Signal Complexity in Schizocosa Ethospecies

Good Vibrations: Signal Complexity in Schizocosa ethospecies

A thesis submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of

Master of Science in the Department of Biological Sciences of the College of Arts and Sciences by

Madeline Lallo B.S. University of Cincinnati

March 2019 Committee Chair: George W. Uetz, Ph.D. Cincinnati, OH

Abstract Communication signals have evolved to convey information from a sender to a receiver through different sensory modalities. These signals may vary in their complexity to ensure successful transmission through the environment and enable receiver discrimination. Sibling wolf spider species, Schizocosa ocreata and S. rovneri, have recently diverged and are reproductively isolated by their behavior during courtship. Males of both species court females using multicomponent vibratory signals that vary in their complexity. The vibratory signal of male S. ocreata represents a complex pattern of stridulation and percussion components, compared to that of S. rovneri, which produces a regular pattern of brief pulses of nearly simultaneous (combined) stridulation and percussion components. I examined the role of signal complexity in species recognition and mate preference using vibratory playback via piezoelectric disc benders of separate individual components (percussion and stridulation) from each male signal. Female S. ocreata and S. rovneri were exposed to either conspecific or heterospecific signals within four treatment groups: complete signal, percussion only, stridulation only, or white noise. The number of female receptivity displays varied significantly among treatment groups for both S. ocreata and

S. rovneri females, however there was no difference in female receptivity to individual components (stridulation and percussion) compared to complete signals. There were significant differences in the number of female receptivity displays for both S. ocreata and S. rovneri when presented with playback of complete conspecific vs. heterospecific vibration signals as females were more receptive to conspecific signals. Each focal species responded differently to treatment groups, with S. rovneri displaying significantly more receptivity displays compared to S. ocreata.

I determined that individual signaling components are redundant and when combined in a complete signal they elicit an equivalent response in terms of number of female receptivity

ii displays. My results show that females of these two ethospecies recognize isolated vibratory signaling components of conspecifics and heterospecifics.

iii

Acknowledgments First and foremost, I want to thank my advisor, Dr. George Uetz, for all the advice, opportunities, and support over the six years I have known him. I will never forget the day I walked into his office, career binder in hand, trying to impress him. He “molded my brain” into the scientist I am today, and I will never be able to repay him. Thank you, George. And special thanks to Kitty Uetz for all her support and baked brie.

Thank you to my committee members, Dr. Nate Morehouse and Dr. Elke Buschbeck, for all their feedback and comments they gave me over the years. And to the Cincinnati Nature

Center and Great Parks of Hamilton County for letting me collect spiders at their parks.

Thank you to the friends I have made over the years – Dr. Brent Stoffer, Dr. Alex Sweger,

Dr. Rachel Gilbert, Tim Meyer, Trinity Walls, and most importantly, Emily Pickett, for all the support and pushing I needed to accomplish this feat. Time with you all has been an unforgettable journey. Special thanks to Olivia Bauer-Nilsen for being there my last year as a lonely graduate student.

To my grandfathers, Jim Lallo and John Cernica, who both passed away during my first year in the program. They were the strongest and smartest men I knew. Thank you so much, you’ve given me something to strive for.

Last, but certainly not least, I would like to thank family. None of this was possible without them, and for that, I am forever grateful. Thank you to my siblings – Nicholas, Anthony, and Vivian, for the late nights of soccer and beer when I needed it the most. My parents gave me immense love and support throughout my graduate school career, so thank you Mom and Dad.

Words cannot express how thankful I am to have you both in my life.

Table of Contents Abstract ……………………………………………………………………………..……..…….. ii Acknowledgments …………………………………………………………………....………….. v List of Tables and Figures ………………………………………………….……………...….. vii Introduction ……………………………………………………………………...……...….…… 8 Study Species and Research Problem …………………………………...…..…….….… 10 Goals and Objectives …………………………………...……………………..……...… 11 Methods …………………………………...………………………………………..………...… 15 Collection and Rearing ………………………………..………………...…..………..… 15 Exemplars …………………………………...……………………………....………...… 15 Calibration and Playback …………………………………...………………………..… 16 Experimental Trials …………………………………...……………………..………..… 17 Data Analysis …………………………………...…………………………...………….. 18 Results …………………………………………………………………...……...…..…….….… 21 Seasonal Differences …………………………………...…..……………………..….… 21 Generalized Linear Models …………………………………...…..…………...….…..… 21 Latency to Move …………………………………...……………………………….…… 22 Latency to Receptivity …………………………………...…..………………………..… 23 Discussion …………………………………...…………………………………….…………..... 38 Conclusion …………………………………………………………………...…..………..…… 43 References …………………………………………………………………...…..………..…… 45

List of Tables and Figures Figure 1: Phylogeny of the ocreata clade of the genus Schizocosa. Figure 2: Vibratory signals of male S. ocreata and S. rovneri. Figure 3: Laser Doppler vibrometer. Figure 4: Experimental trial set up. Table 1: Generalized linear model results. Figure 5: Female receptivity responses to the presence/absence of signaling components. Figure 6. Female receptivity to presence/absence of stridulation and percussion separated by species. Figure 7: Female receptivity to conspecifics and heterospecifics. Figure 8: Number of receptivity displays by focal species. Table 2: Parametric survival analysis of latency to move. Figure 9: Latency to move by focal species. Figure 10: Latency to move to presence/absence of percussion signals. Figure 11: Latency to move to conspecific/heterospecific stridulation signals. Figure 12: Latency to move to all conspecific and heterospecific treatments. Table 3: Parametric survival analysis of female latency to receptivity. Figure 13. Latency to receptivity to presence/absence of stridulation. Figure 14: Latency to receptivity to presence/absence of percussion Figure 15: Latency to receptivity of all treatments.

vii

Introduction

Communication is ubiquitous in the animal kingdom, and signals have evolved to convey information from a sender to a receiver. Some communication signals are more varied or complex, which may ensure there is successful transmission through the environment and allow for receiver discrimination. Signals are used to grab the attention of receivers, which comes with some risk if the receivers are unintended (such as potential predators, competition, other species) but will potentially lead to some reward with intended receivers, such as mates.

Communication signals may relay species information to assist in recognition of conspecifics, which in turn helps ensure behavioral reproductive isolation between closely related species. Pre-mating isolation, (e.g., behavioral isolation between two species prior to copulation and fertilization), enhances fitness, as it prevents potential interspecies hybrids, which are likely sterile and do not contribute to future generations. Examples of this behavioral reproductive isolation can be found in models such as tree frogs (Gerhardt 1974), Heliconius butterflies (Southcott & Kronsforst 2018) and wolf spiders (Stratton & Uetz 1981). Behavioral pre-mating reproductive isolation is predominantly driven by female recognition of species- specific male signals, likely arising from or reinforced by mate choice.

Some animals use a combination of different sensory modes to ensure successful transmission of information. When two or more of these modalities are used simultaneously, it is defined as multimodal communication (Partan & Marler 1999). The use of multimodal communication is prevalent across many animal taxa, ranging from vertebrates – such as primates (Ghazanfar et al. 2005; Leavens et al. 2010), fish (Maruska et al 2012) - to invertebrate taxa such as spiders (Uetz 2000; Uetz & Roberts 2002; Hebets 2008; Uetz et al. 2009), and crayfish (Acquistapace et al. 2002; Crook et al. 2004). There are several different hypotheses

8 explaining the use of multimodal vs. unimodal communication: 1) the multiple messages hypothesis (Møller & Pomiankowski 1993) which states that separate modes are used to send different information about the subject (bright colored plumage for parasite load, acoustic calls for quality/size, etc.); 2) the redundant signals hypothesis (Møller & Pomiankowski 1993) which proposes that these signals are all sending the same information, with regard to mate quality, to the receiver about the signaler; and 3) the species recognition hypothesis, which states that species recognition is more effective when a combination of different traits/modalities are used

(Pfennig 1998).

Invertebrate animals, and spiders in particular, are more frequently becoming recognized models in which to study communication, despite earlier bias toward vertebrate animals (Witt &

Rovner 1982, 2014; Uetz 2000; Huber 2005; Uhl & Elias 2011; Uetz et al. 2016). For example, communication in spiders is critical for mating success, arguably more so than other species.

Spiders need to be able to identify both conspecifics and heterospecifics to avoid potential harm, such as cannibalism. Male spiders must also court female spiders in order to mate, so there is pressure on males to communicate effectively, or else risk being the next meal. Spiders use a variety of signal modes to communicate - visual signals, vibratory signals, chemical signals, or a combination of the three (Stratton & Uetz 1983; Scheffer et al. 1996; Uetz 2000; Stratton 2005;

Hebets et al. 2008; Uhl & Elias 2011; Uetz et al. 2016). Spiders can convey information such as species identification information (Scheffer et al. 1996; Uetz et al. 2009; Elias et al. 2003, 2006), mate quality (Uetz et al. 2002; Rypstra et al. 2003), and mating status (Moskalik & Uetz 2011a, b; Meyer & Uetz 2019) to each other based on visual, vibratory and chemical signals.

Study species and research questions

The genus Schizocosa (family: Lycosidae) is a well-studied taxon of medium sized wolf spiders found all over the United States (Dondale & Redner 1978, 1990; Stratton 2005). Species in this genus exhibit considerable variation with respect to male morphology and courtship complexity, especially within the ocreata clade (Fig. 1). Some species have very prominent multimodal courtship (visual and vibratory: S. ocreata, S. stridulans) while others use a unimodal courtship signal (vibration: S. rovneri, S. uetzi). While not all Schizocosa spiders use visual signals when courting females, they all use vibratory signals (Uetz 2000; Hebets et al.

2013).

The wolf spiders S. ocreata and S. rovneri are recently diverged sibling species that are reproductively isolated by their behavior (Stratton & Uetz 1983, 1986). These two spiders have identical genitalia and were once considered a single species (Uetz & Denterlein 1979; Uetz &

Dondale 1979). However, these spiders are reproductively isolated by their behavior, making them ethospecies (Hollander et al. 1974; Uetz & Denterlein 1979; Stratton & Uetz 1981, 1983).

Although S. ocreata and S. rovneri have the potential to hybridize, they typically do not, owing to differences in their courtship behavior (Stratton & Uetz 1981; Stratton & Uetz 1986). Analysis of phylogenetic relationships places these two species as sharing a node on the cladogram

(Hebets et al. 2013). Hebets, et al. (2013) calculated complexity scores for species in the

Schizocosa clade, ranking S. ocreata as the most complex (with a score of 4) and S. rovneri as the least complex (score of 1). The difference in complexity scores is interesting, given their status as ethospecies, and allows for a “natural experiment” to test the evolution of divergence in signal complexity.

Although sibling species S. ocreata and S. rovneri do not both use visual signals when courting females (only male S. ocreata do, with leg tufts as well as leg tapping and waving displays), both species use vibratory signals. Male vibratory courtship in these two ethospecies is made of two main components: stridulation and percussion (figure 2). Stridulation is the act of rubbing two body parts together (usually a file-and-scraper mechanism), while percussion is produced by the sternum or abdomen of the spider hitting the substrate. Although both S. ocreata and S. rovneri produce vibratory signals with both components, the structure of these components differs, and is species-specific. In male S. ocreata, vibratory signals are arguably more complex and irregular, while those of S. rovneri are more patterned and regular. Given these differences in vibratory signal complexity, questions arise regarding the role of individual components of vibratory signaling in the evolutionary divergence of these two ethospecies.

Goals and objectives

The overall goal of this study was to understand the role(s) of signal complexity and individual vibratory components in species recognition and isolation and thereby gain insight to the evolutionary divergence of these sibling species. To determine if individual vibratory components (stridulation, percussion) were the same in both species, or were species-specific, it was first necessary to identify the essential components of vibratory communication signals in each species, and whether females can discriminate between components of signals from conspecifics and heterospecifics. The objectives of this study can thus be framed as three main questions: (1) Are females receptive to isolated individual vibratory components (stridulation and percussion) of conspecific and heterospecific male signals?; (2) how do female responses to individual components compare to complete signals?; and (3) Are these components species- specific, or interchangeable elements of vibratory courtship? Female receptivity to individual

11 signaling components will help us understand important aspects of the vibratory signal such as a preference for components or preference for heterospecific or conspecific signals. To address these questions, I used digital editing technology to manipulate courtship signal structure and complexity (increased or reduced signal complexity) and examined receptivity responses of female S. ocreata and S. rovneri to isolated vibratory courtship signals of conspecifics and heterospecifics (complete signals compared to simplified signals).

Figure 1. Phylogeny of the ocreata clade of the genus Schizocosa, showing patterns of vibratory signals for each species (from Hebets et al. 2013). Highlighted bars indicate species with leg tufts.

Figure 2. Vibratory signals of male S. ocreata and S. rovneri, with the individual vibratory components of each signal indicated.

Methods

Collection and rearing

Female and male spiders were collected in fall 2016, and both spring and fall of 2017 at

Triple Creek Park in Hamilton, OH (S. rovneri) as well as the Cincinnati Nature Center Rowe

Woods in Milford, OH (S. ocreata). Spiders were collected as immatures and allowed to mature in the lab. Individuals were housed separately in deli dishes with free access to water and fed 2-3 crickets twice a week. Humidity, temperature, and light (L:D 11:13h) were held constant in the lab, simulating early spring conditions to stimulate maturation.

Exemplars

To create audio files of male S. ocreata and S. rovneri components for playback, 25 males of each species (from the fall 2016 group) were recorded using a laser Doppler vibrometer

(LDV) (Polytec LDV 100). To stimulate male courtship, females laid down silk (with chemotactile cues such as pheromones) 24 hours before recordings on filter paper (Fisher

Scientific). The filter paper was placed within an arena in an anechoic chamber upon a small slab of granite to isolate it from outside vibrations. The LDV was placed below the filter paper and arena and focused on a mylar dot attached to underside of the filter paper (Fig. 3). Males were placed on top of the filter paper and were allowed to court for 150 seconds. Recordings were made using SpectraPlus acoustic analysis software (Hanning window, 2048 FFT).

Male signals were analyzed in SpectraPlus to determine mean amplitude for individual components, stridulation rate, and percussion rate, from which population averages were obtained. Amplitude was measured as root mean square values (RMS). A principal component analysis (PCA) of vibration measures for both species was run to determine three individuals that best represent the population average to use as exemplars. Mean values for stridulation

15 amplitude, stridulation rate, percussion amplitude, percussion rate, and mass were all used as components in the PCA analysis. We chose three individuals (of both species) closest to the centroid of a two-dimensional projection of PC1 and PC2 which represented the average for each factor in the PCA. These individuals were determined to be the best representation of what an average male of each species would be. These individuals were then used as the basis for our exemplars and treatment groups.

Three different playback exemplars (i.e., separate individuals) for each species were used to avoid pseudo-replication (Hurlbert 1984; McGregor 2000). These exemplars were created using Audacity® (version 2.1.2, Audacity 2018) recording and editing software. Three treatment groups (complete signal, percussion only, stridulation only) were created from each exemplar spider, along with a white noise treatment group for each exemplar, making 18 total audio files.

The complete signal treatment group exemplars were unmanipulated, while for both the percussion only and stridulation only treatments, components were eliminated. For example, for the percussion only group, stridulations were removed, and for the stridulation only treatment group, percussion was eliminated. Exemplars were each 30 seconds and looped between five- and-a-half to six minutes long to last the whole trial period.

Calibration and Playback

Vibratory playback of the exemplars was through Piezoelectric discbenders, with a signal from an Apple iPod touch™ connected to a Pyle mini 2x40W Stereo Power Amplifier. The

Piezoelectric discbender was attached to parchment paper in the arena. Parchment paper was used because it transmits vibrations in a manner similar to dried leaves (Uetz & Gibson unpubl.).

A plastic circular arena (15cm diameter) was placed atop Bristol board and granite blocks (figure

4). These were used to keep the surface flat and make the arena taller while also isolating the arena from outside vibrations.

Playback exemplars were calibrated using the LDV to determine the correct amplifier volume at which to play the files. Amplitude measurements were taken at different volumes of the amplifier at the discbender to determine the volume that best matched the population average amplitude of living spiders.

Experimental trials

Mature females of both species were selected for playback trials once they were 10-15 days post maturity. Based on work from Norton and Uetz (2005), females are less receptive in the first week of maturity and very receptive after 15 days of maturity, so we chose the middle- aged females (females between 10-15 days post-maturity). Spiders were assigned at random to treatment groups, exemplars, and exposure to conspecific/heterospecific signals. Trials lasted five minutes and were recorded using a Sony Handycam (HDR-XR260) so trials could be scored for receptivity. Female spiders were only exposed to treatment groups once, so each trial was independent of others and there were no repeated measures.

Tapes of female responses were scored blind for receptivity behaviors using version 2.2.6 of VLC media player. Receptivity displays were used as a proxy for mating, as they indicate female willingness to mate and it is well-established that copulation will usually not occur unless one or more of them is displayed (Montgomery 1903; Uetz & Denterlein 1979; Stratton & Uetz

1981, 1983; Scheffer et al. 1996; Delaney M.S. Thesis 1997; Norton & Uetz 2005; Delaney et al.

2007). Female S. ocreata and S. rovneri both present three distinct behaviors (settle, tandem leg extend, slow pivot) when they are willing to mate. More recently, the quantity of female displays has been shown to be associated with latency to copulate, and the sum of receptivity displays has

17 been used as a comprehensive index of receptivity (Uetz & Roberts 2002; Uetz & Norton 2007;

Johns et al. 2009; Rutledge & Uetz 2014). Latency to move and latency to display receptivity were also recorded for all trials.

Data Analysis

Generalized linear models (GLM) were constructed in R studio statistical software

(version 1.1.463) to determine significance. A quasi-poisson was used to account for the zero- inflated and over dispersed data set. To test our hypothesis that individual components will elicit female receptivity responses, full models were created and compared to “null” models with likelihood ratio tests to determine if factors within the model influenced the receptivity behavior of females. After determining significance with the full model, interactions and individual factors were removed and compared to the full model using a likelihood ratio test. If the newly reduced model compared to the full model was significant, it was determined that the removed interaction/factor contributed significantly to the receptivity display of the female (Crawley

2013). This method was used to test hypotheses about the effects of individual components on female receptivity. We did not test for the best model that predicts female receptivity or the signaling components that were more important within the signal. Parametric survival analyses were used to determine significance regarding female latency to move and latency to receptivity.

Figure 3. Laser Doppler vibrometer. Males were placed into an area with filter paper. The

LDV was below the arena fixated on a Mylar® dot.

Figure 4. Experimental trial set up. A circular area was placed on top of granite blocks. A

Piezoelectric discbender was used to transmit vibrations across the filter paper.

Results Seasonal differences

Trials were conducted over two research seasons (spring 2017 and spring 2018). A

Student’s t-test comparing female receptivity scores across two research seasons revealed no significant difference between them (t = -1.8751, df = 407.43, p = 0.06149). Data points from both seasons were then pooled for all remaining analyses.

Generalized Linear Models

I constructed a generalized linear model (GLM) with a quasi-Poisson distribution (in R studio version 1.1.463) for the total number of female receptivity displays as a response to the presence/absence of percussion, stridulation, exposure to heterospecific or conspecific, and focal species (S. ocreata or S. rovneri) (Table 1). The full GLM model was significant (X2 = 1400.05, df = 15, p = 2.20 x 10-16). I then began by removing factors and interactions within the model to determine the significant factors that influence female receptivity. This approach was used to test the hypothesis that male vibratory components will elicit receptivity from females. We did not test for preference of components, or which component is more important within a vibration signal.

After removing interactions from the GLM, I found the only significant interaction term was the interaction of stridulation and percussion (X2 = 132.41, df = 6, p = 8.41 x 10-08).

Breaking down the GLM further, the presence and absence of percussion and stridulation separately are significant (X2 = 189.68, df = 12, p = 7.46 x 10-09; X2 = 181.7, df = 12, p = 2.27 x

10-08; figure 5). It is apparent that regardless of the focal species, females are more receptive to signals that include one of the typical components, whether it be percussion or stridulation.

While females are equally receptive to both percussion and stridulation in isolation, there is no additive or enhanced effect of female receptivity displays when exposed to a complete signal.

Females are just as receptive to an individual signaling component or “simplified signal”, as to a complete signal (Figure 6).

Exposure to conspecific or heterospecific signals did have a significant effect on the total number of receptivity displays (X2 = 150.72, df = 12, p = 1.54 x 10-06; Figure 7). Females are more receptive to male signals of the same species. This was followed by the significant effect of which focal species was being used (X2 = 175.1, df = 12, p = 5.64 x 10-08; Figure 8). Female S. rovneri displayed more receptivity behaviors compared to S. ocreata females.

Latency to move

Data on latency to move (i.e. the time it takes for a spider to move in their arena after starting a trial) were collected when scoring the trials, and a parametric survival analysis was used to analyze the latency to move data (Table 2; Figures 9,10,11,12). Factors included focal species, exposure to conspecific or heterospecific, and the presence or absence of stridulation and percussion. The full model predicted a significant amount of variation in our data (X2 =

36.3223, df = 15, P= 0.0016). A three-way interaction between exposure to conspecific or heterospecific, stridulation, and percussion also had a significant effect on female latency to move (X2 = 5.0223, df = 1, p = 0.025). Females of both species resumed movement more quickly when exposed to white noise signals and more slowly with exposed to simplified or complete signals. A two-way interaction of exposure to conspecific and heterospecific signals with stridulation was also significant (X2 = 5.3127, df = 1, p = 0.0212). Female S. ocreata resumed movement more quickly when exposed to heterospecific stridulation signals. Lastly, presence/absence of percussion was significant (X2 = 7.9821, df = 1, p = 0.0047) as well as focal species (X2 = 14.4028, df = 1, p = 0.0001). Females of both species resumed movement quickly

22 in response when signals were absent of percussion, but female S. rovneri resumed movement more slowly when exposed to complete heterospecific signals compared to S. ocreata.

Latency to receptivity

Females were scored for their latency to a receptivity display (i.e. how long it took a female to display a receptive behavior after exposure to the signal). A parametric survival analysis was used to analyze the latency to receptivity data (Table 3; Figures 13,14,15). The overall model was significant (X2 = 103.4339, df = 15, p = < 0.001). There was also a significant effect of the interaction of stridulation and percussion on female latency to receptivity (X2 =

51.7222, df = 1, p = < 0.001). Individual components, stridulation and percussion, had a significant effect on the latency to female receptivity displays, females were quicker to show receptivity when exposed to a signal with an individual component, regardless of what component it was. The presence/absence of percussion explained a significant amount of the variation in female receptivity (X2 = 36.9597, df = 1, p = < 0.001), as well as the presence/absence of stridulation (X2 = 45.2388, df = 1, p = < 0.001). As stated above, females were quicker to display receptivity to individual component signals, regardless of exposure to conspecific or heterospecific.

Table 1. Generalized linear model results. Each factor was removed from the overall full model and compared to it with a likelihood ratio test to test the hypothesis that individual factors influence female receptivity. Probabilities indicate effect of the interaction/factor on female receptivity displays (significance at α < 0.05 indicated in yellow).

Female Receptivity - GLM family = quasi Poisson X2 df Probability Full model - compared to Null 1400.05 15 2.20 x 10 -16 FocalSpecies*ConHet*Stridulation*Percussion 3.6728 1 0.2708 Con/Het * Strid * Perc 5.3167 2 0.4157 Focal Species * Strid * Perc 11.536 2 0.1489 Focal Species * Con/Het * Perc 3.9194 2 0.5236 Focal Species * Con/Het * Strid 5.8622 2 0.3799 Strid * Perc 132.41 6 8.41 x 10-08 Con/Het * Perc 20.676 6 0.3372 Focal Species * Perc 18.792 6 0.4007 Con/Het*Strid 18.883 6 0.3974 Focal Species * Strid 18.47 6 0.4123 Focal Species * Con/Het 27.448 6 0.1701 Percussion 189.68 12 7.46 x 10-09 Stridulation 181.7 12 2.27 x 10-08 Conspecific/Heterospecific 150.72 12 1.54 x 10-06 Focal Species 175.1 12 5.64 x 10-08

1.8

1.6 1.4 1.2 1

0.8

receptivity Mean 0.6 0.4

0.2 0 Yes No Stridulation?

1.8

1.6 1.4 1.2

1 0.8

Mean receptivity Mean 0.6

0.4

0.2

0 Yes No Percussion?

Figure 5. Female receptivity responses to the presence/absence of signaling components

(pooled for both species) (p = 2.27 x 10-08 ; p = 7.46 x 10-09). Percussion/stridulation “yes” may indicate the component only signal or the complete signal. Percussion/stridulation

“no” indicates either the opposite component signal or white noise.

3 3

2.5 2.5

2 2

1.5 1.5

1 1 Mean receptivity Mean

Mean receptivity Mean 0.5 0.5

0 0 White Noise Percussion Stridulation Complete White Noise Percussion Stridulation Complete Only Only Only Only Treatment Treatment

Figure 6. Female receptivity to presence/absence of stridulation and percussion separated by species (S. ocreata above; S.

rovneri below) (p = 8.41 x 10-08).

2.5

1.5

Mean receptivity Mean 0.5

0 Conspecific Heterospecific

Figure 7. Female receptivity to conspecifics compared to heterospecifics. Females are significantly more receptive to signals of the same species (conspecific) compared to heterospecifics (p = 1.54 x 10-06).

1.8 1.6 1.4

1.2 1 0.8

0.6 Mean receptivity Mean 0.4 0.2 0 S. ocreata S. rovneri

Figure 8. Number of receptivity displays by focal species. Females of S. rovneri display significantly more receptivity compared to S. ocreata (p = 5.64 x 10-08).

Table 2. Parametric Survival Analysis of latency to move (significance at α < 0.05 indicated in yellow).

Parametric Survival Analysis Chi-sq df Probability Full model 36.3223 15 0.0016 FocalSpecies*ConHet*Stridulation*Percussion 0.0001 1 0.9936 Con/Het * Strid * Perc 5.0223 1 0.025 Focal Species * Strid * Perc 0.1420 1 0.7063 Focal Species * Con/Het * Perc 0.5660 1 0.4518 Focal Species * Con/Het * Strid 0.0498 1 0.8234 Strid * Perc 0.3305 1 0.5654 Con/Het * Perc 0.7482 1 0.387 Focal Species * Perc 0.0046 1 0.9461 Con/Het*Strid 5.3127 1 0.0212 Focal Species * Strid 1.0642 1 0.3023 Focal Species * Con/Het 0.0333 1 0.8552 Percussion 7.9821 1 0.0047 Stridulation 2.1058 1 0.1467 Conspecific/Heterospecific 0.1652 1 0.6844 Focal Species 14.4028 1 0.0001

1 0.9 0.8 0.7 0.6 0.5 ocreata 0.4 rovneri

Proportion moved Proportion 0.3 0.2 0.1 0 0 50 100 150 200 250 300 Time (seconds)

Figure 9. Latency to move separated by focal species. Schizocosa ocreata females move more quickly to signals compared to Schizocosa rovneri (X2 = 14.4028, df = 1, p = 0.0001).

1 0.9 0.8 0.7 0.6 0.5 0.4 Percussion- Yes

Proportion moved Proportion 0.3 0.2 Percussion- No 0.1 0 0 50 100 150 200 250 300 Time (seconds)

Figure 10. Latency to move to presence/absence of percussion signals. Pooled data of S. ocreata and S. rovneri female movement to presence/absence of percussion signals. Spiders moved more quickly to signals without percussion compared to signals with percussion. It is important to note that treatments that did not include percussion were stridulation only and white noise signals (X2 = 7.9821, df = 1, p = 0.0047 ).

1 0.9 0.8 0.7 Con-StridY 0.6 0.5 Con-StridN 0.4 Het-StridY 0.3 Het-StridN Proportion moved Proportion 0.2 0.1 0 0 50 100 150 200 250 300 Time (seconds)

Figure 11. Latency to move to conspecific/heterospecific stridulation signals. Pooled results for S. ocreata and S. rovneri female movement to presence/absence of stridulation in conspecific and heterospecific signals. Overall, both species were quicker to move to signals without stridulation. It is important to note that treatment groups without stridulation include percussion only signals and white noise (X2 = 5.3127, df = 1, p = 0.0212).

1 0.9 0.8 0.7 Con-WhiteNoise 0.6 Con-PercOnly 0.5 Con-StridOnly Con-Complete 0.4 Het-WhiteNoise 0.3 Het-PercOnly

Het-StridOnly Proportion moved Proportion 0.2 Het-Complete 0.1 0 0 50 100 150 200 250 300 Time (seconds)

Figure 12. Latency to move to all conspecific and heterospecific treatments. Pooled responses from both species of female latency to move to conspecific and heterospecific signals of all treatment groups. Females of both species moved more quickly to white noise signals over all treatment groups. When presented with complete signals of heterospecifics females were slower to move. And finally, when females were presented with stridulation only signals of heterospecifics, they moved quickly compared to other treatment groups (X2

= 5.0223, df = 1, p = 0.025).

Table 3. Parametric Survival Analysis with Likelihood ratio tests of female latency to receptivity (significance at α < 0.05 indicated in yellow).

Parametric Survival Analysis Chi-sq df Probability Full model 103.4339 15 <0.001 FocalSpecies*ConHet*Stridulation*Percussion 0.1636 1 0.6858 Con/Het * Strid * Perc 0.1513 1 0.6973 Focal Species * Strid * Perc 2.0857 1 0.1487 Focal Species * Con/Het * Perc 1.8731 1 0.1711 Focal Species * Con/Het * Strid 1.8858 1 0.1697 Strid * Perc 51.7222 1 <.0001 Con/Het * Perc 0.0312 1 0.8597 Focal Species * Perc 0.3491 1 0.5546 Con/Het*Strid 0.0051 1 0.9431 Focal Species * Strid 1.6141 1 0.2039 Focal Species * Con/Het 0.0387 1 0.8441 Percussion 36.9597 1 <.0001 Stridulation 45.2388 1 <.0001 Conspecific/Heterospecific 0.0653 1 0.7982 Focal Species 1.3969 1 0.2373

1 Stridulation-Yes 0.9 Stridulation-No 0.8 0.7 0.6 0.5 0.4

0.3 Proportion receptive Proportion 0.2 0.1 0 0 50 100 150 200 250 300 Time (seconds)

Figure 13. Latency to receptivity to presence/absence of stridulation. Pooled responses of S. ocreata and S. rovneri female receptivity to presence/absence of stridulation signals. Female spiders were quicker to show receptivity when a stridulation component is present (X2 =

45.2388, df = 1, p = 0.0001).

1 0.9 0.8 Percussion-Yes 0.7 Percussion-No 0.6 0.5 0.4

Proportion receptive Proportion 0.3 0.2 0.1 0 0 50 100 150 200 250 300 Time (seconds)

Figure 14. Latency to receptivity to presence/absence of percussion. Pooled receptivity responses of females to presence/absence of percussion signals. Females were quicker to show receptivity to signals that included percussion (X2 = 36.9597, df = 1, p = 0.0001).

1 0.9 White Noise 0.8 Percussion Only 0.7 Stridulation Only 0.6 0.5 Complete 0.4

0.3 Proportion receptive Proportion 0.2 0.1 0 0 50 100 150 200 250 300 Time (seconds)

Figure 15. Latency to receptivity of all treatments. Pooled responses of S. ocreata and S. rovneri female latency to receptivity to treatment groups. Females generally did not show receptivity to white noise signals compared to other treatment groups. Female latency to receptivity to percussion only, stridulation only, and complete signals were all very similar, but significantly different than latency to receptivity to white noise signals (X2 = 51.7222, df

= 1, p = 0.0001).

Discussion

In order to better understand the evolution of signaling complexity, I manipulated vibratory signals of the divergent ethospecies S. ocreata and S. rovneri and addressed three main questions: (1) Are females receptive to isolated individual vibratory components (stridulation and percussion) of conspecific and heterospecific male signals; (2) How do female responses to individual components compare to complete signals; and (3) Are these components species- specific, or interchangeable elements of vibratory courtship? In answer to the first question, female S. ocreata and S. rovneri were receptive to individual components of both conspecifics and heterospecifics. Females display receptivity to signals - whether they are simplified or complete signals - from both conspecific and heterospecific males but were more receptive to signals from conspecifics. For the second question, both complete signals and their individual components are sufficient to elicit receptivity from conspecifics, and females were just as receptive to individual components as they were to complete signals (i.e., there was no difference in female responses to complete signals compared to individual components). This suggests that individual components are redundant elements within the male vibratory signal and that when combined result in the same (equivalent) response. For the last question, I found that S. ocreata and S. rovneri females will show receptivity to either components of both species, supporting the idea that individual components may be interchangeable elements within their signals.

Data on latency to move revealed that female S. ocreata and S. rovneri, surprisingly, moved much faster in response to white noise signals rather than simplified or complete signals.

White noise is a novel signal for females; they do not experience it in the wild. However, this noise may appear threatening, which could potentially encourage them to move quickly.

However, it is more likely that females move because white noise does not include any valuable

38 information (i.e., it is un-patterned and broad-spectrum noise), but females “hear” this white noise signal and choose to move. However, S. ocreata females are quicker to move to stridulation-only signals from heterospecifics. Females are much slower to show receptivity (if any at all) to white noise signals as they do not contain any species-specific information.

However, latency to receptivity data of S. ocreata and S. rovneri show that females will display receptivity about 150 seconds (2.5 minutes) into the trial. This could be due to the simplified signals. It may take more time for females to gain an understanding of the male’s condition and quality, so females may take more time to assess them. However, female S. ocreata were much quicker to display receptivity to heterospecific complete signals compared to simplified signals.

This could be due to complete heterospecific signals (i.e., simultaneous stridulation and percussion) being novel.

Female S. ocreata and S. rovneri are receptive to individual components of male vibratory signals, as well as complete signals of conspecific and heterospecific males. However, results from the GLM of overall female receptivity displays reveal these responses to individual vibratory signaling components were less than female responses to complete unimodal and/or multimodal signals in other studies. This may be due to the single-component, unimodal nature of playback used here. In other studies (McClintock & Uetz 1996; Uetz 2000; Uetz et al. 2009;

Kozak & Uetz ms in review; Orr & Uetz M.S. Thesis), females were exposed to complete unimodal or complete multimodal signals. This is the first study to separate out individual components within a unimodal signal in Schizocosa spiders.

If females are equally receptive to individual components, this raises the question - why have multiple components at all? One explanation for this could be that vibratory signals are specifically adapted for their microhabitats (Elias et al. 2010). For example, S. ocreata is found

39 primarily on a complex leaf litter habitat. In contrast, S. rovneri is more commonly found on flood plains with muddy, flattened leaf litter (Scheffer et al. 1996; Uetz et al. 2013; Pickett M.S.

Thesis 2018). The primarily percussive signals of S. rovneri have much higher amplitude (more energy) compared to S. ocreata, which may compensate for living on floodplains with tightly packed litter/mud substrata (Scheffer et al. 1996). While vibratory signals may attenuate more quickly on that substrate, high energy percussion could ultimately reach a greater distance

(Scheffer et al. 1996) compared to the lower amplitude stridulation of S. ocreata in its native substrate. However, despite the low amplitude of stridulation signals created by S. ocreata, they have the potential of being able to travel relatively greater distances depending on leaf connectivity (Uetz et al. 2013; Pickett M.S. Thesis 2018). Consequently, the divergence in signal complexity of these two species results in fairly equivalent signal active space (Pickett, M.S.

Thesis 2018). Moreover, pulses of stridulation within the male signals of S. ocreata and S. rovneri may be transposable in a signal because they are produced by similar structures located within the pedipalps (stridulatory organ) (Rovner 1975; Stratton 2005). This is akin to using the same instrument to produce the same notes, but in a different song.

There is a potential for natural hybridization between S. ocreata and S. rovneri. However, this is generally rare in the wild (Stratton & Uetz 1986). Based on the results found in this study, females could show receptivity to males of the opposite species, based on vibratory components and signals alone. These receptivity scores, however, are much lower compared to Uetz et al.

(2009), which measured female receptivity to multimodal and unimodal male signals, and

Rutledge & Uetz (2009) which looked at female receptivity displays to multimodal conspecific and heterospecific male signals. Females displayed higher rates of receptivity in those studies, which used live spiders rather than playback. This may be a drawback of the current study

40 design, which used vibratory playback through piezoelectric disc benders rather than live spiders.

However, I wanted to focus specifically on individual components of signals and using vibratory playback gave more control over the design.

Studies suggest the possibility of selection on the courtship signals of these male spiders.

Male S. ocreata (as well as females) have cryptic or disruptive coloration within the leaf litter when viewed from above. However, from a lateral or spider eye level view, the coloration is contrasting, making it easier for females to spot males (Clark et al. 2015). This difference in color contrast/conspicuousness would likely result in selection on male courtship display. Visual signals (waving of the forelegs) of male S. ocreata attract mates as well as potential predators with acute vision such as birds and amphibians) (Clark et al. 2016; Rubi et al. 2019). Therefore, it is likely there is selection pressure on males from potential predation which result in a trade-off between their visual and vibratory signals. When visual displays attract unwanted attention, males may rely on vibratory signals, which travel through the leaf litter and may be detected by a female, but not by most predators.

This scenario supports a hypothesis suggested by phylogenetic analysis, i.e., that visual signals have been secondarily lost in S. rovneri (Stratton 2005; Hebets et al. 2013). These spiders live in riverine floodplains of the Midwest and central South, where litter is compressed and open, making it possible for visual signals to be seen not only by females, but by unintended receivers, including potential predators such as toads, birds, as well as other spiders.

Consequently, S. rovneri relies predominately on vibratory signals in courtship. The vibratory signals of S. rovneri are much higher in amplitude compared to S. ocreata, possibly because these signals must travel through densely packed floodplain substrates (as suggested above).

Rather than have two temporally distinctly separate components, the vibratory signal

41 components of S. rovneri are nearly simultaneous, with stridulation and percussion occurring together in a single “body bounce” display.

Future studies should examine the importance of pattern and temporal structure of S. ocreata and S. rovneri and their role in evolutionary divergence. A recent study was published detailing the importance of signal structure and temporal structure in species recognition of the stinkbugs Chinavia ubica and Chinavia impicticornis (Silviera et al. 2019). The authors found that changing the basic structure or temporal patterns of the female calling signals reduced the number of responses of males, supporting the signal recognition hypothesis. Their work with manipulation of the whole signaling pattern across two closely related species would be a logical future step with regard to this study.

This study provides only limited support for the species recognition hypothesis for signal complexity (Pfennig 1998). This hypothesis states that species recognition is more effective when different traits are used. If this were true, elimination of individual components should make it more difficult for female S. ocreata and S. rovneri to identify conspecific males, which is apparently not the case. Individually, components of wolf spider vibratory courtship elicit the same qualitative response from females (receptivity), and when used together there is an equivalent quantitative effect (receptivity score). This may give some support for the idea that pattern may play a more significant role in species recognition. Although individual components may not be species-specific, but pattern might be. This leads to the idea that individually, these components are not distinctly different enough to contain species-specific recognition information themselves, but since they appear redundant, there is stronger support for the redundant/back-up signals hypothesis (sensu Partan & Marler 2005; Hebets & Papaj 2005).

However, this is a reductionist approach (isolating components), and it may be possible that there

42 is some interaction of signaling components and environmental contexts in Schizocosa wolf spiders (Hebets et al. 2016). Future studies should examine the role of pattern and signal complexity in species recognition, as selection on males to communicate effectively through a complex environment (leaf litter) may have led to an increase in signal complexity to overcome that obstacle to effective communication.

Conclusions

Overall, this study provides some insights to the differences in signal complexity and function of vibratory signals in these two species. When presented with individual signal components in isolation (simplified signal), female S. ocreata and S. rovneri were equally receptive as they were to complete signals, suggesting support for the redundant signal hypothesis (Møller & Pomiankowski 1993) over the species recognition hypothesis (Pfennig

1998). The reductionist approach to this question provided greater control over individual aspects of the vibratory signal and allowed for a test of female receptivity to isolated components. Through this approach, it appears that individual components are redundant and sufficient to elicit receptivity from females. In future studies, a systems approach design (sensu

Hebets et al. 2016) might allow for better understanding of the interaction of possible missing factors.

Examining female receptivity responses to individual signaling components of S. ocreata and S. rovneri may also provide insight into the likely direction of signal evolution between these two species, based on phylogenetic interpretation and differences in habitat complexity. In these species, there has likely been selection on male S. ocreata to communicate more effectively through a complex environment (upland leaf litter), which may have led to an increase in

43 multimodal signal complexity to overcome barriers to signal reception. At the same time, selection from predation in the open environment of floodplains may have resulted in a shift to vibratory signals for S. rovneri, with increased amplitude percussion to increase signal range.

Regardless, the likely direction of signal evolution in this case was equally probable toward a gain in complexity for S. ocreata, and a loss of complexity in S. rovneri.

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