 cm. dia.) with a clear removable divider and a filter paper substrate (Fisherbrand P8). We placed females in one half of

44 the arena for approximately 24 hours prior to the trial to deposit chemical cues. Females were then removed and given access to a cricket and water for 1 hour before beginning the trial. We then placed females back into the arena on the opposite side under a circular vial. Males were placed on the female cues and allowed to acclimate for 1 minute. The vial containing the female was then removed, allowing for visual and vibratory signal transmission while blocking direct contact between the male and female. After a 10-minute period, we then removed the clear divider and allowed for direct contact between males and females for another 10 minutes. This process was repeated for a total of 51 male/female pairings.

We recorded all trials using video cameras and laser Doppler vibrometry to allow for detailed analysis following the experiment. Video of each trial was recorded using a Sony

Handycam (HDR-XR260). Vibratory recording was conducted using a Polytec PDV-100 laser

Doppler vibrometer (125 mm/s/V sensitivity, 500 mm/s max, 96-mm standoff distance), connected to an external sound card (Roland QuadCapture) and calibrated with a 1-kHz tone at

50% full-scale. Digital signal processing and analysis of vibratory signals was conducted in

SpectraPLUS-SC sound analysis software (24 kHz sampling rate, 2048 FFT, Hanning window).

Behavior Scoring

We evaluated trials multiple times and updated defined behaviors to ensure consistency in our assessment and characterization of male and female behaviors. All behaviors described and included in our statistical analyses were exhibited in at least five of our total pairings. We quantified individual components of the vibratory signal of males and analyzed the amplitude of the overall signal. We also quantified latency to exhibit relevant behaviors for both males and

45 females. Mating was defined as the male successfully mounting the female and engaging in copulatory palpal insertions.

Statistical Analyses

Our main goal in analyzing these data was to identify which variables from 1) males, 2) females, and 3) both females and males together, were most predictive of mating success. Given that most of our independent variables were continuous (either as counts of behaviors or morphological measurements) and that we were focused primarily on a binary categorical result

(mating/no mating), we utilized stepwise logistical regressions to evaluate which of these continuous variables were most predictive of a positive mating. This model allowed for a broad survey of the relative significance of all continuous variables in our mating trials as they related to mating success. From there we could pinpoint specific variables to analyze in more detail.

We first tested for correlations among similar measurements and used stepwise logistic models across broad variable groupings (morphology, behaviors, etc.) to narrow our analyses to those statistically independent variables most associated with mating success. For example, if several different measurements of overall body size were correlated, we grouped them into a stepwise logistic regression to isolate the strongest predictors of mating success within that group of correlated variables, which we then included in subsequent analyses. Stepwise logistic models were then used to assess male and female behaviors separately in association with successful copulation, and then both male and female variables from previous analyses were combined into a single stepwise logistic model. Additionally, we used linear regressions to assess correlations between predictive male and female behaviors and evaluate relationships between male courtship behaviors and female receptivity responses.

46 RESULTS

Male behaviors were few, and were primarily isolated to the vibratory signaling modality.

Males generated stridulatory pulses and percussive strikes as characterized by Sweger & Uetz

(2016). Male cephalothorax width, which serves as a rigid scalar for overall body size, correlated significantly with male stridulatory amplitude (N=45, F=6.3378, P=0.0156). Both the number of percussive strikes (N=51, χ2=7.025, P=0.0080), and the number of stridulatory pulses (N=51,

χ2=5.483, P=0.0192) were significantly positively related to mating success, as well as the amplitude of stridulatory pulses (N=45, χ2=7.347, P=0.0067). Male latency to begin courtship was also significantly positively associated with successful mating (N=51, χ2=14.667, P<0.001).

The only noticeable visual behavior was a rubbing of the first pairs of legs against one another, which is characteristic of grooming behavior. Though infrequent relative to other behaviors, this grooming behavior was also significantly associated with mating success (N=51, χ2=11.703,

P<0.001). We also found that overall male size (as a measurement of prosoma width) was significantly associated with mating success (N=51, χ2=5.273, P=0.0217). A stepwise logistic model of male behavior found that the two variables most significantly related to mating success were stridulatory pulse amplitude and latency to begin courtship.

Females exhibited a range of primarily visual behaviors during successful trials, which are described in Table 1. Neither female size nor body condition related significantly to mating success, nor did they correlate with any female behaviors. Of the numerous behaviors exhibit by females, only tap (N=51, χ2=60.59, P<0.001), pivot (N=51, χ2=8.49, P=0.0036), and approach

(N=51, χ2=7.88, P=0.0050) were found to be significantly associated with a successful mating. A stepwise logistic model of female behavior found overwhelmingly that tap was the main predictor of mating success.

47 We also found a significant correlation (N=51, F=12.740, P<0.001) between the amplitude of male stridulatory pulses and the number of taps produced by females in response

(Fig. 1), both of which are individually strongly associated with mating success for males and females respectively.

48 DISCUSSION

Male courtship is simple in structure, containing two primary components that are alternated repeatedly, matching our previous findings from males in isolation (Sweger & Uetz

2016). However, they did exhibit an additional grooming behavior that, while infrequent relative to the two other behaviors, occurred much more frequently than in recordings of isolated males.

We believe the statistical significance of this behavior is likely an anomaly, as the behavior was very infrequent relative to other behaviors. Anecdotal observations suggest that males in multiple lycosid species engage in grooming behaviors, and that these behaviors appear to increase in direct encounters with females (Sweger & Uetz personal observation). Interestingly, this study found that male body size (measured as cephalothorax width) correlated significantly with male stridulatory amplitude, a result which directly contradicts our own previous findings (Sweger &

Uetz 2016). We largely believe that this new result is a more accurate reflection of the relationship, as this result drawn from a larger sample size. Moreover, previous work has shown similar relationships in other lycosid species (Gibson & Uetz 2008).

Females show a variety of discrete visual behaviors during male courtship, though not all appeared to be significant predictors of mating success. To some extent, the exact relationship between every individual behavior and mating success is still unclear. While individual male and female behaviors, when isolated, are significantly related to mating success, our stepwise logistic models found that latency and vibratory amplitude were the strongest predictors from the male perspective, possibly relating to the energetic state of the male, and only the number of tap events were the strongest predictors from the female perspective.

49 The relationship between the relevant male and female variables may provide some insight into sexual selection in this species. Females appear to prefer "louder" males that begin courtship as quickly as possible. Previous work in wolf spiders have shown that a male's latency to begin courtship is often significantly associated with mating success (Scheffer et al. 1996), though it is unclear how much male courtship latency continues to be part of a female's evaluation once courtship has begun. Thus, following the onset of courtship, the amplitude of a male's vibratory signal could be the primary means of evaluating a male's potential as a mate, and females show a corresponding increase in their receptive behaviors as male amplitude increases.

The significance of male signal amplitude in predicting mating could suggest a selection pressure that could drive acoustic signal emergence and relevance. Previous work (Sweger &

Uetz 2016) has shown that the amplitudes of the vibratory and acoustic components are tightly correlated, as one would expect for any species producing a mechanical signal (Caldwell 2014).

Given the consistent, albeit often anecdotal, mentions of an audible signal in this species, this relationship could be important in explaining why this species may produce such a strong signal.

It is possible that female preferences for "louder" males may be driving selection for a stronger male signal, which would lead to an increased acoustic component.

Quantification of the courtship display is important for future studies of the mating system and sexual selection in G. gulosa. Sexual selection studies frequently make use of specific behaviors as metrics for mating success. The data presented here will hopefully allow future studies to utilize male and female behaviors as metrics for mating success in experimental studies of the ecologically-relevant variables in this species, which may further elucidate the role of airborne sound in the evolution of this communication system.

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Allard, H. A. 1936. “The drumming spider (Lycosa gulosa Walckenaer).” Proceedings of the

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Biological Reviews 82(1): 27-48.

Gibson, J. S., & Uetz, G. W. 2008. “Seismic communication and mate choice in wolf spiders: components of male seismic signals and mating success.” Animal Behaviour 75(4): 1253-1262.

Harrison, J. B. 1969. “Acoustic behavior of a wolf spider, Lycosa gulosa.” Animal Behaviour

17(1): 14-16.

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52 Huber, B. A. 2005. “Sexual selection research on spiders: progress and biases.” Biological

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53 Scheffer, S. J., Uetz, G. W., & Stratton, G. E. 1996. “Sexual selection, male morphology, and the efficacy of courtship signalling in two wolf spiders (Araneae: Lycosidae).” Behavioral Ecology

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'purring' wolf spider, Gladicosa gulosa (Araneae: Lycosidae).” Bioacoustics 25(3): 293-303.

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230.

54 Uetz, G. W., Roberts, J. A., Clark, D. L., Gibson, J. S., Gordon, S. D. 2013. “Multimodal signals increase active space of communication by wolf spiders in a complex litter environment.”

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78(2): 299-305.

Witt, P. N., & Rovner, J. S. 1982. Spider Communication: Mechanisms and Ecological

Significance. Princeton University Press.

55 TABLES & FIGURES

Table 1. Female behaviors quantified during mating trials.

Behavior Definition

a smooth and slow raising of left or right first leg well above the Wave prosoma and subsequent lowering, occasionally resulting in a tap

a sharp percussive strike of one leg, occasionally following a wave, Tap though often independent

Orient rotation of the body to face the courting male

forward movement oriented towards the courting male, generally about Approach 1-2 inches

movement directly backwards while facing the courting male, generally Backup covering the same distance as an approach

a full 360-degree rotation of the body, sometimes accompanied by Pivot waves and taps

56

Figure 1. Linear regression of male stridulatory amplitude against female tapping behavior.

57 Chapter 3- The role of the substrate in the production of acoustic and vibratory signals in the wolf spider Gladicosa gulosa

58 ABSTRACT

Among modalities for animal communication, vibration has been largely ignored until recently, although its prevalence in arthropods and connection with airborne sound may shed light on its role in the evolution of acoustic communication systems. The environment also frequently plays a role in the connection between vibration and airborne sound. Spiders, while adept at vibration production and reception, have no means of directly perceiving airborne sound and thus provide an opportunity to study the relationship between these two modalities. Of particular note is the species Gladicosa gulosa, males of which have been previously documented as creating airborne sound during courtship. I examined the role of substrate in the production of the vibration and airborne sound in this species. I recorded males across a series of vibration-conductive and non-conductive substrates and found that both the vibratory and airborne signal were consistently present when males were courting on vibrating substrates, and consistently absent when males were courting on non-vibrating substrates. These results provide a clear understanding of the pivotal role that substrate plays in sound production among this particular (and possible other) spider species.

59 INTRODUCTION

Vibratory signaling is a relatively understudied mode of communication in animals, and only recently have the prevalence and importance of vibration as a modality been gaining attention in the behavior community (Cocroft & Rodriguez 2005; Hill 2008; Cocroft et al. 2014;

Hill & Wessel 2016). Despite the lack of extensive research into vibratory signaling relative to other modalities, it is estimated that over 200,000 species of arthropods alone may utilize this mode of communication, with a suggested nearly 150,000 utilizing it as the exclusive mode for conspecific communication (Cocroft & Rodriguez 2005). For insects and spiders in particular, vibration can be a useful signaling method that avoids the size and frequency relationships that normally limit airborne acoustic communication and may be a more clandestine way of communicating in the presence of possible eavesdroppers (Ewing 1989; Hill 2008; Cocroft et al.

2014). In animal behavior and sensory ecology, interest in “biotremology” (vibratory communication) is growing, and research is beginning to shed new insights into other modes of communication (Hill & Wessel 2016). Of particular interest is the evolutionary and biophysical relationship between vibration and airborne sound (Caldwell 2014).

The distinction between vibration and airborne sound is more or less one of transmission medium, though many animals use separate organs for direct reception of airborne sound and vibration (Hill 2008; Bradbury & Vehrencamp 2011). Moreover, in many cases the medium for vibratory signaling is plant matter, which can often result in a simultaneous airborne sound as a byproduct (Mazzoni et al. 2014). This makes the two signaling modalities more closely linked than any other modes of animal communication, and the signaling environment frequently plays a role in the use of these modalities (Caldwell 2014). This biophysical link, as well as the prevalence and complexity of vibratory signals among arthropods, increasingly suggests that

60 vibration may have been a precursor to acoustic signaling in many arthropod species. Insects that have evolved acoustic signaling, such as many Orthopteran species, utilize similar mechanisms as vibratory species and have evolved additional membranes to couple with the air and transmit the vibrations into the air, creating audible sound (Ewing 1989).

Spiders represent a unique group of animals with relation to the connection between vibration and airborne sound. They are well-adapted for reception and production of vibrations, but unlike most insects, they lack any organs for direct perception of airborne sound (Barth 2002;

Foelix 2011). While this would suggest that acoustic signals would serve little purpose in spider species, there are still a few species in which airborne signals seem to exist (Harrison 1969;

Kronestedt 1996; Sweger & Uetz 2016). The drumming wolf spider Hygrolycosa rubrofasicata and the purring wolf spider Gladicosa gulosa both have documented courtship displays that are audible at a distance, though the mechanisms of signal production are somewhat different in these species. While Hygrolycosa has an audible drumming behavior (Kotiaho et al. 1996),

Gladicosa produces audible stridulation (Sweger & Uetz 2016) via a file-and-scraper mechanism similar to those found in other wolf spiders and crickets- mechanisms that typically require a membrane to couple with the air to effectively generate an airborne signal (Ewing 1989). While no such membrane appears to exist in spiders, males of this species court in dried leaf litter, and it is possible that dried leaves may act in a similar, albeit much less effective, fashion.

The goal of this project was to evaluate the role of the substrate in both the airborne and substrate-borne signals in the wolf spider Gladicosa gulosa. Given the established atypical volume of courtship in this species, I sought to test that male G. gulosa were creating a simultaneous acoustic signal during courtship as a direct result of their courtship medium.

Moreover, I hypothesized that natural and artificial substrata that allowed for effective vibratory

61 transmission would similarly allow for production of this simultaneous airborne signal in courting male G. gulosa, and conversely, males courting on substrata that were not conducive to vibration would be unable to generate either signal.

62 METHODS

Spider Collection and Maintenance

Male and female spiders used in this study were collected in a deciduous forest habitat located in New Richmond, OH. Males and females were collected post-maturity in Fall 2013, prior to the established mating season. Each spider was reared in an individual plastic deli cup

(9.5 cm dia.) on a 13:11 light/dark cycle and controlled humidity (65-75%). Each spider was provided with 2-3 crickets twice per week, and water ad libitum. Both males and females were fed 24-hours prior to use in any trial.

Trial Preparation

A series of small, rectangular Plexiglas arenas (13 x 7 cm) were set up on six different substrates: filter paper, parchment paper, dried leaves, granite, wood, and soil. Each substrate was wiped down with 70% ethanol between trials to eliminate any lingering cues from prior trials. A female G. gulosa was chosen at random and placed into the arena to deposit chemical cues for 1 hour. After an hour, the female was removed and a male was placed into the arena.

Vibratory and acoustic recording of the male took place over a 5-minute period.

Signal Recording and Analysis

Vibratory and acoustic signals were recorded simultaneously. Vibrations were recorded using a laser Doppler vibrometer (PDV-100, 125 mm/s/V sensitivity) placed above the arena.

Airborne acoustic signals were recorded using an iSEMcon omni-directional microphone (1/4" front end, 125 dB max, 20 cm standoff distance). Digital signal processing of both signals was conducted through SpectraPLUS-SC (24 kHz sampling rate, 2048 FFT, Hanning window), with

63 independent scaling and calibration for each signal. A live video feed was used to confirm active courtship behavior in each trial. Root-mean-square values for amplitude were measured and analyzed for each individual across each treatment using a repeated measures ANOVA.

64 RESULTS

Both vibratory and airborne signals varied dramatically by substrate type, though the overall pattern of signal production was largely matched in both modalities. There was a highly significant effect of substrate type on vibratory signal amplitude (N = 24, F = 259.4812, P <

0.0001) (Fig.1) as well as airborne signal amplitude (N = 24, F = 61878.92, P < 0.0001) (Fig.2).

In both cases, each signal modality was significantly attenuated on granite, wood, and soil- substrates that are not typically conductive to small vibrations. Paper, parchment, and leaves all had significantly higher signal amplitude than the non-vibrating substrates, with a few graded differences in amplitude among them in both modalities.

65 DISCUSSION

Recordings of male courtship on vibration conductive and non-conductive substrates suggest that the leaf litter substrate typically used by males during courtship is almost entirely responsible for the production of the airborne component of this signal. Male signal production, both in the vibratory modality and the airborne modality, was consistently produced on any substrate conductive of vibration (filter paper, parchment paper, leaves) and was consistently

(and almost entirely) attenuated on substrates that were not conductive of vibration (granite, wood, soil). The absence of any airborne signal production when a substrate does not readily conduct vibrations suggests that this species has no other means of generating airborne sound aside from vibrating its substrate. These results confirm that there is not necessarily a unique sound production mechanism in G. gulosa; the mechanisms behind its vibratory signal production are the same as those of other Nearctic lycosids. However, its habitat helps facilitate the considerable volume of the sounds it produces via airborne transmission of the vibration.

The nature of these substrata, specifically leaves, allows for the efficient transmission of vibratory signals, and it is likely that G. gulosa males are readily adapted for transmission of vibrations through leaves and similarly conductive substrates. Previous work has shown that other co-occurring lycosid species actively prefer a leaf substrate for courtship and can alter signaling modalities when the substrate is less conductive to vibrations (Gordon & Uetz 2011).

The efficacy of a leaf litter habitat for vibratory courtship could mean that several species have adapted specifically for signaling in this habitat. I have found previously that in G. gulosa that the relative amplitude of vibratory signaling is strongly correlated with the amplitude of its acoustic byproduct (Sweger & Uetz 2016), and that female responses to higher amplitude vibrations may select for exaggerated signals. In this case, the strong vibration produced in this

66 species has led to an acoustic byproduct primarily because of a coupling with an efficient membrane such as a leaf.

The relative importance of this new modality is still not fully understood, nor is it clear if the signal is detected at any relevant levels by female conspecifics (see Chapter 4). However, the audible courtship in this species is clearly a result of a combination of exaggerated vibratory signals transmitted through a leaf, which acts as an efficient coupler with the air, resulting in an audible byproduct. Future studies will examine the potential for this same mechanism to exist in other Nearctic lycosids, as no part of the basic biology of G. gulosa suggests the mechanisms for signal production are unique to this species. Still, the circumstances that have facilitated the emergence of this linked acoustic signal may provide an ideal opportunity to examine the evolution of acoustic signal emergence from a vibratory precursor.

67 REFERENCES

Barth, F. G. 2002. A Spider’s World: Senses and Behavior. Springer Science.

Bradbury, J. W., & Vehrencamp, S. L. 2011. Principles of Animal Communication. 2nd ed.

Sinauer Associates.

Caldwell, M. S. 2014. “Interactions between airborne sound and substrate vibration in animal communication.” In: Cocroft, R. B., Gogala, M., Hill, P. S. M., & Wessel, A. (eds). Studying

Vibrational Communication. Springer, Berlin. pp. 65-92.

Cocroft, R. B., & Rodriguez, R. L. 2005. “The behavioral ecology of insect vibrational communication.” BioScience 55(4): 323-334.

Cocroft, R. B., Gogala, M., Hill, P. S. M., & Wessel, A. (eds.). 2014. Studying Vibrational

Communication. Springer, Berlin.

Ewing, A. W. 1989. Arthropod Bioacoustics: Neurobiology and Behavior. Cornell University

Press.

Gordon, S. D., & Uetz, G. W. 2011. “Multimodal communication of wolf spiders on different substrates: evidence for behavioural plasticity.” Animal Behaviour 81(2): 367-375.

Harrison, J. B. 1969. “Acoustic behavior of a wolf spider, Lycosa gulosa.” Animal Behaviour

17(1): 14-16.

Hill, P. S. M. 2008. Vibrational Communication in Animals. Harvard University Press.

Hill, P. S., & Wessel, A. 2016. “Biotremology.” Current Biology 26(5): R187-R191.

68 Kotiaho, J., Alatalo, R. V., Mappes, J., & Parri, S. 1996. “Sexual selection in a wolf spider: male drumming activity, body size, and viability.” Evolution 50(5): 1977-1981.

Kronestedt, T. 1996. “Vibratory communication in the wolf spider Hygrolycosa rubrofasciata

(Araneae, Lycosidae).” Revue Suisse de Zoologie 341-354.

Mazzoni, V., Eriksson, A., Anfora, G., Lucchi, A., & Virant-Doberlet, M. 2014. “Active space and the role of amplitude in plant-borne vibrational communication.” In: Cocroft, R. B., Gogala,

M., Hill, P. S. M., & Wessel, A. (eds). Studying Vibrational Communication. Springer, Berlin:

125-145.

Sweger, A. L., & Uetz, G. W. 2016. “Characterizing the vibratory and acoustic signals of the

'purring' wolf spider, Gladicosa gulosa (Araneae: Lycosidae).” Bioacoustics 25(3): 293-303.

69 TABLES & FIGURES

7.00

6.00

5.00

4.00

3.00

2.00 Amplitude (mm/s RMS) (mm/s Amplitude 1.00

0.00 Paper Parchment Leaf Granite Wood Soil Substrate

Figure 1. Vibratory signal amplitude (in mm/s RMS) across varying vibrating and non- vibrating substrates. Bars represent mean amplitude in mm/s RMS, with error bars indicating standard error of the mean.

70 0.0025

0.002

0.0015

0.001 Amplitude (Pa. RMS) (Pa. Amplitude 0.0005

0 Paper Parchment Leaf Granite Wood Soil Substrate

Figure 2. Airborne acoustic signal amplitude (in Pa. RMS) across varying vibrating and non-vibrating substrates. Bars represent mean amplitude in Pascals RMS, with error bars indicating standard error of the mean.

71 Chapter 4- Behavioral responses to isolated acoustic signals in the purring wolf spider Gladicosa gulosa

72 ABSTRACT

Animals utilize a variety of modalities in conspecific communication, and numerous ecological and evolutionary factors influence the adaptation and emergence of new signaling modalities. The connection between vibratory and acoustic communication, as well as their relative prevalence in many animal species, suggests that acoustic signaling may emerge from a vibratory precursor. Spiders provide a unique opportunity to study this connection, as they possess sophisticated sensory systems for the detection of vibration, but no known mechanism for directly perceiving airborne sound. However, the "purring" wolf spider Gladicosa gulosa produces an audible sound during courtship, and anecdotal reports suggest that conspecifics will respond to this sound. Previous studies have shown that there is a measureable acoustic signal in this species, though it is still unknown if the signal is detected or results in any meaningful change in behavior. I presented males and females of this species with a variety of acoustic stimuli and on varying substrates and evaluated behavioral responses. While most showed non- significant responses, females did show preferences for male conspecific signals when on a paper substrate. The response of females only occurred on paper substrates, which could suggest that the means of detection of the acoustic component could be via induced transmission in the substrate. These data suggest that acoustic signals alone are capable of eliciting a behavioral response, and there may be a functional role for acoustic signals in this species.

73 INTRODUCTION

Animal behavior is mediated by intraspecific communication across a variety of modalities (Bradbury & Vehrencamp 2011). Conspecific communication can take the form of visual ornaments (Rosenthal et al. 2001), visual displays (Osorio & Vorobyev 2008), acoustic calls (Wiley & Richards 1978; Gerhardt & Huber 2002), vibrations (Hill 2008), chemical volatiles (Wilson & Bossert 1963), and even electrical signals (Hagedorn & Heiligenberg 1985).

Many species show evidence of multi-modal communication, often using more than one mode of signaling in sequence or simultaneously (Partan & Marler 1999). While the examples of communication across various modalities or through multiple modalities simultaneously are numerous and frequently obvious, the evolutionary mechanism through which new modalities emerge may not be as clear. This is partially due to the complex series of variables that impact communication, often simultaneously, and the variety of viable hypotheses for the emergence of new signals (Endler 1992).

Conspecific communication signals can be shaped by the consequences of agonistic interactions, the success or failure of courtship signals, the noise and obstacles of the environment, and even the physical constraints of the animal itself (Bradbury & Vehrencamp

2011). In some cases, these factors can even influence the modalities used in intraspecific signaling. Some species alternate between modalities, utilize multiple modalities, or can even shift to a potentially new mode of signaling in order to optimize effective communication

(Hebets & Papaj 2005; Bro-Jørgensen 2010). This process likely varies in difficulty, largely depending on the sensory abilities of the signaler and receiver (Endler & Basolo 1998) as well as the relative connection of the new modality to an existing mode of signaling.

74 Two of the most closely linked modalities in animal signaling are substrate-borne vibration and airborne sound (Caldwell 2014). While many animals utilize separate sensory organs for perception of vibration and sound, there is an inextricable physical connection between the two modalities, with the medium of mechanical wave transmission serving as the only physical distinction. Particularly in arthropods, there is increasing evidence that many acoustically communicating species may have adapted acoustic signals from vibratory precursors

(Cocroft & Rodriguez 2005; Cocroft et al. 2014), and among a variety of signaling modalities the emergence of one of these modalities from the other would likely be an easier evolutionary transition than many less closely associated modes of communication.

Within the context of the relationship between substrate-borne vibration and airborne sound, spiders provide a unique “umwelt” through which to evaluate this connection. Spiders possess extremely sensitive and well-adapted systems for reception of vibration (Barth 2004), and many species have consequently co-opted vibration as a primary means of conspecific communication in agonistic and courtship interactions (Barth 2002; Herberstein 2004; Foelix

2011). However, spiders lack any tympanum or other sensory organ for direct reception of airborne sound. While there are several examples of spiders producing audible acoustic signals during conspecific communication, such as the drumming wolf spider Hygrolycosa rubrofasciata (Kohler & Tembrock 1987; Kronestedt 1996; Kotiaho et al. 1996, 1999, 2004;

Parri et al. 1997, 2002) and the purring wolf spider Gladicosa gulosa (Harrison 1969; Sweger &

Uetz 2016), it is generally assumed that direct perception of these signals is unlikely and that the audible signal is an artifact of exaggerated vibratory signaling.

Still, the extraordinary sensitivity of spider mechanoreceptors may yet allow for a reception of airborne sound (Barth 2004), and species producing audible conspecific signals

75 would be likely candidates for adaptation to this new modality. Recent work has suggested that not only are the current sensory organs capable of detection of airborne sound, but that acoustic stimuli produce consistent neurological responses in the CNS (Shamble et al. 2016). The question then remains if these responses to stimuli result in meaningful shifts in behavior that could be acted on by selective forces. It is thus imperative to test behavioral responses to relevant acoustic stimuli in species that might show the potential for utilization of airborne acoustic signaling.

The objective of this project was to expose males and females of the "purring" wolf spider Gladicosa gulosa to a variety of acoustic stimuli under varying sensory contexts and assess behavioral responses in order to evaluate and contextualize the relevance of possible acoustic signals. If isolated conspecific acoustic signals are not only perceived in this species, but are also cognitively recognized, males or females should show varied behavior when presented with conspecific signals relative to control stimuli. Additionally, the context in which behavioral responses may or may not present might provide insight into the mechanism through which these signals are primarily received.

76 METHODS

Spider Collection and Maintenance

Individuals were collected from a deciduous forest habitat near New Richmond, OH and were reared with controlled lab conditions (13:11 light:dark, ~23-25 degrees, relative humidity

65-75%.) and provided food and water ad libitum. Individuals were fed two to three appropriately sized crickets the day before trials to control for hunger. Randomized repeated measures trials were run on three consecutive days, with additional water and food provided between trials.

Playback Trials

We conducted a total of eight independent sets of trials using isolated acoustic cues in an anechoic chamber. Four sets of trials involved placing males and females on a two different substrates while presenting varying types of acoustic stimuli. An additional set of four manipulations involved placing males and females on two different substrates while presenting the same type of acoustic stimulus at varying amplitudes. Within each set of trials, a repeated measures designed was used such that each focal individual was exposed to all playback stimuli.

Each focal individual was placed at the center of a rectangular polycarbonate plastic arena with a granite base (10 x 10 x 20 cm), with one speaker at either end (Boss BRS40 4” Dual

Cone). The entire apparatus was built within an anechoic acoustic chamber. Speakers were attached to an amplifier (Pyle PTA2) to control stimulus amplitude, and were isolated from the rest of the arena to avoid direct vibratory cues. The granite base of the arena (cleaned with ethanol to eliminate prior cues) or provided with a clean paper substrate. We then provided individuals with one of three acoustic stimuli, chosen at random- a conspecific cue (spider), an

77 acoustically neutral stimulus (white noise) and a control (blank). The stimulus was randomly presented from one side of the arena, and individual responses were recorded for 10 minutes. We analyzed movement latency and time spent in each of four areas of the arena (1- closest to the stimulus, 4- farthest from the stimulus).

Playback Stimuli

Two series of three stimuli were created for playback trials. The first set consisted of three independent acoustic stimuli. The “blank” stimulus consisted of a blank control, the “white noise” stimulus consisted of an flat frequency-weighted white noise stimulus calibrated to match intensity of a conspecific signal, and a the “spider” stimulus consisted of a conspecific male signal (chosen at random from six representative recordings from a prior study). The second series of stimuli consisted of only conspecific male signals (again chosen at random from six representative recordings), though of increasing amplitudes. The “mean” stimulus reflected the average amplitude of the male courtship signal, the “high” stimulus reflected the amplitude of the upper 95% interval in male signal distribution, and the “amplified” stimulus reflected an artificial amplitude outside the normal range of male signals at a similar interval beyond the

“high” group. The amplitude of each artificial stimulus was calibrated to be consistent across each playback speaker, and to match an accurate amplitude that would reflect the source intensity of a natural stimulus.

Statistical Analyses

Trial data was analyzed across each of the eight separate experiments using repeated measures ANOVA for evaluation of time spent in association with the acoustic stimulus, and

78 Log-Rank tests to evaluate latency to the onset of movement and total proportion responding to the stimulus in each group.

79 RESULTS

Overall, most groups showed no significant changes in behavior in response to stimuli based on sex, substrate, or stimulus type across the eight experiments. Table 1 summarizes the response levels based on varied stimulus type, and Table 2 summarizes the response levels based on varied conspecific stimulus amplitude. Males generally showed a low latency and higher level of activity overall, with no significant differences in association or latency based on either stimulus type or substrate across the four experiments involving males. Moreover, no focal spiders (male or female) showed significant differences in association or latency based on stimulus type when the arena substrate was granite.

Only one group showed a significant difference in response rate by stimulus type. When females were presented with varied stimuli on a vibration-conductive paper substrate, they showed marked differences in behavior. Latency to begin movement as well as proportion that moved during the 10-minute trial (N=22, χ2 = 12.6697, P= 0.0018) varied significantly when the stimulus was a conspecific male signal (Fig. 1). They also showed a graded response in time spent in association with the stimulus source based on the stimulus type (N=22, F= 4.2520, P=

0.0185) with the conspecific signal showing the strongest response (Fig. 2). In the experimental series involving conspecific signals of increasing amplitude, females showed a slightly higher overall level of response and lower latency, but responses were not significantly different based on stimulus.

80 DISCUSSION

While the majority of the results in this series of experiments were non-significant, females did show significant alteration of behavior based on the type of acoustic stimulus presented, with a higher level of response to conspecific male signals. This suggests that the acoustic component of the male signal may have relevance to communication in this species.

Previous anecdotal observations in this species have suggested the possibility of male-male eavesdropping and even possible “chorusing” behavior (Davis 1904; Lahee 1904; Allard 1936), though the results of this study do not support either of these phenomena. My results suggest that males do not appear to show any response to signals from other males, nor do they alter behavior regardless of substrate or stimulus type. It seems that males maintain a relatively high and consistent level of activity regardless of the circumstances presented, at least those presented in this study.

Of particular interest is that female responses to male signals occurred specifically when focal spiders were on a paper substrate. This suggests that the substrate plays a role in the perception of airborne signals, and follow-up recordings using laser Doppler vibrometry show an induced vibration in the paper substrate from the airborne signals (Fig. 3). Previous studies in

Schizocosa wolf spiders have shown that individuals exhibit changes in behavior to acoustic predator cues when on similarly vibration-conductive substrates (Lohrey et al. 2009). Taken together with the responses in this study, it seems that females are likely perceiving acoustic signals via the same sensory organs used to detect vibration, namely slit sensillae and lyriform organs.

81 Studies in other spider species have shown a remarkable degree of sensitivity in other mechanoreceptors, such as trichobothria (Barth et al. 1993), which is well within the range of these acoustic stimuli (Barth 2002b), as well as direct physiological responses in the central nervous system to airborne sound (Shamble et al. 2016). However, this study emphasizes the consequent behavior that may or may not result from perception of these types of acoustic cues.

Evaluating the potential for adaptation and utilization of the acoustic component of the G. gulosa signal lies in the behavioral responses of the receiver, which can ultimately shape the use of this signal in the communication system.

In this particular case, conspecific male signals induced a higher likelihood for movement in females. Female spiders were more likely to move and had a lower latency to initial movement when presented with a male courtship signal. While there was no evidence of female receptivity responses, the shift in female movement could potentially create an advantage for males and ultimately provide an adaptive framework for the evolution of this signal. Since male lycosid courtship is largely persuasive, rather than attractive, any signal that allows for a higher likelihood for a male to locate and/or attract the attention of a female would be advantageous.

Additional studies are necessary to fully investigate the possible utility of this signal, though it is possible that an airborne sound could serve to increase the overall active space of a male’s courtship signal. Though responses to acoustic signals in this species are not as dramatic as stated in earlier anecdotal literature, previous work (Chapter 2, 3 in this dissertation) confirms that acoustic signals are indeed present, and this study shows that there is some level of consistent and relevant behavioral response.

82 REFERENCES

Allard, H. A. 1936. “The drumming spider (Lycosa gulosa Walckenaer).” Proceedings of the

Biological Society of Washington 49: 67-68.

Barth, F. G. 2002. A Spider’s World: Senses and Behavior. Springer Science.

Barth, F. G. 2002. (b) “Spider senses–technical perfection and biology.” Zoology 105(4): 271-

285.

Barth, F. G. 2004. “Spider mechanoreceptors.” Current Opinion in Neurobiology 14(4): 415-

422.

Barth, F. G., Wastl, U., Humphrey, J. A., & Devarakonda, R. 1993. “Dynamics of arthropod filiform hairs. II. Mechanical properties of spider trichobothria (Cupiennius salei Keys.).”

Philosophical Transactions of the Royal Society of London B 340(1294): 445-461.

Bradbury, J. W., & Vehrencamp, S. L. 2011. Principles of Animal Communication. 2nd ed.

Sinauer Associates.

Bro-Jørgensen, J. 2010. “Dynamics of multiple signalling systems: animal communication in a world in flux.” Trends in Ecology & Evolution 25(5): 292-300.

Caldwell, M. S. 2014. “Interactions between airborne sound and substrate vibration in animal communication.” In: Cocroft, R. B., Gogala, M., Hill, P. S. M., & Wessel, A. (eds). Studying

Vibrational Communication. Springer, Berlin. pp. 65-92.

83 Cocroft, R. B., & Rodriguez, R. L. 2005. “The behavioral ecology of insect vibrational communication.” BioScience 55(4): 323-334.

Cocroft, R. B., Gogala, M., Hill, P. S. M., & Wessel, A. (eds.). 2014. Studying Vibrational

Communication. Springer, Berlin.

Davis, W. T. 1904. “Spider calls.” Psyche 11(6): 120.

Endler, J. A. 1992. “Signals, signal conditions, and the direction of evolution.” The American

Naturalist 139: S125-S153.

Endler, J. A., & Basolo, A. L. 1998. “Sensory ecology, receiver biases and sexual selection.”

Trends in Ecology & Evolution 13(10): 415-420.

Foelix, R. F. 2011. Biology of Spiders. 3rd Edition. Oxford University Press.

Gerhardt, H. C., & Huber, F. 2002. Acoustic Communication in Insects and Anurans: Common

Problems and Diverse Solutions. University of Chicago Press.

Hagedorn, M., & Heiligenberg, W. 1985. “Court and spark: electric signals in the courtship and mating of gymnotoid fish.” Animal Behaviour. 33(1): 254-265.

Harrison, J. B. 1969. “Acoustic behavior of a wolf spider, Lycosa gulosa.” Animal Behaviour

17(1): 14-16.

Hebets, E. A., & Papaj, D. R. 2005. “Complex signal function: developing a framework of testable hypotheses.” Behavioral Ecology & Sociobiology 57(3): 197-214.

84 Herberstein, M. E. 2004. Spider Behaviour. Cambridge University Press.

Hill, P. S. M. 2008. Vibrational Communication in Animals. Harvard University Press.

Kohler, D., & Tembrock, G. 1987. “Akutische signale bei der wolfspinne Hygrolycosa rubrofasciata (Arachnida: Lycosidae).” Zoologischer Anzeiger 219(3-4): 147-153

Kotiaho, J., Alatalo, R. V., Mappes, J., & Parri, S. 1996. “Sexual selection in a wolf spider: male drumming activity, body size, and viability.” Evolution 50(5): 1977-1981.

Kotiaho, J., Alatalo, R. V., Mappes, J., & Parri, S. 1999. “Sexual signalling and viability in a wolf spider (Hygrolycosa rubrofasciata): measurements under laboratory and field conditions.”

Behavioral Ecology & Sociobiology 46(2): 123-128.

Kotiaho, J., Alatalo, R. V., Mappes, J., & Parri, S. 2004. “Adaptive significance of synchronous chorusing in an acoustically signalling wolf spider.” Proceedings of the Royal Society of London

B 271(1550): 1847-1850.

Kronestedt, T. 1996. “Vibratory communication in the wolf spider Hygrolycosa rubrofasciata

(Araneae, Lycosidae).” Revue Suisse de Zoologie 341-354.

Lahee, F. H. 1904. “The calls of spiders.” Psyche 11(4): 74

Lohrey, A. K., Clark, D. L., Gordon, S. D., & Uetz, G. W. 2009. “Antipredator responses of wolf spiders (Araneae: Lycosidae) to sensory cues representing an avian predator.” Animal Behaviour

77(4): 813-821.

85 Osorio, D., & Vorobyev, M. 2008. “A review of the evolution of animal colour vision and visual communication signals.” Vision Research 48(20): 2042-2051.

Parri, S., Alatalo, R. V., Kotiaho, J., & Mappes, J. 1997. “Female choice for male drumming in the wolf spider Hygrolycosa rubrofasciata.” Animal Behaviour 53(2): 305-312.

Parri, S., Alatalo, R. V., Kotiaho, J. S., Mappes, J., & Rivero, A. 2002. “Sexual selection in the wolf spider Hygrolycosa rubrofasciata: female preference for drum duration and pulse rate.”

Behavioral Ecology 13(2): 615-621.

Partan, S., & Marler, P. 1999. “Communication goes multimodal.” Science 283(5406): 1272-

1273.

Rosenthal, G. G., Flores Martinez, T. Y., García de León, F. J., & Ryan, M. J. 2001. “Shared preferences by predators and females for male ornaments in swordtails.” The American

Naturalist 158(2): 146-154.

Shamble, P. S., Menda, G., Golden, J. R., Nitzany, E. I., Walden, K., Beatus, T., Elias, D. O.,

Cohen, I., Miles, R. N., & Hoy, R. R. 2016. “Airborne acoustic perception by a jumping spider.”

Current Biology 26(21): 2913-2920.

Sweger, A. L., & Uetz, G. W. 2016. “Characterizing the vibratory and acoustic signals of the

'purring' wolf spider, Gladicosa gulosa (Araneae: Lycosidae).” Bioacoustics 25(3): 293-303.

Wiley, R. H., & Richards, D. G. 1978. “Physical constraints on acoustic communication in the atmosphere: implications for the evolution of animal vocalizations.” Behavioral Ecology &

Sociobiology 3(1): 69-94.

86 Wilson, E. O., & Bossert, W. H. 1963. “Chemical communication among animals.” Recent

Progress in Hormone Research. 19: 673-716.

87 TABLES AND FIGURES

Table 1. Summary data of latency to movement and time spent in close association with the stimulus source for males and females on granite and paper substrates.

Paper Substrate

Latency (s)

Blank White Noise Spider N χ2 P

Male 87.5 + 13.3 83.5 + 18.1 83.6 + 15.9 25 0.2388 0.8875

Female 305.7 + 47.3 324.4 + 50.9 131.5 + 31.2 22 12.6697 0.0018**

Time Spent at Stimulus (s)

Blank White Noise Spider N F P Male 125.6 + 22.0 139.1 + 22.0 145.6 + 21.2 25 0.2221 0.8014 Female 25.5 + 11.2 76.7 + 22.8 99.8 + 19.4 22 4.252 0.0185*

Granite Substrate

Latency (s)

Blank White Noise Spider N χ2 P

Male 200.6 + 38.3 245.4 + 38.7 213.2 + 39.4 28 0.5996 0.741 Female 232.5 + 41.5 314.1 + 47.1 359.9 + 55.5 21 0.3603 0.8351

Time Spent at Stimulus (s)

Blank White Noise Spider N F P Male 91.6 + 17.8 98.3 + 21.3 121.7 + 19.0 28 0.6623 0.5184 Female 55.2 + 19.4 74.6 + 30.5 42.5 + 21.6 21 0.4427 0.6444

88 1.2

1

0.8

0.6

0.4 Blank Proportion Responding Proportion Spider

0.2 White

0 0 100 200 300 400 500 600 700 Time (s)

Figure 1. Latency to movement for females on a paper substrate in response to three stimulus types, showing a proportion of females beginning movement over a 10-minute trial period.

89 140 A AB B 120

100

80

Time (s) Time 60

40

20

0 Blank White Spider

Stimulus Type Figure 2. Time spent in close association with the acoustic stimulus across three stimulus types over a 10-minute trial period for females on a paper substrate, including post-hoc analysis. Bars indicate mean time spent in close proximity to the stimulus in seconds, error bars represent standard error of the mean, and capital letters indicate individual post-hoc relationships among groups.

90

Figure 3. Spectrograms of laser Doppler vibrometer recordings from the paper substrate of the arenas used in playback trials (Freq. range 300-5500 Hz; 30-s recording). A) Blank. B)

White noise stimulus. C) G. gulosa signal. The recordings above show clear vibratory reproductions of each acoustic signal induced in the paper substrate.

91 Chapter 5- A comparison of vibratory and acoustic signal production in co-occurring lycosid species

92 ABSTRACT

Communication studies of focal animal species can be improved by providing a broader context of the physical and morphological relationships that exists within other related and co- occurring species. This is particularly true when examining relationships between closely-linked modalities, or even multimodal communication. The use of different or new signaling modalities can be better understood when viewed through the context of the broader communication strategies in a larger taxonomic group. Given the prevalence and importance of vibration in spiders, the uniquely audible courtship of Gladicosa gulosa will be similarly aided by a broader understanding of the relative strength of the G. gulosa signal compared to other lycosids.

Moreover, the relationships between vibration and airborne sound in other co-occurring wolf spider species may shed light on the nature of vibration and airborne sound in the Lycosidae. In this study, I simultaneously recorded the vibratory and airborne signals of four co-occurring lycosid species and assessed the amplitude of their signals when scaled for body size. I found that while G. gulosa had a significantly higher amplitude signal in both modalities than both larger and smaller species, the relationship of vibratory and airborne signals was consistent across species. This work underscores the unique strength of the G. gulosa signal, and provides a context for the evolution of acoustic signaling in wolf spiders.

93 INTRODUCTION

In any study involving animal communication, a broader perspective on the production and possible reception of signals across related taxa helps frame the phenomena being studied in a focal species. The evolution of any communication phenomenon unique within a given species can be more thoroughly understood by elucidating the possible aspects of the physical and social environment that may shape it, which may be clarified further by looking at how they impact (or do not impact) other co-occurring species. The varying use of certain modalities, or the emergence of multimodality, across related species may help discern the relative role of the physical and social environments in selecting for signal type. Moreover, examination of the prevalence of more derived modalities relative to ancestral forms of communication may help isolate the circumstances through which new modalities, or multimodality, emerge across related taxa.

For arthropods, vibratory signaling is a fairly well-documented, widespread, and ancestral modality for conspecific communication (Witt & Rovner 1982). Spiders in particular are ideal models for studying vibratory communication, as their sensory systems are well- developed and highly sensitive to vibration (Hill 2001; Cocroft & Rodriguez 2005). They also have a series of relatively simple mechanisms for production of vibration, and since they do not possess any known means of directly perceiving or using acoustic signals, they lack additional complex organs for sound coupling or production (Barth 2002, 2004; Foelix 2011). Broadly speaking, spiders produce vibrations through 1) tremulation, which involves a shaking of the entire body which transfers a vibration into their substrate, 2) percussion, which involves striking the body (in whole or part) against the substrate directly, or by striking to body parts together, and 3) stridulation, which involves a "file-and-scraper" mechanism located on opposing surfaces,

94 usually at a body joint. While tremulation and percussion, for the most part, do not require additional structures in order to be functional mechanisms of vibratory signal production, the plectrum and pars stridens of many spiders are generally adapted specifically for the production of vibratory signals, making them ideal for studying the communication of related species.

Many North American wolf spider species, which number well over 200, utilize stridulation in conspecific courtship displays (Dondale & Redner 1990). Rovner (1975) discovered that the mechanism for this stridulation was a "file-and-scraper" organ located at the tibio-tarsal joint of the pedipalps. These structures along with production of stridulatory signals, appear to be present in nearly all North American lycosid species. This presents an opportunity to investigate unique features of stridulatory signals in focal species with the Nearctic lycosidae, such as those of the purring wolf spider Gladicosa gulosa. Previous studies have shown that the vibratory signals of this species, in particular those from the stridulatory mechanism, produce an accompanying acoustic component (Davis 1904; Lahee 1904; Allard 1936; Harrison 1969;

Sweger & Uetz 2016). While there are few, if any, references to similarly audible courtship in other lycosid species, the mechanism for production of this signal does not appear to be unique.

However, to date no study has confirmed the relative amplitude of vibratory and acoustic signals across co-occurring genera of Lycosidae.

The goal of this study was to record and analyze both vibratory and acoustic signal production in several genera of wolf spiders (Araneae: Lycosidae), and assess the amplitude of both modalities in relation to overall body size, testing gross morphology as a possible explanation for the strength of the G. gulosa signal. Consequently, this study should additionally provide context for the uniquely audible nature of G. gulosa courtship. Since the mechanisms within this focal system are similar to other related species, this could possibly verify if signal

95 strength is in any way related to gross body size metrics. Given the references to the audible courtship of G. gulosa in the literature, and the comparative lack of any references to audible courtship in other Nearctic lycosid species, I would hypothesize that the amplitude of the signals in G. gulosa is unlikely to be related strictly to body size, though the potential for production of similar signals in other lycosids may still exist.

96 METHODS

Spider Collection and Maintenance

The following species were used in this study: Schizocosa ocreata, Gladicosa gulosa,

Rabidosa rabida, Tigrosa aspersa. Male and female spiders of each species were collected at two different sites- at a private property near New Richmond, OH and at the Cincinnati Nature

Center in Milford, OH. All spiders used in this study were housed individually in deli containers in a stable climate (65-75% humidity, 25°C) on a springtime light cycle (13:11 hr light/dark).

Each spider was provided with 2-3 crickets of appropriate size (pinhead crickets for Schizocosa ocreata, 1/8" crickets for Gladicosa, Rabidosa, and Tigrosa species). Individual spiders were fed

24 hrs prior to use in any trial.

Gross Anatomical Measurements

All focal males used in this study were weighed and imaged for gross anatomy measurements prior to trials, and euthanized following recording trials for record maintenance.

Mass was recorded as an average of three measurements using a RadWag® microbalance

(AS220/C/2), and the cephalothorax was imaged and measured using an OMAX® dissecting scope and the ImageJ® photo analysis program. In addition, a sub-sample of the focal individuals were dissected at the tibio-tarsal joint for imaging of the plectrum of the stridulatory mechanism. Successfully dissected plectra were mounted in wax and imaged using an OMAX® compound scope and the ImageJ® program.

97 Spider Signal Recording

Prior to each recording trial, a female conspecific was placed on a clean filter paper substrate for 1 hour to deposit chemical cues that elicit male courtship. Following removal of the female, males were placed on the same substrate within a rectangular arena and were recorded for any courtship signals produced for up to 5 minutes, or until sufficient samples of courtship were produced. Recording of vibratory signals was conducted using a laser Doppler vibrometer

(Polytech PDV-100, 125 mm/s/V sensitivity, 96 mm standoff distance) positioned beneath the substrate. Any airborne acoustic signals were recorded via microphone (iSEMcon omni- directional, 1/4" front end, 125 dB max, 20 cm standoff distance) placed above the arena. Both signals were digitally processed via a external sound card (Roland QuadCature) calibrated to a 1 kHz test tone (50% FS for LDV, 94 dB for microphone) and a laptop running the SpectraPLUS acoustic analysis software package. Following recording, SpectraPLUS was used to analyze the amplitude of corresponding vibratory and acoustic signals during stridulatory courtship events.

98 RESULTS

Each of the representative species had at least one element of vibratory courtship that involved stridulation, and all males produced recordable vibratory and acoustic signals, regardless of amplitude. The amplitude of male stridulatory signals, when scaled for body size, showed a significant difference across taxa, both in the vibratory modality (F3,19 = 10.3734, P =

0.0006) (Fig. 1) and the acoustic modality (F3,19=7.2692, P= 0.0031) (Fig. 2). Post-hoc analyses show that while intermediates vary based on modality, G. gulosa males produced the highest amplitude courtship in both modalites (Fig. 1, Fig. 2). Additionally, across all species recorded, there was a significant correlation between vibratory signal amplitude and acoustic signal amplitude (R2= 0.78, P<0.0001) (Fig. 3).

Plectrum area scaled for overall body size in the subset of dissected males varied significantly across genera, with a general downward trend in proportional plectrum size as overall body size increased (F3,15= 8.8177, P=0.0048) (Fig. 4).

99 DISCUSSION

The primary result of this experiment was that G. gulosa courtship is demonstrably stronger in amplitude than several other lycosid species, regardless of size. When scaled for overall body size, signal strength was far from even across species, and in both vibratory and acoustic contexts, G. gulosa emerged as the highest amplitude signal. This confirms earlier anecdotal reports of audible courtship in this species, while also reinforcing why similar reports do not seem to exist for many other lycosid species. However, given the limited range of species sampled in this study, an expanded survey investigating similar courtship across the more than

200 North American lycosid species is need to confirm the context of this species' courtship.

Still, among its co-occurring taxa, G. gulosa has a quantifiably stronger signal.

Expanding this study is also likely to reinforce the other major finding of this study- the connection between vibratory and acoustic amplitude for all focal species. This result may not be surprising, given the inextricable physical link between these two forms of mechanical waves, but having this result confirmed again through quantitative recordings within the family of the focal species only serves to provide a more robust lens through which to view the evolution of signaling in G. gulosa. Recognizing that the potential for acoustic signaling may not be unique to

G. gulosa allows future studies to more thoroughly investigate exactly how this species came to set itself apart from its relatives.

The results of this study confirmed several hypotheses regarding the context through which the "unique" courtship of Gladicosa gulosa should be viewed. The amplitude of the signal in both vibratory and acoustic modalities, especially in relation to overall size, sets G. gulosa apart among its co-occurring genera within the Lycosidae, and may possibly set it apart from

100 most species within the family. However, the results show clearly that there is evidence for the same potential among each of the species we recorded, as both the mechanism for signal production and the amplitude correlation among the modalities were identical across all species.

Ultimately, this serves not only to contextualize the phenomenon of audible courtship signals in

G. gulosa, but to frame the entire question of emergent acoustic signals within wolf spiders.

Rather than a unique morphological or behavioral adaptation within this species, it seems more apparent that the phenomenon of audible courtship in this species may arise from a direct and observable connection to pre-existing courtship mechanisms in the Lycosidae. Future studies will continue to expand on the data presented here, adding additional individuals and species to the question of acoustic and vibratory signaling of wolf spiders, though I believe that even the limited data presented in this survey make a compelling case that each addition will continue to reinforce the same conclusions, ultimately making for a strong system for studying emergent acoustic behavior.

101 REFERENCES

Allard, H. A. 1936. “The drumming spider (Lycosa gulosa Walckenaer).” Proceedings of the

Biological Society of Washington 49: 67-68.

Barth, F. G. 2002. A Spider’s World: Senses and Behavior. Springer Science.

Barth, F. G. 2004. “Spider mechanoreceptors.” Current Opinion in Neurobiology 14(4): 415-

422.

Cocroft, R. B., & Rodriguez, R. L. 2005. “The behavioral ecology of insect vibrational communication.” BioScience 55(4): 323-334.

Davis, W. T. 1904. “Spider calls.” Psyche 11(6): 120.

Dondale, C. D., & Redner, J. H. 1990. The Insects and Arachnids of Canada Part 17. Canadian

Government Publishing.

Foelix, R. F. 2011. Biology of Spiders. 3rd Edition. Oxford University Press.

Harrison, J. B. 1969. “Acoustic behavior of a wolf spider, Lycosa gulosa.” Animal Behaviour

17(1): 14-16.

Hill, P. S. 2001. “Vibration and animal communication: a review.” American Zoologist 41(5):

1135-1142.

Lahee, F. H. 1904. “The calls of spiders.” Psyche 11(4): 74

102 Rovner, J. S. 1975. “Sound production by nearctic wolf spiders: a substratum-coupled stridulatory mechanism.” Science 190(4221): 1309-1310.

Sweger, A. L., & Uetz, G. W. 2016. “Characterizing the vibratory and acoustic signals of the

'purring' wolf spider, Gladicosa gulosa (Araneae: Lycosidae).” Bioacoustics 25(3): 293-303.

Witt, P. N., & Rovner, J. S. 1982. Spider Communication: Mechanisms and Ecological

Significance. Princeton University Press.

103 TABLES AND FIGURES

3.00 A B AB A

2.50

2.00

1.50

1.00 Amplitude (Velocity mm/s RMS) mm/s (Velocity Amplitude 0.50

0.00 Schizocosa Gladicosa Rabidosa Tigrosa Genus

Figure 1. Amplitude (velocity in mm/s RMS) of male vibratory courtship signals across four genera, scaled for body size. Bars indicate mean amplitude in mm/s RMS, error bars represent standard error of the mean, and capital letters indicate individual post-hoc relationships among groups.

104 0.00018 AB B A A

0.00016

0.00014

0.00012

0.0001

0.00008 Amplitude (Pa.) Amplitude 0.00006

0.00004

0.00002

0 Schizocosa Gladicosa Rabidosa Tigrosa Genus

Figure 2. Amplitude (in Pascals) of male acoustic courtship signals across four genera, scaled for body size. Bars indicate mean amplitude in Pascals RMS, error bars represent standard error of the mean, and capital letters indicate individual post-hoc relationships among groups.

105 0.00095

0.00085

0.00075

0.00065

0.00055

Acoustic Signal (Pa. (Pa. RMS) Signal Acoustic 0.00045

0.00035

0.00025 0.0000 5.0000 10.0000 15.0000 20.0000 Vibratory Signal (mm/s RMS)

Figure 3. Linear regression of vibratory signal amplitude against acoustic signal amplitude, both scaled for body size, across all trials spiders.

106 0.0016 A B B C 0.0014

0.0012

0.001

0.0008

Plectrum Area Plectrum 0.0006

0.0004

0.0002

0 Schizocosa Gladicosa Rabidosa Tigrosa Genus

Figure 4. Plectrum area scaled for overall body size across the four genera.

107 Chapter 6- Conclusion: An evolutionary framework for the emergence of an adaptive acoustic signal in Gladicosa gulosa

108 GENERAL CONCLUSIONS

The initial curiosity of courtship in Gladicosa gulosa was drawn from the anecdotal works of early arachnologists which suggested, albeit without experimental evidence, the possibility of a courtship phenomenon completely atypical of spiders, ranging from a far-field acoustic signal to the possibility of male chorusing behavior (Davis 1904; Lahee 1904; Allard

1936; Kaston 1936; Harrison 1969). The goal of this thesis was to begin the long process of thoroughly investigating the true nature of these phenomena, reducing each piece of the signal and behavior to its component parts and attempting to dissect whether or not each observation could be supported through experimental evidence. The data presented in this thesis in part lays to rest some of the less credible suggestions about this species. Mostly, it provides the groundwork for what I believe could be a framework for demonstrating the evolution of an adaptive acoustic signal in G. gulosa, though numerous additional studies are needed to truly confirm such a phenomenon. Moreover, regardless of the outcome of investigations into the acoustic potential of this species, this thesis should raise additional questions about the communication system of this species, providing launching points for its value in studying the ecology of wolf spider behavior.

In this concluding chapter, I provide brief summaries of the preceding chapters, highlighting the most salient results as they relate to the broader question of potential acoustic signaling, and then provide what I believe to be a possible framework for the emergence of an adaptive acoustic signal. I will then provide a series of directions for future studies. It is my hope that the conclusions drawn in this thesis will inspire additional investigation beyond my own studies, and an eventual confirmation and reinforcement of the value of this species as a model system, for acoustic communication and any number of additional behavioral phenomena.

109 Chapter 1 provides a fundamental quantitative investigation and characterization of the signal, to a degree of clarity that was previously unpublished for this species. The characterization of the signal itself, at its most basic level, provides the beginnings of any understanding for the origin of the acoustic component of this male signal, as well as quantitative confirmation of its existence beyond that proposed by Harrison (1969). The acoustic signal is structurally similar to the vibratory signal and time-locked with the production of the vibratory signal. That result, taken with the absence of any apparent organs adapted for detecting the airborne signal directly, lead to the conclusion that airborne sound produced in this species is almost certainly a by-product of the high amplitude of the vibratory signal.

However, within this conclusion is an important factor that contributes to the overall evolutionary framework for this species' communication- vibratory and acoustic signal amplitudes are significantly correlated. The normal distribution of vibratory amplitudes of signaling males corresponds to a similar distribution of acoustic signals, and males that produce a stronger vibratory signal reliably produce a stronger acoustic signal.

Chapter 2 provides a large data set related to relatively simple methods of quantifying behaviors during the live courtship and mating interactions of males and females. Part of the value of this chapter is establishing previously undescribed behaviors during these interactions and statistically evaluating their individual value as metrics for mating success. These results can thus be used to justify future experimental manipulations without requiring the live presence of both males and females.

Still, the most salient result from this chapter with regard to the potential for acoustic signaling lies in the relationship between the most predictive behaviors of males and females.

110 Male vibratory signaling amplitude is significantly correlated with a corresponding tapping behavior from females, both of which individually predict mating success in this system. Given that male acoustic signal amplitude correlates with vibratory signal amplitude, the possibility of female responses driving a stronger vibratory signal is important in the emergence of an acoustic signal.

Chapter 3 replicates, to some extent, previous studies investigating the role of conductive substrates in the effective communication of wolf spiders, though it additionally focuses on the continued connection between vibratory and acoustic signals. Though no morphological evidence exists for the presence of an adapted structure on the spider's body for radiating sound from a stridulatory organ (similar to those found on orthopterans, for example), the results of this chapter confirm that in the absence of a properly vibrating substrate, both modalities are fully attenuated. Males can and do produce a measureable acoustic signal on a variety of substrates, but only on those substrates that readily conduct the vibratory signal.

While the results of this chapter are somewhat unsurprising given previous studies, the role of the substrate provides a compelling argument for the contribution of the physical environment to the potential for a relevant acoustic signal. While sexually selective forces may be the best explanation for a driving force behind signal strength, an accommodating habitat is without a doubt a necessary part of the overall evolution of the signal.

Chapter 4 provides quite possibly the most convincing results as pertain to the actual value of the acoustic signal, while simultaneously debunking myths about possible chorusing behavior. Isolated acoustic signals presented across a variety of contexts to both males and females result in a demonstrable pattern of behavioral shifts. Males do not react at all to any

111 acoustic stimuli, suggesting that regardless of potential for reception of the signals, their informational value seems to be largely ignored by males, or at the very least masked by the high level of activity in exploring males. Females, however, showed a marked change in their behavior under very specific circumstances. Females were more likely to move, and began said movement more readily when in the presence of male conspecific signals on a vibration- conductive substrate. This demonstrates that 1) females likely detect acoustic signals via induced vibration, and 2) females likely discriminate between conspecific signals and other non-relevant acoustic stimuli. While no females showed receptivity behaviors like those described in chapter

2, the change in movement patterns is not irrelevant to this mating system.

Wolf spider courtship is largely persuasive rather than attractive, and it is generally up to males to locate a female on his own, since signals are not designed to draw females to a male from a distance. Thus, any mechanism through which females become more exploratory and are more likely to be located by males may serve to increase male fitness. Moreover, while recent studies make a compelling case that spiders are sensitive enough to detect airborne sound, in the absence of evidence that detection of sound results in significant changes to behavior, the relevance of sound perception is still lacking when discussing conspecific communication. This chapter, first and foremost, demonstrates that isolated acoustic signals result in meaningful and measureable changes in behavior.

Chapter 5 attempts to pull the scope back from the focus on G. gulosa and investigates whether the supposed phenomena of its courtship could exist in its co-occurring lycosid relatives.

While the survey itself is relatively small, the results are a compelling suggestion that the

"unique" nature of G. gulosa courtship lies strictly in its comparative strength, and not in its mechanistic or biophysical characteristics. The value of these results is thus two-fold: it confirms

112 that the audible nature of G. gulosa is not only measurable in isolation, but across related species independent of size, and it confirms that an evolutionary framework for the emergence of this signal is not species-specific, as the same patterns and mechanisms, when give the proper selective forces, could drive a similar signal in other species.

113 EVOLUTIONARY FRAMEWORK

Perhaps the most appropriate context in which to view this series of conclusions is through the lens provided by the ornithologist R. Haven Wiley, who has proposed a view of the evolution of communication that centers on the role of "noise" in all contexts as the driver for efficiency and quality in communication for both senders and receivers (Wiley 2013, 2015).

Wiley suggests that noise drives optimal signaling, though "optimization" may not have the same definition for senders and receivers. Signals in their current form are a combine result of forces driving effective signal transmission from senders (in this case, males) and decision-making accuracy of receivers (in this case, females). The resulting signal occupies a normal distribution that straddles the overlap with background noise and the response threshold of receivers (Fig. 1).

When viewed in conjunction with the unique biology of spiders, Wiley’s proposed framework may help explain the possible emergence of adaptive acoustic signaling arising from vibratory signaling of Gladicosa gulosa. Undoubtedly, these results as well as numerous other publications have demonstrated an intrinsic link between vibration and acoustic signals

(Caldwell 2014), so in the context of Wiley's framework, any spider species utilizing a vibratory signal on a conductive substrate, may simultaneously produce an acoustic signal (even as a by- product). Because it would be perceived as induced vibration, the acoustic signal may be shaped by the same factors as the vibratory signal, even in the absence of direct detection. This is supported by the relationships demonstrated in chapters 1, 3, and 5. However, in the context of

G. gulosa signaling, a correlation between male signal strength and female response may lead to sexual selection for "louder" male vibration. Thus, exaggeration of this vibratory signal would result in simultaneous exaggeration of the acoustic signal.

114 The potential value of the acoustic signal lies in the aforementioned "threshold" for female response, or more likely a series of thresholds: one for raw sensory perception, another for identification and detection, and yet another for qualitative mate assessment. While the latter threshold is likely set at the highest level for optimal accuracy in signal assessment, the threshold for species identification and detection of courting males may remain lower. Consequently, as both signals shift towards higher amplitude, the induced vibrations of an acoustic signal may pass through this threshold (Fig. 2). The resulting phenomenon would then lead to females showing a detection/identification response to isolated signals without showing direct receptivity, as shown in chapter 4.

The series of studies comprising this thesis only scratch the surface of the depth of questions that still surround the intriguing nature of the communication system in G. gulosa and its value in studying the evolution of communication. Undoubtedly, the previous chapters serve to raise more questions than they ultimately answer. In the final concluding paragraphs, I will outline my thoughts on a few of the most promising directions for the research that will hopefully continue on this species. Regardless, this species has the potential to serve as focal system for answering intriguing questions across a variety of contexts in the study of communication.

115 FUTURE DIRECTIONS

Adaptive Value of the Acoustic Signal

Given the framework and over-arching questions driving this thesis, the most obvious route for continued work would be to begin the long process of investigating hypotheses that might ultimately demonstrate adaptive value for the acoustic signal. Chapter 4 begins that process, though it does not provide data that can conclusively demonstrate adaptive value. While the isolated acoustic signal is detectable and produces relevant changes in behavioral that could be potentially advantageous, it appears to be perceived via the same mechanism that the vibratory signal is detected, and at most ranges of interaction between males and females, the acoustic signal would be entirely masked by the primary vibration. However, vibratory signals are heavily dependent on contiguous substrates, and they attenuate rapidly at breaks in the substrate. Thus, the transfer of some of the signal energy into an airborne signal that can then travel to another vibrating substrate could potentially extent the range of the overall signal.

While I have already begun collecting data on this possibility, there are no doubt numerous field and lab experiments needed to fully confirm this possible range extension hypothesis.

Mechanism of Amplification

The previous chapters have confirmed, I believe, that the amplitude of G. gulosa courtship is uniquely strong, even in the context of other species. It is clearly generated via the same vibratory mechanisms present in other lycosids, and the substrate plays a vital role in its production. Still, a physiological, anatomical, or energetic explanation for how this species manages to produce such an exaggerated signal remains to be found. While gross anatomy and organ size do not seem to be factors, any number of other hypotheses could explain the

116 phenomenon. My own possible speculations range from the relatively straightforward (possibly a stronger energetic investment) to the more far-fetched (possible constructive wave interference in the two alternating organs). From a mechanistic viewpoint, this question may have considerable value given that stridulatory organs are common across arthropods.

The Third Element

As briefly mentioned in chapter 1, there is a subtle, but consistent element produced during courtship that was not taken into consideration when focusing on the questions of acoustic signaling in this species. Still, the regularity and considerably smaller amplitude of this signal are intriguing. It is not clear how this signal is produced, or more importantly, why it is produced.

The explanation may end up fairly mundane, but it remains a substantial curiosity, and one that could potentially shed light on other aspects of this species’ communication.

Patterning

Again, a point that is briefly considered in chapter 1 is the regular, and seemingly predictable, pattern of the elements in the construction of the signal in this species. The two primary signal elements are very regular in their production, and the bouts of courtship are surprisingly predictable in their timing and composition. This may create an opportunity for an entire series of experiments investigating the role of pattern in species identification and female preference.

Ecological Trade-Offs

One factor that has not yet been discussed throughout this dissertation is the evolutionary cost of producing this signal. While the value of this high-amplitude vibration and its

117 corresponding acoustic byproduct is becoming increasingly clear, this fails to consider constraints or disadvantages that may be conferred on louder males. From an ecological perspective, the fact that spiders do not seem to directly perceive sound does not preclude the fact that several of their predators do. The responses of predatory species, and what influences they have on providing a possible ceiling for the exaggeration of this signal, remains an interesting area of investigation.

118 REFERENCES

Allard, H. A. 1936. “The drumming spider (Lycosa gulosa Walckenaer).” Proceedings of the

Biological Society of Washington 49: 67-68.

Caldwell, M. S. 2014. “Interactions between airborne sound and substrate vibration in animal communication.” In: Cocroft, R. B., Gogala, M., Hill, P. S. M., & Wessel, A. (eds). Studying

Vibrational Communication. Springer, Berlin. pp. 65-92.

Davis, W. T. 1904. “Spider calls.” Psyche 11(6): 120.

Harrison, J. B. 1969. “Acoustic behavior of a wolf spider, Lycosa gulosa.” Animal Behaviour

17(1): 14-16.

Kaston, B. J. 1936. “The senses involved in the courtship of some vagabond spiders.”

Entomologica Americana 16(2): 97-167.

Wiley, R. H. 2013. “Signal detection, noise, and the evolution of communication.” In: Brumm,

H. (Ed.) Animal Communication and Noise. Springer Berlin Heidelberg. pp. 7-30.

Wiley, R. H. 2015. Noise Matters: The Evolution of Communication. Harvard University Press.

119 TABLES & FIGURES

Figure 1. Theoretical distribution of probability of response from a receiver receptor across a range of receptor activity for a signal, in this case the vibratory signal of G. gulosa.

Redrawn from Wiley (2015; Fig 7.3 p. 140).

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Figure 2. Theoretical distribution of receptor activity (or response from a receiver) for a second signal, in this case, the acoustic byproduct signal of G. gulosa. Further adapted from Wiley (2015; Fig 7.3 p. 140). Any selective force driving exaggeration of the vibration should lead to a corresponding shift in the acoustic signal, possibly into the receiver’s threshold of response.

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