The Role of Male Vocal Signals During Male-Male Competition and Female Mate Choice in Greater Prairie-Chickens (Tympanuchus cupido)

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

By

Jennifer Ann Hale, B. S.

Graduate Program in Evolution, Ecology, and Organismal Biology

The Ohio State University

2013

Thesis Committee:

Dr. Jacqueline Augustine, Advisor

Dr. Douglas Nelson, Advisor

Dr. Andrew Roberts

Dr. William Mitchell Masters

Copyright by

Jennifer Ann Hale

2013

Abstract

In many taxa, vocal communication plays an integral role in aggression, territorial defense, and female choice. The acoustic structure of vocalizations is influenced by physical constraints on the vocalizer, suggesting a potential for discrimination among individuals. In the lek-mating Greater Prairie-Chicken (Tympanuchus cupido), male vocalizations are an integral part of the ritualized display. We investigated whether variation among vocal characteristics of individual male Greater Prairie-Chickens plays a role during female choice and male-male competition. Vocal characteristics varied among males but were fairly consistent for each male, suggesting that vocalizations might be used by prairie-chickens to identify individuals. Female choice was evaluated by comparing characteristics of vocalizations produced by reproductively successful and unsuccessful males, and successful males were found to vocalize at a relatively lower mean frequency. Playbacks of familiar and unfamiliar males were conducted on the lek to assess the role of vocalizations during male-male competition. Males responded to the prairie-chicken treatments by vocalizing at a faster rate and approaching the playback speaker, but they did not respond more strongly to the vocalizations of unfamiliar males than familiar males. Our results suggest that variation is present ii among the vocalizations of individual male Greater Prairie-Chickens and that this variation could be used by females during mate choice, but signal variation does not

appear to be used by males to discriminate among familiar individuals and strangers.

However, vocalization elicits an aggressive response in males that hear it, regardless of the individual that has produced it. Vocalization likely functions as a way of announcing that a territory is occupied and defended, but it may also serve as a way of advertising to male or female conspecifics or as a signal that is secondary to other forms of communication.

iii Acknowledgments

I would like to thank my advisor, Dr. Jackie Augustine, for her invaluable assistance, mentorship, and guidance during all stages of this project. I would also like to thank my co-advisor, Dr. Doug Nelson, for his great help and feedback along the way. My other committee members, Dr. Andy Roberts and Dr. Mitch Masters, contributed a great deal of expertise that strengthened the project. I am grateful to Konza Prairie Biological Station, Rannell’s Ranch Preserve, and the private landowners who allowed me to conduct research on their properties. Funding in the form of an OSU-Lima Research and Special Projects Grant, an NAOC Student Travel Award, and travel money from the department of Evolution, Ecology, and Organismal Biology at The Ohio State University made it possible for me to conduct fieldwork in Kansas and travel to scientific conferences. Finally, I would like to thank my parents, my boyfriend Marc, and many friends and family members for their unfailing kindness, patience, and good humor. I am so very fortunate to have had their support over the years and cannot thank them enough.

iv Vita

2010 ...... B.S. Biology, Arizona State University 2010-2013 ...... Graduate Teaching Assistant, Department of Evolution, Ecology, and Organismal Biology, The Ohio State University

Fields of Study

Major Field: Evolution, Ecology, and Organismal Biology

v Table of Contents

Abstract ...... ii Acknowledgments ...... iv Vita ...... v List of Tables ...... vii List of Figures ...... viii Introduction ...... 1 Methods ...... 5 Results ...... 11 Discussion ...... 17 Literature Cited ...... 23 Appendix A: Tables ...... 28 Appendix B: Figures ...... 36

vi List of Tables

Table 1: Results of principal component analysis using vocal characteristics ...... 28 Table 2: Results of a linear mixed model analysis of the relationship between vocal characteristics of 56 male Greater Prairie-Chickens and date, year, and female presence with male ID as a random effect ...... 29 Table 3: Results of discriminant function analyses including 5 vocalizations from each of 45 males ...... 30 Table 4: Results of principal component analysis using behavioral characteristics ...... 31 Table 5: Results of a linear mixed model analysis of the relationship between environmental factors and behavior for 74 males with male ID as a random effect ...... 32 Table 6: Coefficients of variation for vocal, behavioral, and morphological characteristics of male Greater Prairie-Chickens near Manhattan, Kansas in 2011 and 2012 ...... 33 Table 7: Summary statistics for differences in response measures before and during treatments in 10 playback trials, each including 4 treatments: owner, neighbor, stranger, and control ...... 34 Table 8: Results of principal component analysis using playback responses ...... 35

vii List of Figures

Figure 1: Sonogram of a Greater Prairie-Chicken call, with several structural elements identified ...... 37 Figure 2: Territories of 7 male Greater Prairie-Chickens at the Kreider lek in 2012 near Manhattan, Kansas ...... 38 Figure 3: Mean differences ± SE in the average boom rate (# booms/min) of male Greater Prairie-Chickens before and during playback treatments .. 39 Figure 4: Mean differences ± SE in the average faceoff rate (# faceoffs/min) of male Greater Prairie-Chickens before and during playback treatments . 40 Figure 5:,,,,,,,,,,,,,l Mean differences ± SE in the proportion of time spent in faceoffs by male Greater Prairie-Chickens before and during playback treatments ...... 41 Figure 6: Mean differences ± SE in the closest distance of approach to the playback speaker (m) by male Greater Prairie-Chickens before and during playback treatments ...... 42

viii Introduction

Animal vocalizations play an integral role in signaling aggression, territory defense, and male quality. The acoustic structure of vocalizations is influenced by physical constraints on the vocalizer (Bradbury and Vehrencamp 2011). Thus, physically different individuals may produce different vocalizations. If this is the case, vocalization might be a useful signal for individual recognition as well as allowing for both males and females to assess the fitness of an individual. Auditory signals can also be used to communicate information about spatial location. When an individual’s signals repeatedly originate from a particular location, they may help to define the signaler’s territory (Bee and Gerhardt 2001). Thus, male vocalizations may be a valuable way of establishing territory boundaries. Vocal signaling may also be a mechanism for males to defend their territories by warning other males away before it becomes necessary to escalate to a physically aggressive response. If their territory is encroached upon, males may engage in physical battles with intruders (Apollonio et al. 1992, Arita and Kaneshiro 1985), and those who can successfully maintain their territories may experience higher reproductive fitness (Ryder et al. 2008). Some bird species such as the Dusky Grouse (Dendragapus obscurus, formerly Blue Grouse), Ochre-bellied Flycatcher (Mionectes oleaginous), and Song Sparrow (Melospiza melodia) can recognize the vocalizations of individual neighbors and will respond more strongly to playbacks of vocalizations of a stranger than of a neighbor (Falls and McNicholl 1979, Westcott 1997, Burt et al. 2001). Additionally, when an individual signals from a location outside of its usual territory, it may provoke a higher than usual aggressive response from its neighbors, as in the Song Sparrow (Akçay et al. 2009, 2010). These results are all consistent with the “dear enemy” effect, where more aggression is directed towards strangers than to familiar neighbors (Fisher 1958). If 1 males are able to distinguish between neighbors and intruders, they may be better able to effectively allocate energy to territory defense (Ydenberg et al. 1988). Males must evaluate the relative threat of a stranger as compared to a neighbor when deciding how aggressively to respond (Stamps 1987, Temeles 1994). Vocalizations can also be directed towards females and may be one of the characteristics they use to assess male fitness, in addition to “extravagant” traits such as ornate tail feathers, more vigorous displays, and higher levels of aggression (Beehler and Foster 1988, Fiske et al. 1998). In this case, male vocalizations would influence female mate choice. Vocal characteristics that females assess may include the vocal display rate and spectral call properties such as dominant frequency and note lengths (Höglund and Alatalo 1995). Lek-mating systems are useful for studying the role of vocalizations in the context of female choice and male-male competition simultaneously among many different males and females. Lek-mating animals display a particular form of reproductive behavior in which females provide all parental care and select males for copulation only. Lek-mating males defend territories without food resources or nesting sites at communal breeding sites and attempt to copulate with as many females as possible (Höglund and Alatalo 1995). Females choose mates based on the perceived quality of the males, and males must compete with each other to maintain territories and win copulations. Because of this dynamic, lek mating systems are also characterized by reproductive skew, where a small proportion of males on the lek attain most of the copulations (Höglund and Alatalo 1995). Of lek-mating birds, the grouse are among the best-studied taxa, but only a few descriptions of the role of vocalizations in mate selection exist for any grouse species. In Greater Sage-Grouse (Centrocercus urophasianus), the structure of male acoustic displays (loud volume and long inter-pop intervals) has been associated with male mating success (Gibson 1996). Loud, low- frequency booming vocalizations have been found to be audible at greater distances than higher-frequency vocalizations in Lesser Prairie-Chickens (Tympanuchus pallidicinctus; Butler et al. 2010), and one can infer that greater audibility allows an

2 individual to signal to a higher number of potential competitors and mates. In the Greater Sage-Grouse, the most successful males were those that adjusted their vocal display rate according to female distance (Patricelli and Krakauer 2010). In the Greater Prairie-Chicken (T. cupido), a lek-mating grouse, behavioral traits such as display and aggression are the strongest predictors of male mating success, and vocalizations are an integral part of the ritualized display (Nooker and Sandercock 2008). In this paper, we explore several potential functions for the vocalizations of male Greater Prairie-Chickens. To establish the role of acoustic signaling in Greater Prairie- Chicken reproduction, several questions must be resolved. Firstly, do vocal characteristics vary among males? If so, then they can potentially be used during female choice and male-male competition. Secondly, are the vocal characteristics correlated with other potentially sexually-selected attributes such as morphology? If vocalizations are a signal of male fitness, they may be associated with other attractive morphological characteristics in the context of multi-modal signaling. We investigated this by examining relationships between the vocal characteristics of individuals and their morphological measurements. Thirdly, do vocalizations play a role in female choice? If females consider vocal signals when selecting mates, vocal characteristics should be correlated with male mating success. Further, they may change in the presence of females to reflect heightened display effort on the part of the males. We investigated this third question 1) by using a multinomial discrete choice model to determine whether females use male vocal characteristics when selecting a mate, and 2) by exploring whether the vocal characteristics of an individual male’s vocalizations are different in the presence versus absence of females. Lastly, are vocal signals used for recognizing individuals during male-male competition? If vocal signals contain information about the identity or location of a male, we expect variation in response to manipulation of these variables as predicted by the “dear enemy” effect. We performed playback experiments in which vocalizations of familiar and unfamiliar males were played in an adjacent territory. We observed whether vocal and behavioral responses of males varied when presented with four playback treatments: an adjacent

3 neighbor played from his own territory, a non-adjacent neighbor played from outside of his territory, a stranger from a neighboring lek, and a control (co-occurring grassland bird species).

4 Methods

OBSERVATIONAL DATA

Individual marking and morphological measurements Data collection took place at six locations on native, cattle-grazed grasslands near Manhattan, KS that were between 0.9 – 34.3 km apart from each other. The field research period coincided with the annual breeding season, which begins in early March, peaks around April 10, and then declines through late May (McNew et al. 2011). Females are typically present on the lek from late March to mid-April, and males display most actively in their presence (Nooker and Sandercock 2008). Displaying generally starts half an hour before sunrise and continues for about three hours. Males were captured using drop nets set up above the lek each morning prior to the arrival of the birds. Captured males were placed into separate burlap holding bags before being individually marked. Each male was given three colored leg bands in a unique combination and a numbered aluminum leg band. Each male’s tail was also marked with colored nontoxic permanent markers in a unique two-color combination. Morphological data were also collected (mass and tarsus, head, tail, comb, wing, and pinnae lengths).

Behavioral observations For each focal observation period, one male was watched for ten minutes, unless visual obstruction, a predation attempt, or other unpredictable event occurred. Observations shorter than five minutes were discarded. The few observations shorter or longer than 10 minutes were converted to a 10-minute standard by calculating the 5 number of behaviors per minute and multiplying by 10 minutes. We counted the number of boom vocalizations, flutter jump displays, fights, and number of males engaged during fighting. We also calculated time spent displaying or fighting.

Vocal characteristics The “boom” vocalization that males produce during display consists of two notes, with a lower-frequency or sometimes silent gap in between and a brief peak in frequency occurring during the first note (Figure 1). Using SIGNAL (Version 4, Engineering Design), we calculated the dominant frequencies of the first and second notes and the peak frequency, location of the peak (time elapsed between the start of the first note and occurrence of the peak/total duration of the first note), the relative amplitude of the two notes and peak, and length of the first and second notes.

Statistical analyses Principal component analyses were performed to assess which factors were primarily responsible for variation among males in vocal characteristics and behavior. Vocalizations were recorded throughout the season and in the presence and absence of females to assess consistency, and effects of year, date, and female presence on vocal characteristics and behavior were explored using linear mixed models in Minitab (Version 16, Minitab Inc.) with male ID as a random effect. We performed discriminant function analyses using JMP (Version 9.0.0, SAS Institute Inc.) to assess whether individual males tend to produce individually distinctive vocalizations. Correlations between vocalization and morphological measurements were examined using least squares models and mixed stepwise linear regression models. Correlations between vocal characteristics and mating success were also examined using multinomial discrete choice models in SAS (Version 9.3, SAS Institute Inc.). Coefficients of variation were calculated in JMP to assess the relative variability of vocal, behavioral and morphological characteristics among males. The raw data for these vocal and behavioral characteristics (not the PC scores) were used to calculate a

6 mean for each male, and then the per-male means were used to calculate the total mean and standard deviation of all males for that characteristic or measurement. Coefficients of variation (SD/mean) were calculated for only two behavioral measures (proportion of time spent displaying and proportion of time spent fighting) because the other behavioral measures had means of < 0.1, and coefficients of variation are suggested only for means that are not close to 0. Because the coefficients of variation are calculated using the mean as the denominator, when the mean is close to 0 the coefficient of variation approaches infinity and is not resilient to small changes in the mean (UCLA: Statistical Consulting Group 2013).

PLAYBACK EXPERIMENTS

Data collection To assess male responses to other males’ vocalizations, 10 sets of playbacks were conducted from April 5-30, 2012, at four leks. A total of 26 male prairie-chickens were observed. Leks were visited sequentially, with the original order of the visitation determined randomly. One four-treatment set of playbacks was conducted per day. Playbacks began at sunrise, a time when males are typically active on the lek and the light is bright enough to enable video recording. Each set of playbacks included three treatments of prairie-chicken ‘boom’ vocalizations and a control treatment. The control was a neutral stimulus, a common bird species local to the area (the contact call of an Upland Sandpiper, Bartramia longicauda; from the Borror Laboratory of Bioacoustics at The Ohio State University). The three prairie-chicken playback treatments represented males of varying familiarity to the focal males: a territory owner within his territory (in which the speaker was placed for a single day’s playback treatments and which bordered on the territories of the focal males), a neighbor with a nonadjacent territory to the speaker, and a stranger from a nearby lek (Figure 2). The four playback treatments were played in a random order, and the order of treatments was not repeated on a lek. Vocal tracks were controlled using an iPod touch (Version 6.0, Apple

7 Inc., Cupertino, California, USA) from within a blind where the observer was seated, amplified using an amplifier (PL-P model, Nagra, Switzerland), and played from a speaker (PH3-S model, Audix, Wilsonville, Oregon, USA) on the territory of a single male for each set of playbacks. Speaker placement was varied between subsequent days so that data could be collected from different focal males each time. The speaker was placed flat on the ground, facing skyward, wrapped in a plastic bag for protection from the dew. To ensure that sound was not being distorted when played back, we examined spectrograms of vocalizations directly recorded from Greater Prairie-Chickens and the same vocalizations recorded when played from the speaker wrapped in its bag. Additionally, the vocalizations emitted from the speaker during playbacks sounded indistinguishable to the observer from the vocalizations produced by males on the lek. The speaker was not placed facing the focal males because multiple males were observed each day and it was not possible to have multiple speakers facing each male and yet maintain constant volume. Tracks were played at a volume of approximately 106 dB at 10 cm, the average volume of the “boom” call of the Lesser Prairie-Chicken, a closely related species for which sound intensity data have been analyzed and made available (Butler et al. 2010). The playback volume was tested using dB Meter Pro (Performance Audio, Salt Lake City, Utah, USA) on an iPad (Version 5.0.1, Apple Inc., Cupertino, California, USA). Each prairie-chicken playback treatment included 18 “boom” vocalizations collected from a single individual in 2012 (or 18 contact calls in the case of the control treatment), and the order of these vocalizations within each treatment was also randomized to minimize any potential bias. One call was played every 10 seconds, the typical length of time between successive calls of a displaying male prairie-chicken (unpub. data), for a period of approximately 3 minutes. During each playback trial, the behavior of up to 3 focal adjacent neighbors was observed. Multiple males were observed to improve sample size, and each focal male was observed only during a single 4-trial set of playbacks to avoid pseudoreplication. All playback trials were filmed with a Sony HDR-CX160 video camera with 30x optical zoom from a blind approximately 4.5 m

8 from the edge of the lek. In addition, male identity, the activities of the focal males, and location of the males relative to labeled stakes covering the area of the lek in a grid with 6.10 m2 squares were described aloud during the recording as movement occurred. We allowed 10 minutes between each trial to allow the males’ response threshold to return to normal after each stimulus. Males typically resumed their normal activities very quickly after a playback trial. If a playback trial was interrupted by females or predators, the recording was paused and restarted 10 minutes after the distraction ended.

Statistical analyses Male response to the playbacks was quantified using four response measures: boom rate (number of booms per minute), faceoff rate (number of stereotypic aggressive faceoff interactions engaged in per minute), proportion of time spent in faceoffs, and closest distance to speaker (m). All statistical analyses were done using JMP. The consistency of male response was evaluated using a repeated measures ANOVA to check for any effect of a trial’s order in the sequence for a set of playbacks or for any effect of the immediately preceding treatment. Possible date-dependent effects were investigated using a linear mixed model with male ID as a random effect. We used a 2-tailed t-test to investigate whether differences occurred in the averaged response measures of the 26 males between the approximately 180-second pre-playback period and the approximately 210-second during-playback period for the three treatments in which prairie-chicken vocalizations were played, and a separate t-test for the control treatment. The difference in response measures before and during the control treatment was evaluated separately to determine whether a heterospecific vocalization would also elicit a response. A significant change in activity level between the pre- playback and during-playback periods indicates a response of males to the playback. Significant responses to the grouped prairie-chicken treatments were further investigated using 2-tailed t-tests on the full complement of treatments to determine which treatments elicited a response. We used a principal component analysis on the response data to assess which response measures were the primary sources of variation

9 among males. Male response measures were also evaluated among the three prairie-chicken playback treatments to determine whether response to the vocalizations of other males was affected by the familiarity of the male or by whether the caller was the owner of the territory in which the speaker was located. For each response measure, a repeated measures ANOVA was performed to compare the change in response between pre- playback and during-playback among treatments. Another repeated measures ANOVA was used to determine whether averaged response measures of each male for the three prairie-chicken treatments differed between males who were observed to achieve one or more copulations during the field season and those who were not observed to do so. Repeated measures ANOVAs were also used to assess whether there was any effect of the order in which a playback treatment was presented during the set of playbacks, or whether any of the four treatments affected the responses to the treatment immediately following it. An additional repeated measures ANOVA was used to compare male activity during the four pre-playback periods to determine whether the “baseline” activity level was consistent throughout the morning.

10 Results

OBSERVATIONAL DATA

Variation in Vocalizations Between March 16-April 30, 2011 and March 22-April 18, 2012, 622 vocalizations were recorded from 56 males. A PCA was performed to determine which vocal components were primarily responsible for variation among the males sampled. It was found that three vocal principal components (VPC1, VPC2, and VPC3) explained variation in the first note, overall frequency, and second note, respectively (Table 1). The three principal components accounted for 73.5% of the variation in vocalization measurements. Various aspects of the vocalizations were influenced by female presence, date, and year (Table 2). The first principal component, VPC1 (1stnote), was not affected by year but decreased with date and increased with female presence. The second principal component, VPC2 (Frequency), was not affected by year but decreased with date and increased with female presence. The third principal component, VPC3 (2nd note), was not affected by female presence but was affected by year and decreased with date. The coefficient of determination r2 was very small for most correlations (< 0.05), with the exception of VPC1 and date (r2 = 0.19). Year and female presence explained little of the variation in vocalizations (i.e. annual and female proximity-related variation was low), and because multiple vocalizations were recorded for each male throughout the season, we calculated an average PC score for each male. We performed discriminant function analyses using 5 vocalizations from each of 45 males (Table 3). Only males with 5 or more recorded vocalizations were included in the analysis. A discriminant function analysis using the original set of 7 vocal 11 characteristics correctly classified an average of 87.4% of vocalizations produced by an average of 7.5 males per lek. A discriminant function analysis using the three vocal PC scores summarizing 73.5% of the variation in vocal structure correctly classified an average of 65.6% of the vocalizations per lek. Both analyses resulted in a greater likelihood of assigning vocalizations to the correct individuals than would be probable due to chance, with an average chance expectation of 13.3% with 7.5 males.

Variation in Behavior A total of 327 separate focal observations were obtained from 74 males between March 17-April 25, 2011 and March 20-April 30, 2012. A PCA using the behavioral data revealed that two behavioral principal components (BPC1 and BPC2) were associated with aggression and display behavior, respectively (Table 4). These two principal components accounted for 75.0% of the variation in male behavior. The first principal component, BPC1 (Display), was not affected by observer but was affected by year, increased with female presence, and decreased with increasing date and time since sunrise (Table 5). The second principal component, BPC2 (Aggression), was not affected by date or year but was affected by observer, increased with female presence, and decreased with increasing time since sunrise. Because observers were rotated among leks, and observations occurred throughout the morning and season, there was no systematic bias when conducting male observations. Additionally, the coefficient of determination r2 was low for observer, year, date and time since sunrise (< 0.05). Therefore, we did not correct for observer, year, date, or time since sunrise. Because significant differences were found when females were present, behavioral PC scores were separated by female presence and averaged for each male to obtain four measurements of behavior (BPC1 Aggression with females, BPC1 Aggression without females, BPC2 Display with females, and BPC2 Display without females).

Coefficients of Variation

12 We calculated the coefficients of variation (CV) for vocal characteristics, behavioral characteristics in the presence and absence of females, and morphological measurements to determine which traits vary among males and might most easily be used to differentiate individuals (Table 6). Many of these male attributes showed a moderate or high degree of variation. The vocal characteristics with the highest degree of variation among males were the frequency difference between the first and second notes (CV = 96.6%) and the length of the gap between the notes (CV = 40.3%). When females were present, there was much higher variation in the amount of time males spent fighting than was spent displaying (CV = 80.9% and 21.0%, respectively). However, when females were absent, there was higher variation in the amount of time males spent displaying than in time spent fighting (69.5% and 60.6%, respectively). Of the morphological measurements, the greatest amount of variation among males was found in comb area (CV = 19.1%).

Vocalization and Body Size There was no association between vocal principal components and measures of body size (tarsus length, wing length, and comb area), with the exception of an increase st 2 in VPC1 (1 note) with increased wing length (F1, 28 = 6.13, p = 0.02, r = 0.18).

Otherwise, VPC1 (tarsus: F1, 28 = 0.41, p = 0.53; comb: F1, 28 = 0.89, p = 0.35; overall 2 model F3, 28 = 2.48, p = 0.08, r = 0.21), VPC2 (tarsus: F1, 28 = 0.07, p = 0.79; wing: F1, 28 < 2 0.01, p = 0.97, comb: F1, 28 < 0.01, p = 0.96; overall model F3, 28 = 0.03, p = 0.99, r < 0.01), and VPC3 (tarsus: F1, 28 = 0.24, p = 0.63; wing: F1, 28 = 0.10, p = 0.75, comb: F1, 28 = 0.17, p 2 = 0.68; overall model F3, 28 = 0.14, p = 0.94, r = 0.01) were not related to our measures of body size. In a mixed stepwise model with p = 0.05 to enter and 0.1 to leave, wing length was again associated with VPC1 (F1, 30 = 8.67, p < 0.01), but no other associations were found between vocal PC scores and morphology.

Factors affecting Male Mating Success Twenty-eight copulations by 13 out of 74 males were observed (i.e. 17.6% of

13 males were observed to copulate). When vocal PC scores were compared when females were present and absent, VPC1 (F1, 55 = 9.47, p < 0.01) and VPC2 (F1, 55 = 4.98, p = 0.03) were found to increase in the presence of females, but VPC3 (F1, 55 = 3.64, p = 0.06) was unchanged by the presence of females. VPC2 (Frequency) was an attribute related to female choice in a multinomial discrete choice model (t = -2.93, df = 1, p < 0.01), but VPC1 (t = 0.11, df = 1, p = 0.91) and VPC3 (t = -1.43, df = 1, p = 0.15) were unrelated to female choice. Males that copulated had a lower value for VPC2 (successful: -0.37, unsuccessful: -0.13), which corresponds with a lower frequency first note of their vocalization relative to the frequencies of the second note and frequency peak.

Male response to playback treatments In response to prairie-chicken playback treatments, males increased their vocalization rate and approached the speaker (Table 7). Boom rate increased during prairie-chicken treatments (Figure 3; t = 3.87, df = 25, p < 0.01), but did not change during the control (t = -0.50, df = 25, p = 0.62). Faceoff rate did not change during the prairie-chicken treatments (Figure 4; t = 0.91, df = 25, p = 0.37) but decreased during the control treatment (t = -3.22, df = 25, p < 0.01). The proportion of time spent in faceoffs was also unchanged for the prairie-chicken treatments (Figure 5; t = -0.41, df = 25, p = 0.69) but decreased during the control treatment (t = -3.75, df = 25, p < 0.01). Males approached significantly closer to the speaker during the prairie-chicken treatments (Figure 6; t = -4.69, df = 25, p < 0.01) than during the control treatment (t = 0.11, df = 25, p = 0.92). A PCA was performed using the four response measures for the playback experiments (Table 8). The analysis revealed that playback PPC1 was associated with distance measures, PPC2 with aggressive measures, and PPC3 with display measures. These three principal component scores account for 81.3% of the variation in changes of male behavior.

Consistency of male playback response

14 Male response was not affected by date. Thus, our results do not suggest a seasonal effect on PPC1 (F1, 25 = 0.35, p = 0.55), PPC2 (F1, 25 = 0.49, p = 0.49), or PPC3 (F1,

25 = 0.04, p = 0.85) in response to hearing the vocalizations of another male. We did not detect bias caused by whether a given playback treatment came first, second, third, or fourth in the sequence of treatments (i.e. no bias caused by the order in which a treatment occurred). We found no significant effect of order on PPC1 (F3, 74 =

0.54, p = 0.66), PPC2 (F3, 74 = 1.04, p = 0.38), or PPC3 (F3, 74 = 0.42, p = 0.74). Additionally, male activity during the pre-playback periods was compared by order to determine whether the “baseline” activity level of males differed throughout the morning. We found no significant effect of order on PPC1 (F3, 74 = 0.13, p = 0.94), PPC2 (F3, 74 = 1.39, p

= 0.25), or PPC3 (F3, 74 = 0.52, p = 0.67) during the pre-playback periods.

No effect of the immediately preceding trial was found on PPC1 (F4, 73 = 1.97, p =

0.11), PPC2 (F4, 73= 0.62, p = 0.65), or PPC3 (F4, 73 = 1.22, p = 0.31). Thus, no playback trial was found to affect responses to the subsequent trial.

Comparison of male response among playback treatments For the three prairie-chicken vocalization treatments, we compared the difference in pre-playback and during-playback response measures of males among treatments to determine whether any treatment provoked a relatively heightened response. We found no significant differences in PPC1 (F2, 75 = 0.39, p = 0.68), PPC2 (F2,

75 = 0.71, p = 0.50), or PPC3 (F2, 75 = 0.74, p = 0.48).

Male playback response and mating success Some differences in response were found between males who were observed to achieve at least one copulation during the 2012 field season and males who were not observed mating. Increases in PPC2 (F1, 24 = 5.54, p = 0.03), aggressive behavior, during prairie-chicken playbacks were greater for reproductively successful males when compared to males who were not observed copulating. However, differences between

15 during-playback and pre-playback times in PPC1 (F1, 24 = 1.03, p = 0.32) and PPC3 (F1, 24 = 3.06, p = 0.09) were similar between successful and unsuccessful males.

16 Discussion

In this paper, we demonstrate that Greater Prairie-Chicken vocalizations vary among individuals and may relate to wing length, a measure of body size. Because of this variation, male vocalizations seem to play a role during female mate choice, with more successful males having lower frequency vocalizations. Males also respond by increasing their rate of vocalizations to male playbacks, regardless of the individual identity of the playback male.

Discriminant function analyses suggest that vocal characteristics may allow vocalizations to be assigned to individual males with a likelihood of correct classification that is greater than the probability due to chance. In a variety of bird species in which individual recognition has been observed, such as the Bald Eagle (Haliaeetus leucocephalus), European Nightjar (Caprimulgus europaeus), Great Bittern (Botaurus stellaris), Alder Flycatcher (Empidonax alnorum), and Spotted Antbird (Hylophylax naevioides), correct assignment percentages of 80% or more are commonly reported (Eakle et al 1989, Rebbeck et al 2001, Puglisi and Adamo 2004, Lovell and Lein 2004, Bard et al 2002). The vocal characteristics we measured allowed vocalizations to be assigned to the correct Greater Prairie-Chicken male an average of 87.4% of the time. This suggests that Greater Prairie-Chicken vocalizations may contain a level of individually distinctive information that is comparable to vocalizations from species where individual recognition has been observed.

We found only one association between male vocalizations and morphological traits. Vocal VPC1 (length of first note) increased with increasing wing length, suggesting that this vocal characteristic may be partially dependent on body size. Body size has been found to affect the production of vocalizations in a variety of bird species; in 17 particular, larger body size has been associated with lower frequency vocalizations (Barbraud et al. 2000, Bertelli and Tubaro 2002, Soma et al. 2008) and longer notes

(Badyaev and Leaf 1997).

In several lek-mating bird species, vocal characteristics have been shown to correlate with mating success (Gibson 1996, Höglund and Lundberg 1987). In the Greater Sage-Grouse, low-frequency vocalizations in particular have been associated with reproductive success for males (Gibson and Bradbury 1985). It has been suggested that low-frequency vocalizations may be a way that grassland birds can reduce excess attenuation of their vocal signals in this environment, potentially increasing the number of females who receive the signal and visit a male’s territory (Morton 1975). The ability to produce low-frequency vocalizations is also constrained by morphology, and may thus be associated with other aspects of male quality (Bradbury and Vehrencamp 1998). These factors may explain why male Greater Prairie-Chickens with relatively lower- frequency vocalizations experience greater mating success.

Male prairie-chickens responded more to playbacks of male prairie-chicken vocalizations than to control playbacks of a co-occurring species. Vocalizations may allow males to gather information about the locations of other males on the lek, because they were found to approach the playback speaker more closely during playbacks of prairie-chickens than during the pre-playback period or control treatment. Males known to have achieved at least one copulation showed a greater increase in aggressive response measures during playbacks of other males than individuals not observed copulating. However, we found no evidence that a greater level of response to prairie-chicken vocalizations occurs when the individual played is a stranger or a neighbor out of his own territory than when the owner of the territory is played. Unexpectedly, we found a decrease in aggressive activity when the control treatment was played; this may be because the contact call of the Upland Sandpiper is relatively high-pitched with a narrow bandwidth, characteristics shared by alarm calls in many

18 bird species (Marler 1955). It may be that the sandpiper calls elicited vigilance behavior from the prairie-chickens, temporarily interrupting their fighting.

One question that arises in light of our results is why we failed to find evidence of a difference in aggressive response depending on the identity of the caller during playbacks, despite prior evidence that vocalizations may be distinctive enough to allow some level of discrimination among individual males. The most likely explanation is that Greater Prairie-Chickens are not a system for which the “dear enemy” phenomenon would be adaptive. Temeles (1994) provided a review concerning the dear enemy phenomenon, and found that the phenomenon tended to be associated with species where a male had more to lose from a stranger (i.e. both territory and mates) than from a neighbor (i.e. mates alone, since the neighbor has already established his territory). Species that do not show the dear enemy phenomenon react to neighbors just as intensely as they do to strangers because both neighbors and strangers represent the same threat: loss of both mates and territory (Temeles 1994). Greater Prairie-Chickens risk losing both territory and mating opportunities to neighbors and strangers alike, and thus it may not be adaptive for them to respond with less aggression towards an intruding neighbor than to a stranger. Anecdotally, we often observed physical combat occurring between the same sets of neighboring males on a daily basis throughout the breeding season. Another possible reason why species may not show the dear enemy phenomenon is that inter-individual distances are small. In the same review of the dear enemy phenomenon, it was found that colonial seabird species that do not show the phenomenon have a shorter mean nearest nest distance than species that do show the phenomenon (Temeles 1994). The territories defended by male Greater Prairie-Chickens are very close to each other, and even a small encroachment by a neighbor may result in a significant territory loss for the owner.

In numerous lek-mating bird species such as the Black Grouse (Tetrao tetrix) and the White-bearded Manakin (Manacus manacus), male aggressive behavior is an important predictor of mating success (Alatalo et al. 1996, Höglund et al. 1997, Shorey 19 2002). The Greater Prairie-Chicken appears to be no exception; socially dominant males achieve a significant majority of copulations on the lek, and aggressive behavior is a strong predictor of male mating success (Ballard and Robel 1974, Nooker and Sandercock 2008). In our experiments, reproductively successful males showed a greater increase in playback PPC2 (aggression) during prairie-chicken playbacks than did reproductively unsuccessful males. If male response to another male’s vocalizations plays a role in male-male dominance interactions, males who take part more actively in these interactions may be more attractive to females.

Because males tended to approach the speaker during prairie-chicken playbacks, it is likely that they may gather information about the location of a signaler upon hearing a vocalization. Vocal signaling may be an important way of establishing and maintaining territory boundaries for territorial bird species. If there is a cost associating with producing such signals, and the costs of signaling or benefits of winning aggressive interactions vary among males, then vocal signaling could be a reliable way of signaling aggressive intent (Enquist 1985). In such varied species as the Broad-tailed Hummingbird (Selasphorus platycercus), Red-winged Blackbird (Agelaius phoeniceus), and Ochre-bellied Flycatcher, experimentally muted males’ territories are intruded upon at far-greater rates than vocally intact males, and many experience the loss of their territories upon being unable to sing (Miller and Inouye 1983, Peek 1972, Smith 1979, Westcott 1992). In a speaker replacement study of Song Sparrows where territory owners were removed and some individuals’ songs were played from their territories, encroachments occurred only on the silent territories (Nowicki et al. 1998). Male Greater Prairie-Chickens may use vocalization in a similar way to warn other males that a particular territory is occupied and being defended.

Vocal signals may also play a role in attracting females to visit a lek. In sage- grouse, playbacks of male vocal displays attract females to the speaker, and some females recall where the speaker was located and return to the same spot in subsequent visits (Gibson 1989). Females in a variety of lek-mating species such as 20 Black Grouse, Blue-crowned Manakins (Lepidothrix coronata), and Ruffs (Philomachus pugnax) preferentially visit larger leks, where they may select from a greater number of males. Additionally, males in these species may benefit by having an increased number of opportunities for copulation due to the increased number of visitations by females (Alatalo 1992, Durães et al. 2009, Lank and Smith 1992). Previous experiments in Greater Prairie-Chickens have shown that playbacks of male vocalizations attract females to visit a lek (Silvy and Robel 1967) and that aggregations of 11-15 males experience higher rates of female visitation per male than do smaller aggregations (Hamerstrom and Hamerstrom 1960). Thus, vocal signaling may not only be a way to broadcast information to females about the location of a male, but may also entice females to visit by providing valuable information about the number of males present on the lek.

It may also be that Greater Prairie-Chicken vocalization is best studied in the context of visual or other signals rather than in isolation. Complex signals are ones in which more than one signal is produced at a time, often across multiple sensory modalities (Hebets and Papaj 2005). The loss of a signal in one sensory modality may reduce the effectiveness of the entire complex signal (Elias et al. 2005). If vocalization is part of a complex signal, then it is possible that our study was unable to elicit normal responses from males due to a lack of other cues. Alternately, vocal signals may be of secondary importance when visual signals are available. Given these possibilities, our results should be interpreted with the understanding that the responses we were able to elicit might have been heightened or otherwise modulated with the inclusion of other signal modalities (such as a visual signal) in our playback trials. Future research should explore the alternate potential functions of vocalization described here. To assess whether male aggressive response to other males’ vocalizations is intensified by visual cues, our playback methodology could be adapted to include a robotic model of a prairie-chicken that would enter the owner’s territory during some playbacks, so that responses to the playbacks could be compared with and

21 without a visible male intruder. However, one limitation of this type of experiment would be a potential confounding effect of using a model bird that is not the male from which vocalizations were recorded; it is possible that presenting vocal signals from one individual and visual signals from another may hinder individual recognition, as opposed to presenting only vocal signals from each male as in our experiment. Because Greater Prairie-Chickens are a vulnerable species (BirdLife International 2010), care should be taken to choose experimental methodologies that will limit any harmful impacts on their reproductive success. Temporary muting or speaker replacement experiments may be appropriate ways to assess the potential role of vocalization in establishing territory boundaries in this species. In muting experiments, some territory owners would be temporarily silenced from calling; similarly, in speaker replacement experiments, territory owners would be removed from the lek and some would be replaced by a speaker playing the territory owner’s vocalizations. If vocalization plays a role in warning intruders away from a male’s territory, we would expect to see reduced encroachment on the territories of owners whose vocalizations were being emitted from the speakers or by the males themselves in comparison with the silent owners’ territories. Our predominant findings were that vocalizations of Greater Prairie-Chickens vary among males, and may be used in the context of both female mate choice and male-male competition. Females tend to choose males with lower frequency vocalizations. In the context of male-male competition and territory defense, male Greater Prairie-Chickens respond to the vocalizations of other males by vocalizing at a higher rate and approaching the source of the sound. We found no indication that males responded with more aggression when presented with vocalizations of strangers or of neighbors outside their normal territory boundaries than with territory owners within their usual territories. Thus, we have found no evidence that the dear enemy phenomenon operates in this species. Taken together, our findings suggest that vocalization likely serves a purpose in female mate choice and male-male interactions in

22 Greater Prairie-Chickens, and future work should explore its potential use in attracting females to a lek and in establishing territory boundaries.

23 Literature Cited

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26 McNew, L. B., Gregory, A. J., Wisely, S. M., and Sandercock, B. K. "Reproductive Biology of a Southern Population of Greater Prairie-Chickens." Studies in Avian Biology 39 (2011): 209-221. Miller, S. J. and Inouye, D. W. “Roles of the wing whistle in the territorial behavior of male Broad-tailed Hummingbirds (Selasphorus platycercus).” Animal Behaviour 31 (1983): 689-700. Morton, E. S. “Ecological sources of selection on avian sounds.” The American Naturalist 109 (1975): 17-34. Nooker, J. K., and Sandercock, B.K. "Phenotypic correlates and survival consequences of male mating success in lek-mating Greater Prairie-Chickens (Tympanuchus cupido)." Behavioral Ecology and Sociobiology 62 (2008): 1377-1388. Nowicki, S., Searcy, W. A., and Hughes, M. “The territory defense function of song in Song Sparrows: A test with the speaker occupation design.” Behaviour 135 (1998): 615-628. Patricelli, G. L., and Krakauer, A. H. "Tactical allocation of effort among multiple signals in Sage Grouse: An experiment with a robotic female." Behavioral Ecology 21 (2010): 97-106. Peek, F. W. “An experimental study of the territorial function of vocal and visual display in the male Red-winged Blackbird (Agelaius phoeniceus).” Animal Behaviour 20 (1972): 112-118. Puglisi, L. and Adamo, C. “Discrimination of individual voices in male Great Bitterns (Botaurus stellaris) in Italy.” The Auk 121 (2004): 541-547. Rebbeck, M., Corrick, R., Eaglestone, B., and Stainton, C. “Recognition of individual European Nightjars Caprimulgus europaeus from their song.” Ibis 143 (2001): 468- 475. Ryder, T. B., McDonald, D. B., Blake, J. G., Parker, P. G., and Loiselle, B. A. “Social networks in the lek-mating Wire-tailed Manakin (Pipra filicauda).” Proceedings of the Royal Society of Biological Sciences 275 (2008): 1367-1374. Shorey, L. “Mating success on White-bearded Manakin (Manacus manacus) leks: Male

27 characteristics and relatedness.” Behavioral Ecology and Sociobiology 52 (2002): 451-457. Silvy, N. J. and Robel, R. J. “Recordings used to help trap booming Greater Prairie- Chickens.” The Journal of Wildlife Management 31 (1960): 370-373. Smith, D. G. “Male singing ability and territory integrity in Red-winged Blackbirds (Ageliaus phoeniceus).” Behaviour 68 (1979): 193-206. Soma, M., Hasegawa, T., and Okanoya, K. “Genetic and developmental effects, and morphological influences on the acoustic structure of individual distance calls in female Bengalese Finches Lonchura striata var. domestica.” Journal of Avian Biology 39 (2008): 101-107. Stamps, J. A. “The effect of familiarity with a neighborhood on territory acquisition.” Behavioral Ecology and Sociobiology 21 (1987): 273-277. Temeles, E. J. “The role of neighbors in territorial systems: When are they ‘dear enemies’?” 47 (1994): 339-350. UCLA: Statistical Consulting Group. “FAQ: What is the coefficient of variation?”. Downloaded from http://www.ats.ucla.edu on 3/26/2013. Westcott, D. “Inter- and intra-sexual selection: The role of song in a lek mating system.” Animal Behavior 44 (1992): 695-703. Westcott, D. A. “Neighbours, strangers and male-male aggression as a determinant of lek size.” Behavioral Ecology and Sociobiology 40 (1997): 235-242. Ydenberg, R. C., Giraldeau, L. A., and Falls, J. B. “Neighbours, strangers, and the asymmetric war of attrition.” Animal Behaviour 36 (1988): 343-347.

28 Appendix A: Tables

Table 1: Results of principal component analysis using vocal characteristics. Data include 622 vocalizations from 56 male Greater Prairie-Chickens recorded in 2011 and 2012 near Manhattan, Kansas. Principal component loadings > 0.5 shown in bold.

Vocal Characteristics VPC1 VPC2 VPC3

Length of 1st note 0.54 0.16 -0.29

Length of 2nd note -0.06 0.37 0.77

Length of gap -0.31 0.06 0.15

Location of peak 0.56 0.13 -0.07

Frequency difference of note 2 – note 1 -0.28 0.52 -0.39

Amplitude difference of note 2 – note 1 0.41 0.39 0.29

Frequency difference of peak – note 1 -0.21 0.63 -0.24

% Variation Explained 35.9 21.7 15.9

29 Table 2: Results of a linear mixed model analysis of the relationship between vocal characteristics of 56 male Greater Prairie-Chickens and date, year, and female presence with male ID as a random effect.

VPC1 (1st Note) VPC2 (Frequency) VPC3 (2nd Note)

Date F1, 55 = 19.96 F1, 55 = 17.21 F1, 55 = 7.47 p< 0.01, r2 = 0.19 p< 0.01, r2 = 0.04 p = 0.01, r2 = 0.01

Year F1, 55 =1.22 F1, 55 = 0.44 F1, 55 = 4.40 p = 0.27, r2 = 0.06 p = 0.51, r2 = 0.02 p = 0.04, r2< 0.01

Female F1, 55 = 9.47 F1, 55 =4.98 F1, 55 = 3.64 Presence p< 0.01, r2< 0.01 p = 0.03, r2< 0.01 p = 0.06, r2 = 0.01

30 Table 3: Results of discriminant function analyses including 5 vocalizations from each of 45 males. For each lek, one analysis was conducted using vocal PC scores, and another was conducted using the raw data that had been used to calculate the PC scores.

Lek Number of Males % Correctly % Correctly Classified Classified Vocal PC Scores Raw Vocal Data Hess 14 46.5 73.2 Konza 7 61.8 79.4 Kreider 9 58.7 91.3 Olson 4 80.0 100.0 Poole 7 71.4 85.7 Rannell 4 75.0 95.0

31 Table 4: Results of principal component analysis using behavioral characteristics. Data were collected during 327 focal observations of 74 male Greater Prairie-Chickens in 2011 and 2012 near Manhattan, Kansas. Principal component loadings ≥ 0.5 shown in bold.

Behavioral Characteristics BPC1 BPC2

Proportion of time spent displaying 0.53 0.25

Proportion of time spent fighting -0.51 0.08

# of face offs -0.22 0.65

# of boom vocalizations 0.49 0.32

# of flutter jumps 0.33 0.13

# of males engaged in conflicts -0.24 0.63

% Variation Explained 46.2 28.8

32 Table 5: Results of a linear mixed model analysis of the relationship between environmental factors and behavior for 74 males with male ID as a random effect.

BPC1 (Display) BPC2 (Aggression)

Date F = 5.73 F = 4.03 1, 73 1, 73 p = 0.02, r2< 0.01 p = 0.05, r2< 0.01

Year F1, 73 =5.89 F1, 73 = 3.44

p = 0.02, r2< 0.01 p = 0.07, r2< 0.01

Female F1, 73 = 199.61 F1, 73 = 69.53

Presence p< 0.01, r2 = 0.36 p< 0.01, r2 = 0.18

Time Since F1, 73 = 10.17 F1, 73 = 4.63 Sunrise p< 0.01, r2 = 0.06 p = 0.03, r2 = 0.03

Observer F1, 73 = 1.35 F1, 73 = 3.20 p = 0.26, r2< 0.01 p = 0.04, r2 = 0.01

33 Table 6: Coefficients of variation for vocal, behavioral, and morphological characteristics of male Greater Prairie-Chickens near Manhattan, Kansas in 2011 and 2012.

Vocal CV% Behavioral CV% CV% CV% No Morphological CV% Characteristics Characteristics Females Females Measures (n=56 males) (n=74 males) (n=42 males) Length 1st Note 9.8 Display/Total 33.6 21.0 60.6 Mass 4.7 Length 2nd Note 12.8 Fighting/Total 49.4 80.9 69.5 Tarsus Length 9.0 Length Gap 40.3 Wing Length 1.7 Location of Peak 6.9 Comb Area 19.1 Freq. Diff. Notes 96.6 Amp. Diff. Notes 19.5 Freq. Diff. Peak 9.8 and 1st Note

34 Table 7: Summary statistics for differences in response measures before and during treatments in 10 playback trials, each including 4 treatments: owner, neighbor, stranger, and control. Data were collected from 26 male prairie-chickens during March and April 2012 near Manhattan, Kansas. The t-statistics indicate whether there was a significant change in response between the pre-playback and during-playback periods. P-values < 0.05 shown in bold.

n = 26 Owner Neighbor Stranger Control

Boom Rate t = 1.84, t = 2.37, t = 2.41, t = 0.42, (#/min) p = 0.08 p = 0.03 p = 0.02 p = 0.67

Faceoff Rate t = 1.43, t = 1.06, t = -0.97, t = 3.05, (#/min) p = 0.17 p = 0.30 p = 0.34 p < 0.01

Prop Time in t = 1.50, t = -1.36, t = -0.90, t = 2.60,

Faceoffs p = 0.15 p = 0.19 p = 0.38 p = 0.01

Closest t = -3.83, t = -1.21, t = -1.77, t = -0.04,

Distance (m) p < 0.01 p = 0.24 p = 0.09 p = 0.97

35 Table 8: Results of principal component analysis using playback responses. Each measure indicates a change in response between the pre-playback period and the during-playback period for prairie-chicken vocalization treatments. Data include responses of 26 male Greater Prairie-Chickens to 10 playback trials conducted in 2012 near Manhattan, Kansas. Principal component loadings > 0.5 presented in bold.

Response measures PPC1 PPC2 PPC3

Change in boom rate 0.36 -0.42 0.75

Change in faceoff rate 0.35 0.55 0.42

Change in proportion of time in faceoffs 0.16 0.69 -0.03

Change in closest distance -0.57 0.20 0.43

Change in distance traveled 0.62 -0.06 -0.27

% Variation Explained 35.2 30.3 15.8

36 Appendix B: Figures

Figure 1: Sonogram of a Greater Prairie-Chicken call, with several structural elements identified. The vocalization depicted here is one of 622 recorded from 56 male Greater Prairie-Chickens in 2011 and 2012 near Manhattan, Kansas.

37

Figure 2: Territories of 7 male Greater Prairie-Chickens at the Kreider lek in 2012 near Manhattan, Kansas. The X represents placement of the playback speaker on the territory of male 6 on 19 Apr 2012. Thus, on this day male 6 was the owner of the territory on which the speaker was placed, and his vocalizations were used for the Owner playback treatment. Three neighbors adjacent to his territory (3, 4, and 5) were used as the focal males, whose responses were measured before and during each playback treatment. The vocalizations of another male (2) whose territory was non- adjacent to that of male 6 were used for the Neighbor playback treatment. The other two playback treatments were vocalizations from a male from another lek (the Stranger treatment) and from an Upland Sandpiper (the Control treatment). Playbacks and video recordings were controlled from within the blind.

38

Figure 3: Mean differences ± SE in the average boom rate (# booms/min) of male Greater Prairie-Chickens before and during playback treatments. Data were collected from 26 males during March and April 2012 near Manhattan, Kansas.

39

Figure 4: Mean differences ± SE in the average faceoff rate (# faceoffs/min) of male Greater Prairie-Chickens before and during playback treatments. Data were collected from 26 males during March and April 2012 near Manhattan, Kansas.

40

Figure 5: Mean differences ± SE in the proportion of time spent in faceoffs by male Greater Prairie-Chickens before and during playback treatments. Data were collected from 26 males during March and April 2012 near Manhattan, Kansas.

41

Figure 6: Mean differences ± SE in the closest distance of approach to the playback speaker (m) by male Greater Prairie-Chickens before and during playback treatments. Data were collected from 26 males during March and April 2012 near Manhattan, Kansas.

42