Behavioral strategies and signaling in interspecific aggressive interactions in gray tree frogs

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1 Behavioral Ecology The official journal of the ISBE International Society for Behavioral Ecology Behavioral Ecology (014), 5(3), doi: /beheco/aru016 Original Article Behavioral strategies and signaling in interspecific aggressive interactions in gray tree frogs Michael S. Reichert and H. Carl Gerhardt Division of Biological Sciences, University of Missouri, 15 Tucker Hall, Columbia, MO 6511, USA Received 4 September 013; revised 16 December 013; accepted 16 January 014; Advance Access publication 17 February 014. Interspecific aggression has important consequences for ecological processes and the evolution of behavioral strategies. We examined interspecific aggressive interactions in the gray tree frog species, Hyla chrysoscelis and Hyla versicolor. These species call side by side in the same ponds, and acoustic interference occurs because of the similar spectral characteristics of their vocalizations. The aggressive calls of these species, although very similar, were statistically distinguishable: calls of H. chrysoscelis were more strongly amplitude modulated than those of H. versicolor. We used playbacks and staged interactions to characterize the behaviors of males exposed to heterospecific competitors or their signals. Males readily responded with aggression to heterospecific individuals and playbacks of their calls. These behaviors were qualitatively similar in both intraspecific and interspecific interactions, but there were some significant differences. First, males of H. chrysoscelis were more aggressive toward playbacks of conspecific advertisement calls than toward those of H. versicolor. There were no significant differences in this respect in H. versicolor. Second, interspecific interactions were usually more escalated than intraspecific interactions and more likely to end with the loser moving away from its opponent. Although neither species had an advantage in staged interactions, behavioral responses were asymmetrical because H. versicolor was more likely than H. chrysoscelis to initiate physical contact. Previous studies showed that H. versicolor suffers from a greater reduction in attractiveness than H. chrysoscelis in the presence of heterospecific call overlap. Thus, the asymmetries in aggressive behavior between the species may be related to differential costs of heterospecific competition. Key words: acoustic communication, aggression, anuran, interspecific competition, playback. Introduction Competition is a major channel through which selection operates because the outcomes of competitive interactions determine the distribution of resources among individuals (Milinski and Parker 1991). In general, competition among conspecifics is common because of high overlap in resource use (Peiman and Robinson 010). Nonetheless, under certain ecological conditions, individuals of different species may also engage in competitive interactions with one another (Orians and Willson 1964; Ebersole 1977; Mikami and Kawata 004). When, for example, species compete for a common food resource or display territory, individuals of different species may exert indirect negative effects on one another (i.e., exploitative competition; Schluter 000) or directly interfere with one another via aggressive behavior (i.e., interference competition or heterospecific aggression; Peiman and Robinson 007). Address correspondence to M.S. Reichert, who is now at Institut für Biologie der Humboldt-Universität zu Berlin, Abt. Verhaltensphysiologie, Invalidenstr. 43, Berlin, Germany. michael.s.reichert@hu-berlin.de. Although heterospecific aggression is apparently widespread in animals, it has received less attention than other forms of interspecific competition and both the mechanisms and consequences of this behavior remain poorly understood (Grether et al. 009, 013; Peiman and Robinson 010). Because animal contests are often mediated by signaling interactions (Maynard Smith and Price 1973; Maynard Smith and Parker 1976), the study of the aggressive signals used in interspecific contests is likely to be a fruitful approach to addressing the major outstanding questions in the understanding of heterospecific aggression. A longstanding question of interest is whether heterospecific aggression affects patterns of signal evolution. Cody (1969, 1973) proposed that when animals display interspecific territoriality, there should be selection for the convergence of aggressive signal characteristics to facilitate heterospecific aggressive interactions. This hypothesis was disputed, however, and other researchers proposed that patterns of similarity in aggressive signal characteristics across species may be related to common ancestry, perhaps with selection for conservation of similar signal properties where species ranges overlap (Gerhardt and Schwartz 1995). Alternatively, selection might disfavor heterospecific aggression so The Author 014. Published by Oxford University Press on behalf of the International Society for Behavioral Ecology. All rights reserved. For permissions, please journals.permissions@oup.com

2 Reichert and Gerhardt Interspecific aggression in tree frogs 51 that most observed instances are caused by receivers inabilities to discriminate conspecifics from heterospecifics (Murray 1971, 1976, 1981). Despite this controversy, several studies have now documented interspecific aggressive interactions and provide evidence that such behavior is adaptive and influences the evolution of signal structure (e.g., Martin and Martin 001; Adams 004; Peiman and Robinson 007; Tobias and Seddon 009; Laiolo 01). The occurrence of heterospecific aggressive interactions also raises important questions for the understanding of strategic contest behavior. This topic has received a great deal of recent attention in the literature on studies of intraspecific contests (reviewed by Arnott and Elwood 009) but is largely unexplored for interspecific contests (Tanner and Adler 009; Lehtonen et al. 010; Green and Field 011). Species that engage in both intraspecific and interspecific contests provide ideal study systems to understand the mechanisms that shape the dynamics and outcome of contests. First, as noted above, selection may favor certain aggressive signal characteristics to facilitate or reduce heterospecific aggression. However, selection from conspecific aggressive interactions also will act on aggressive signals, and the strength and direction of these sources of selection may not be equivalent. A reduction in overall levels of heterospecific aggression might result if competitors signals are unrecognizable (e.g., Tynkkynen et al. 004), but an increase might equally well be predicted if these signals are recognizable but less efficient as a means of contest assessment than the corresponding conspecific signal. Second, even if species utilize identical aggressive signals, the means by which these signals are assessed may differ for each species and therefore limit the utility of interspecific aggressive communication. For instance, the same signal may be used in one species to signal a low level of threat and in another to signal a high level of threat. Thus, it is important to measure variation in not only the structure of signals of interacting heterospecifics but also the responses of receivers to conspecific and heterospecific signals. Finally, individuals of the different species may differ in characteristics that determine the outcome of contests, which offers opportunities to test theoretical models of contest behavior. For instance, the negative consequences of the presence of a heterospecific may be asymmetric between the species studied. This pattern may generate an asymmetry in costs, which, according to many models, could lead one species to be more likely to win contests (Maynard Smith and Parker 1976; Hammerstein and Parker 198). All of these questions can be addressed by examining the dynamics of contests and response to signals of individuals engaged in intraspecific and interspecific aggressive interactions. In this study, we examined aggressive signals and behaviors of cryptic species of gray tree frogs, Hyla chrysoscelis and Hyla versicolor, in staged and simulated heterospecific and conspecific interactions. As in many anurans, mate attraction and reproduction takes place in or near ponds and other small bodies of water, where individuals of different species often gather in large numbers at the same time during the breeding season (Duellman and Pyles 1983; Schwartz and Wells 1983a; Donnelly and Guyer 1994). Competition between species may occur over physical space needed to attract mates and raise offspring, and interspecific aggressive behavior has been documented frequently in anurans (Wells 1980; Brzoska 198; Schwartz and Wells 1984, 1985; Given 1990; Gerhardt and Schwartz 1995; Shimoyama 1999). Male gray tree frogs do not defend territories but nonetheless engage in aggressive interactions with other males to defend a calling space, a buffer between itself and nearby males, which reduces acoustic interference in that area and presumably increases its chances of female attraction (Wells and Schwartz 007). Importantly, acoustic interference from not only conspecifics but also heterospecifics can reduce a male s chances of attracting a mate (Marshall et al. 006). Thus, individuals are likely to respond aggressively to both conspecifics and heterospecifics that intrude on their calling space. The ranges of our study species include large areas of sympatry. Our choice of the species of gray tree frogs was in part based on their frequent activity in mixed species choruses. Although there is some evidence that males of the different species occupy slightly different microhabitats within mixed choruses (Ptacek 199), there is nonetheless considerable overlap within ponds, and males of the species often call side by side (Gerhardt et al. 1994; see also Marshall et al. 006). These species also have a close evolutionary relationship; H. versicolor is a tetraploid species that is thought to have arisen by allopolyploidy from ancestral H. chrysoscelis and extinct, closely related lineages (Holloway et al. 006). A few interspecific hybrids have been found even though hybrid offspring have reduced viability (Gerhardt et al. 1994). The species of gray tree frogs also offer an interesting contrast in their signal repertoires. Although their advertisement calls are distinctive and there is evidence for how these differences evolved (e.g., Keller and Gerhardt 001), the aggressive calls of the species are much more similar to one another. If this similarity facilitates interspecific aggressive behavior, then future comparisons of allopatric and sympatric populations may allow us to determine if conservation or convergence is the cause for aggressive-call similarity. Here, we provide data that address 3 important aspects of interspecific aggressive behavior. First, we analyzed the characteristics of the aggressive calls of both species to determine if there are any differences between the species aggressive calls for the populations under study. Second, we used playbacks to determine whether males were more likely to respond aggressively to a simulated conspecific or heterospecific competitor. Third, we staged interactions between heterospecifics to determine if they would interact aggressively with one another, and if so, if their behavior in these interactions differed from that observed in previous studies of intraspecific aggressive interactions in H. versicolor. Methods Aggressive-call analyses We recorded and analyzed the aggressive calls of males of each species to determine if there were species differences in aggressivecall characteristics. This study was not designed as an exhaustive survey of geographic variation in aggressive calls and hence we assert that the differences we observed may be due both to differences between the species and differences in the geographic location and species composition of the recorded individuals natural habitats. Nevertheless, these analyses are valuable because they provide an initial overview of the potential for variation in aggressive calls in these species. Male H. versicolor were captured from local ponds in Boone County, MO; these populations are currently allopatric with respect to H. chrysoscelis although isolated populations of the latter species occur within 0 km. Male H. chrysoscelis were captured from sympatric populations located in Phelps County, MO. Additional H. chrysoscelis were captured from an allopatric population from Liberty County, FL. Calls to be analyzed were selected from a subset of recordings of males calling during the staged interactions described in Experiment, along with recordings of males that were prompted to give aggressive calls in response to the

3 5 Behavioral Ecology playback of a conspecific advertisement call. When males gave sufficient numbers of aggressive calls, we analyzed the first 10 such calls given during the recording that were sufficiently free from background noise interference to permit the analysis of fine-temporal structure. Not all males gave 10 aggressive calls, but all recordings included in the data set contained measurements from at least 3 calls per male. We analyzed temporal and spectral characteristics of each aggressive call using the Raven Pro sound analysis program (V 1.3, Cornell Laboratory of Ornithology) and custom-designed software (Signan, created by G. Klump and modified by D. Poleet). We were particularly interested in fine-temporal differences in call structure. Thus, in addition to measuring call duration, we measured rise times (the time from the start of the call to its peak amplitude) and fall times (the time from peak amplitude to the ending of the call) of each call. Because absolute rise and fall times depend on the total duration of the call, a characteristic that varies between individuals, we converted these into the percentages of the total call duration in which the call was rising and falling, respectively. These measures allowed us to compare overall differences in call shape between the species in a standardized manner. In addition, we examined the possibility that the pattern of amplitude modulation may differ in the aggressive calls of these species. Aggressive calls are usually not composed of clearly separate pulses; nonetheless, there is substantial variation between calls in the depth of amplitude modulation (Figure 1). Because we could not count pulses in most cases, we quantified amplitude modulation by measuring the number of times the amplitude of the call dropped below 50% of the peak amplitude. By definition, all calls drop below 50% of the peak amplitude at their beginning and end, thus we subtracted one from this number to obtain a quantity somewhat comparable to the pulse number that is often measured for advertisement calls. Figure 1 Waveform displays of typical aggressive calls. All calls were recorded from individuals engaged in staged interspecific interactions. (a) Hyla chrysoscelis, (b) Hyla versicolor, and (c) a pair of calls given by H. chrysoscelis. Note that the calls of H. chrysoscelis have much greater amplitude modulation than the call of H. versicolor. In H. chrysoscelis, this amplitude modulation is usually incomplete, as in (a). However, in some cases, as in (c), the amplitude modulation is of sufficient depth that the aggressive call is composed of clearly separate pulses.

4 Reichert and Gerhardt Interspecific aggression in tree frogs 53 In addition to these temporal characteristics, we measured the values of the major frequency peaks in the call, the low-frequency peak and the (dominant) high-frequency peak. For those male H. chrysoscelis that were recorded in staged interactions, we determined the extent to which they reduced the frequency of their aggressive calls relative to that of their advertisement calls. Such behavior has already been shown to be an important component of aggressive interactions in H. versicolor (Reichert 013a; Reichert and Gerhardt 013a). Thus, we also measured the frequency peaks of advertisement calls of these males and subtracted the average frequency value for the aggressive call from that of the advertisement call to obtain an average amount by which males adjusted their aggressive-call frequencies. Our primary aim was to determine if the aggressive calls of the species are acoustically distinct. To this end, we entered the call characteristics into a discriminant function analysis (DFA) that determined the extent to which the species calls were statistically distinguishable from one another. We performed separate DFAs for all possible combinations of the 3 populations under study. Thus, our analyses also allowed us to assess whether there were any differences in aggressive-call characteristics between H. chrysoscelis from widely separated populations. Independent samples t-tests were then used to determine which of the individual call characteristics included in the DFA were significantly different. In addition, we performed a principal components analysis to reduce the number of variables and then plotted the first components against one another to illustrate differences in call characteristics between the species. The latter analyses were only performed with males from Missouri because the recordings from Florida H. chrysoscelis males could not be corrected for body mass (see below). The H. chrysoscelis males included in this analysis from the Phelps County, MO, population were on average heavier than the H. versicolor males from the Boone County, MO, population. Both the frequency characteristics and the duration of aggressive calls are correlated with body mass (Reichert 013a; Supplementary Table 1). Thus, except where noted below, prior to statistical analyses, we adjusted these characteristics to the average body mass (6.35 g) of the individuals included in this analysis. To do this, we calculated linear regressions for each call characteristic on body mass for each species and used the parameters of the regression equation to adjust each individual s call characteristics relative to its body mass to that expected for an average-massed individual (Platz and Forester 1988). This ensured that our comparisons reflected differences between the species that were not biased by the mass differences between the samples. Mass data were unavailable for H. chrysoscelis from the Florida population; thus, we present only uncorrected data for these individuals and limit the extent to which they were included in analyses. Although many characteristics of the advertisement calls of these species are influenced by temperature, such effects are much weaker or nonexistent in the aggressive-call characteristics we measured in this study (Gayou 1984; Reichert 013a; unpublished data). For this reason, we did not adjust call characteristics for temperature in our analyses. Recordings of 5 male H. chrysoscelis from Missouri, 14 males from Florida, and 34 male H. versicolor from Missouri were used for these analyses. Behavioral experiments Frog capture and experimental setup All males used in behavioral experiments originated from Boone County, MO (H. versicolor) or Phelps County, MO (H. chrysoscelis); males from the Florida population were not used in these experiments. In all cases, males were housed in an animal care facility in a greenhouse located near the campus of the University of Missouri. On each day of testing, we released males in the afternoon into an indoor artificial pond that had environmental conditions (simulated rainfall, artificial chorus noise) conducive to male chorusing (see also Schwartz et al. 001). Experiments were performed at night during the peak period of chorusing activity (000 to 000 hours; tests were carried out from April to July, ). Males that called well in the artificial pond were placed in cages atop platforms scattered about the pond. Males selected for testing could then be transported on these platforms to testing arenas located outside of the pond. Once males were placed in the arena, the cage was removed. Thus, males were potentially free to move off of the platform. Nonetheless, males generally remained in place while calling except when challenged by another nearby male (see Experiment : staged aggressive interactions). Experiment 1: aggressive-threshold measurements We tested whether male H. versicolor and H. chrysoscelis differed in their aggressive responses to a playback simulating an approaching conspecific or heterospecific male. Our experimental design followed the approach of Rose and Brenowitz (1991) to measure aggressive thresholds, defined as the lowest amplitude at which a male gave an aggressive response to the playback stimulus. Thus, lower aggressive thresholds indicated an increased aggressive responsiveness to the playback stimulus. Our playback stimuli were 1) a synthetic H. versicolor advertisement call with call characteristics (pulse number: 18; call duration: 0.9 s; frequency of the low-frequency peak: 1100 Hz; frequency of the high-frequency peak: 00 Hz; call repetition rate: 5 s) based on averages obtained from field recordings of calling males from our study population and ) a synthetic H. chrysoscelis advertisement call with a pulse rate (approximately 40 pulses/s) somewhat lower than the average (pulse number: 34; call duration: 0.9 s; frequency of the low-frequency peak: 1100 Hz; frequency of the high-frequency peak: 00 Hz; call repetition rate: 5 s). Synthetic calls were used rather than natural exemplars to ensure that differences in males responses were not related to any idiosyncratic characteristics of the particular stimuli chosen but rather can be interpreted as a difference in response to calls with typical characteristics of the species (McGregor et al. 199). We recorded the stimuli to a compact disk and broadcast them through a Mineroff SME-AFS speaker. The sound pressure level (SPL) of the loudest playback stimulus was set to 107 db SPL at 10 cm from the speaker using a digital sound level meter (Radio Shack ). This amplitude approximates that expected between males interacting at a typical distance (10 cm) at which aggressive interactions take place (Gerhardt 1975; Reichert and Gerhardt 011). This was also the highest amplitude at which our playback system emitted undistorted signals. We placed the platforms of individual males so that they would be positioned approximately 10 cm from the speaker, but small movements by the frogs prior to testing sometimes prevented us from exposing males to exactly the same maximum SPL. The playback stimulus was designed such that it began at an SPL 30 db below the maximum. The SPL of the playback was increased by 3 db every minute until it reached the maximum SPL at the 10th minute. Playback tests took place approximately 6 m away from the artificial pond and thus the background chorus was audible throughout the playbacks, although at this distance, it was substantially quieter than the playback (see also Christie et al. 010). The background chorus noise fluctuated often. Although we did not attempt to control for this or measure absolute amplitudes, we randomized the playback stimulus, and during time periods when

5 54 Behavioral Ecology both species were available, the species of the focal male, in order to ensure that chorus background noise amplitude did not bias our results. The randomization also ensured that results were robust to any changes in aggression over the course of the season, although no such changes have been observed (Marshall VT and Gerhardt HC, unpublished observations). We began playbacks once the frog resumed spontaneous calling after being transported from the artificial pond. We recorded males calling responses throughout the playback onto a digital-audio recorder (Tascam DR-680) with a microphone (Sennheiser ME-67) suspended directly above the calling male. We continued playbacks until either the male gave an aggressive call or until the 11-min playback period had elapsed. For males that gave an aggressive call, we measured the SPL of the playback stimulus at the male s position at the time they first gave an aggressive call to determine the aggressive threshold. Thus, males that did not give an aggressive call at any point during the playback did not contribute to our measurements of aggressive thresholds. We compared aggressive thresholds for each species using nonparametric Wilcoxon Mann Whitney tests and also compared the proportion of individuals of each species that gave an aggressive call at some point during the playback using chi-square tests. We emphasize that these threshold estimates are not equivalent to those that would be obtained with psychophysical procedures, which would take into account the effects of subthreshold playbacks. Rather, our playbacks simulated the approach of calling individuals to one another. We obtained responses from 61 male H. versicolor and 38 male H. chrysoscelis for this experiment. Experiment : staged aggressive interactions We used the techniques described by Reichert and Gerhardt (011) to stage interactions (n = 0) between male H. versicolor and H. chrysoscelis. We selected a male of each species that was calling well in the artificial pond and brought them to an arena located approximately 3 m away from the pond. Each male was placed on a wheeled platform on opposite ends of a 1.8-m long runway. Once both males resumed calling, we gradually pulled the platforms toward one another by means of ropes attached to each platform until the platforms abutted one another. At this point, males were calling at extremely close range, which, in interactions between conspecific H. versicolor, would often lead to contests involving aggressive calling and in some cases, physical combat (Reichert and Gerhardt 011). We considered an interaction to have taken place when both males called at least once after the platforms had been pulled toward one another. In these competitive interactions, there was always a clear winner and loser. We defined losers as individuals that either escaped from the interaction by moving at least 1 platform length away while their opponent (the winner) remained in place and called or remained in place but stopped calling for at least 5 min while their opponent (the winner) continued to call. We recorded audio and video throughout the interaction using digital-audio recorders (Marantz PMD-660 and PMD-661), directional microphones (Sennheiser ME-66, ME-67 and ME-80), and a digital camcorder (Sony DCR-SR85). All staged interactions were performed at night (000 to 000 hours) under ambient light conditions within a glass-roofed greenhouse from May to June 010. After each interaction had taken place, we measured the mass of each male. We tested several hypotheses with these data. First, we tested whether one species had an advantage over the other in contests. Second, we tested whether one species was more likely to instigate aggressive behaviors. That is, we examined the recordings of each interaction and determined which individual was the first to give an aggressive call, and, when physical combat took place, which individual initiated physical contact. Third, we made several comparisons of contest characteristics between these interspecific interactions and intraspecific interactions between male H. versicolor (n = 173) that had been recorded for previous studies using the same methodology described here (Reichert and Gerhardt 011, 01, 013b). We compared the proportions of intraspecific and interspecific contests that terminated at each of 4 mutually exclusive stages, listed here in order of increasing escalation: both males only gave advertisement calls, only 1 of the males gave aggressive calls, both males gave aggressive calls, and males engaged in physical fighting. Likewise, we compared average contest duration between interspecific and intraspecific interactions. Contest duration was defined as the amount of time between the point at which both males had first called after their platforms were pulled toward one another and the time at which the loser gave its final call; we were unable to measure interaction duration for one interspecific interaction because of an equipment malfunction. Finally, we compared the behavior of losers of interspecific and intraspecific interactions. At the conclusion of the interaction, losers either moved away from the contest winner or remained in place but ceased calling. Thus, we tested whether there were differences in the occurrence of these behaviors when the competitors were of the same or different species. Differences in qualitative behavioral measurements were compared using Fisher s Exact tests because of the small sample size for some categories of interspecific interactions. Results Aggressive-call analyses Descriptive statistics for the characteristics of aggressive calls for each species are given in Table 1. Aggressive calls of both species showed a broadly similar structure. In both species, the calls consist of a brief burst of sound with major frequency peaks, and calls are organized into bouts in which several calls are given in rapid succession followed by a relatively longer pause. As in H. versicolor (Reichert and Gerhardt 013a), the frequency peaks of the aggressive calls of H. chrysoscelis were lower than the respective peaks in their advertisement calls (mean ± standard deviation [SD] difference between the frequency peak for advertisement calls and the frequency peak for aggressive calls, uncorrected for body mass; lowfrequency peak: 9 ± 69 Hz; high-frequency peak: 4 ± 99 Hz; n = 14 males from the Phelps County population calling in staged interactions). Nonetheless, the DFA revealed that the aggressive calls of the species could be distinguished quite well: in comparisons between the Missouri populations, 96.0% of H. chrysoscelis aggressive calls and 97.1% of H. versicolor aggressive calls were assigned by the analysis to the correct species (canonical correlation: 0.90; Wilks s lambda: 0.19; χ 6 = 89. 4, P < 0.001). The amplitude modulation of the calls had the largest influence on the discriminant function (Table ). The aggressive calls of H. chrysoscelis were much more likely to contain amplitude modulation than were those of H. versicolor. Indeed, in exceptional cases, the aggressive calls of H. chrysoscelis were composed of distinct pulses (Figure 1c); this was never the case for H. versicolor. Nonetheless, when we removed this variable from the DFA, the classification success was again high (84.0% correct classifications for H. chrysoscelis and 88.% correct classifications for H. versicolor; canonical correlation: 0.77; Wilks s

6 Reichert and Gerhardt Interspecific aggression in tree frogs 55 Table 1 Descriptive statistics and t-tests comparing the call characteristics of each species Call characteristic Mean (SD) Hc-MO Mean (SD) Hc-FL Mean (SD) Hv-MO t P Amplitude modulation 6. (.3) 6.3 (.1) 1.7 (1.1) 9.98 <0.001 Call duration uncorrected (s) 0.1 (0.04) 0.1 (0.04) 0.15 (0.04) 3.94 <0.001 Call duration mass corrected (s) 0.10 (0.03) 0.16 (0.04) 6.84 <0.001 Low-frequency peak uncorrected (khz) 1.0 (0.07) 1.04 (0.07) 1.06 (0.07) Low-frequency peak mass corrected (khz) 1.06 (0.04) 1.0 (0.04) High-frequency peak uncorrected (khz).08 (0.17).15 (0.18).1 (0.17) High-frequency peak mass corrected(khz).17 (0.10).03 (0.11) 5.0 <0.001 Proportional rise time 0.67 (0.14) 0.69 (0.11) 0.73 (0.13) Proportional fall time 0.4 (0.11) 0. (0.11) 0.19 (0.08) Mean and SD of each of the 6 measured call characteristics for Hyla chrysoscelis from a sympatric population in Phelps County, MO (Hc-MO; n = 5), H. chrysoscelis from an allopatric population in Liberty County, FL (Hc-FL; n = 14), and Hyla versicolor from an allopatric population in Boone County, MO (Hv; n = 34). Definitions of call characteristics are given in Methods. The proportional rise and fall times represent the proportion of the call duration from the beginning of the call to its peak amplitude and from the peak amplitude to the end of the call, respectively. Because the peak amplitude of some calls was a plateau rather than a single point, the sum of the proportional rise and fall times does not add up to 1. The values for call duration and both frequency peaks are presented both as the original measurements and as values after a correction for male body mass (see Methods). Body mass measurements were not made for the Florida males and therefore only raw values of calls from this population are presented. Also shown are test statistics and P values for independent samples t-tests comparing the mean value of each call characteristic between the Missouri populations of the species. Degrees of freedom = 57 for all tests. lambda: 0.41; χ 5 = 48. 5, P < 0.001). Here, the magnitude of the high-frequency peak was the variable that had the largest influence on the discriminant function, and both the low-frequency peak and call duration also contributed heavily (Table ). In fact, when comparisons were made between the species for each individual call characteristic, all the differences were statistically significant except for call rise time (Table 1). A DFA with the full set of 6 predictor variables but with call frequency and duration measurements that were not corrected for body mass gave qualitatively similar results (9% and 97.1% classification success for H. chrysoscelis and H. versicolor calls, respectively). However, the uncorrected mean values were more similar to one another, particularly for the high-frequency peak, than were those that were corrected for body mass (Table 1). In the principal components analysis, the first principal components explained 74.6% of the variance in call characteristics. The first component loaded heavily on all call characteristics except for rise and fall time, whereas the second component was primarily influenced by these latter variables (Table 3). A plot of the first principal components against one another clearly demonstrates the differences between the calls of the species and shows that most of these differences are accounted for by the first principal component, that is, by the extent of amplitude modulation, call duration, and call frequency (Figure ). Although comparisons could not be made for mass-corrected call characteristics between populations of H. chrysoscelis from a sympatric Missouri population and an allopatric Florida population, the uncorrected characteristics were very similar in the populations (Table 1). A DFA analysis classified only 71.8% of calls to the correct population; this analysis did not reach statistical significance (canonical correlation: 0.30; Wilks s lambda: 0.91; χ 6 = 31., P = 0.78). As with H. chrysoscelis males from the sympatric Missouri population, the calls of males from the allopatric Florida population were easily distinguished from those of H. versicolor (uncorrected for body mass; 100% correct classifications for H. versicolor and 9.9% correct classifications for H. chrysoscelis; canonical correlation: 0.89; Wilks s lambda: 0.; χ 6 = 65. 8, P < 0.001). Aggressive thresholds Male H. versicolor did not differ in the likelihood of giving aggressive calls in response to playbacks of conspecific or heterospecific Table Coefficients from a DFA of aggressive calls Call characteristic DFA coefficient (full) Amplitude modulation 0.89 Call duration Low-frequency peak High-frequency peak Proportional rise time Proportional fall time DFA coefficient (reduced) These standardized canonical coefficients show the extent to which each characteristic contributed to the discrimination of the species aggressive calls. The full model included all 6 measured call characteristics. The reduced model excluded the characteristic with the highest explanatory power, amplitude modulation. Even with the reduced model, the calls of these species could be discriminated with few errors (see Results). The values for call duration and frequency peaks were corrected for male body mass prior to calculating the DFA. Values shown are for an analysis of the Missouri populations of each species (see Methods). Table 3 Component matrix for the first principal components (PC1 and PC) of the aggressive-call characteristics Call characteristic PC1 PC Amplitude modulation Call duration (s) Low-frequency peak (khz) High-frequency peak (khz) Proportional rise time Proportional fall time Values for call duration and the frequency peaks were corrected for male body mass prior to the principal components analysis. This analysis included males from the Missouri populations of the species. advertisement calls (13 of 8 males gave aggressive calls in response to the H. versicolor playback; 14 of 33 males gave aggressive calls in response to the H. chrysoscelis playback; χ 1 = 01., P = 0.75). In contrast, male H. chrysoscelis were more likely to give aggressive calls in response to the conspecific playback than to the heterospecific playback (1 of 17 males gave aggressive calls in response to

7 56 Behavioral Ecology Figure Plot of the first principal components of aggressive-call characteristics against one another. Closed circles: Missouri Hyla versicolor; open circles: Missouri Hyla chrysoscelis. the H. chrysoscelis playback; 7 of 1 males gave aggressive calls in response to the H. versicolor playback; χ 1 = 5., P = 0.0). For those individuals that gave aggressive calls, there were no differences in aggressive thresholds when comparisons were made between responses to the stimuli within each species (Figure 3; Wilcoxon Mann Whitney test, H. chrysoscelis: n = 19, U = 3, P = 0.11; H. versicolor: n = 7, U = 7.5, P = 0.37). However, male H. chrysoscelis had lower aggressive thresholds in response to the H. chrysoscelis stimulus than did male H. versicolor (Figure 3; Wilcoxon Mann Whitney test, n = 6, U = 6.5, P = 0.003). Because of movements on the platform, not all males were tested at an identical maximum playback SPL. Nonetheless, there was no evidence that males that failed to respond aggressively to the playback did so because these playbacks were less intense than those in which males responded aggressively. The range of maximum SPLs of playbacks in which males did not give aggressive calls ( db SPL) overlaps with and extends beyond the distribution of SPLs at which aggressive calls were elicited in other males (Figure 3). Staged aggressive interactions Males interacting at close range with a heterospecific readily responded with aggressive behavior, and the structure of aggressive interactions between a male H. versicolor and H. chrysoscelis was qualitatively similar to the structure of interactions between male H. versicolor. As in interactions between conspecifics, interactions between heterospecifics involved 4 distinct levels of escalation, and the types of behaviors exhibited by winners and losers were similar (Reichert and Gerhardt 011). Nonetheless, there were some differences between intraspecific and interspecific aggressive interactions. Interspecific interactions were significantly longer in duration than intraspecific interactions (mean ± SD; interspecific: ± s, n = 19; intraspecific: 11.4 ± s, n = 173; t-test on ln-transformed interaction durations: t 190 =., P = 0.03). This result probably is explained by a tendency for interspecific interactions to reach higher levels of escalation, although distributions of levels of escalation in the types of interaction were not significantly different from one another when all 4 levels of escalation were considered separately (Figure 4; Fisher s Exact test, P = 0.13). Sample sizes were small for some levels of escalation of interspecific interactions. When we pooled the lowest and highest levels of escalation together, the difference in escalation between interspecific and intraspecific interactions approached significance (Fisher s Exact test, P = 0.056). Neither species had a clear advantage in interspecific interactions; 1 of 0 interactions were won by H. versicolor. Also, neither species was more likely to be the first to give aggressive calls in the interaction; in 10 of 19 interactions, the H. chrysoscelis male was the first to give aggressive calls. However, when interactions escalated to physical fighting, males of H. versicolor were much more likely to initiate physical contact; this was the case for 8 of the 9 physical fights between the species. There was a difference in the behavior of losers when comparisons were made between interspecific and intraspecific interactions. In interspecific interactions, losers nearly always moved away from the winner rather than remaining in place and not calling, whereas in intraspecific interactions, losers were more likely to remain in place than to move away (proportion of losers moving away at the end of the contest: interspecific interactions, 18/0; intraspecific interactions, 56/173; Fisher s Exact test, P < ). Losers of physical fights in intraspecific interactions are more likely to move away from the winner than losers at other levels of escalation (Reichert and Gerhardt 011). Nonetheless, the differences between interspecific and intraspecific interactions in loser behavior were not explained by the increased likelihood of interspecific interactions escalating to physical fighting. When physical fights were excluded from the analysis, losers of interspecific interactions were still more likely to move away from their opponent than losers of intraspecific interactions (proportion of losers moving away at the end of the contest: interspecific interactions,

8 Reichert and Gerhardt Interspecific aggression in tree frogs 57 Figure 3 Aggressive thresholds of male Hyla versicolor (Hv) and Hyla chrysoscelis (Hc) in response to advertisement call playbacks of each species. (a) Playback of H. versicolor stimulus. (b) Playback of H. chrysoscelis stimulus. Asterisk indicates a significant difference (P < 0.05) between the species in response to the playback stimulus (see Results). Figure 4 Proportions of interactions that terminated at each level of escalation (ADV: both males only gave advertisement calls, AG1: only 1 of the males gave aggressive calls, AG: both males gave aggressive calls, PF: physical fight). White bars: intraspecific interactions (n = 173; data from Reichert and Gerhardt 011); gray bars: interspecific interactions (n = 0). 10/11; intraspecific interactions, 1/119; Fisher s Exact test, P < ). Heavier individuals did not have an advantage in interspecific interactions (n = 10 of 0 interactions won by the heavier male; mean ± SD mass: H. chrysoscelis losers: 6.45 ± 1.05 g, n = 1; H. versicolor winners: 6.31 ± 0.67 g, n = 1; H. chrysoscelis winners: 6.90 ± 1.30 g, n = 8; H. versicolor losers: 6.90 ± 0.47 g, n = 8). Individuals of neither species were significantly heavier than those of the other species in these contests (paired t-test: t 19 = 0.38, P = 0.71). Likewise, for those interactions in which aggressive calls could be recorded from both males, there were no differences in aggressive-call frequency between winners and losers (frequencies uncorrected for body mass: low-frequency peak: t 13 = 0.49, P = 0.6; high-frequency peak: t 13 = 0.94, P = 0.36). Discussion The results of this study clearly show that males of the species of gray tree frog are responsive to one another s signals and that behavioral contexts that would elicit aggressive behavior in intraspecific interactions also elicit aggressive behavior in interspecific encounters. The acoustic recognition space of males of each species for aggressive signals remains to be determined, but the results obtained here suggest that males will be largely responsive to signals of both species across their range of variation. This similarity in responsiveness contrasts with our finding of acoustic differences between the aggressive calls of the species and in general raises intriguing questions about the strength and direction of, and forces responsible for, changes in aggressive signal characteristics of these species relative to each other. Despite broad similarities between intraspecific and interspecific aggressive interactions, there were some subtle differences that suggest that the consequences of engaging in these types of interactions differ for the participants. Differences in aggressive-call structure Previous studies have described numerous differences in the advertisement calls of H. chrysoscelis and H. versicolor (Gerhardt 001). Indeed, other than chromosome number, differences in advertisement-call characteristics are the only way to reliably distinguish between individuals of these species (Wasserman 1970). Likewise, we found several differences in their aggressive calls. Intriguingly, the greatest difference between the species aggressive calls was found for amplitude modulation, which is also one of the most divergent characteristics of their advertisement calls (Gerhardt 001). Individuals of H. chrysoscelis tended to have clearly amplitude-modulated aggressive calls, whereas those of H. versicolor rarely showed any notable amplitude modulation. Nonetheless, although the aggressive calls of these species were statistically distinguishable, there was some overlap between aggressive-call characteristics of the species and the calls could not always be unequivocally classified as belonging to one species or the other. In contrast, at a given temperature, advertisement calls of these species are apparently always different in pulse rate and fine-temporal structure (Gerhardt and Doherty 1988; Gerhardt 001). That aggressive calls show more structural overlap than advertisement calls may indicate that selection toward species-specific signal characteristics is stronger in advertisement calls than in aggressive calls because the costs of mismating are higher than those associated with misdirected aggression toward a heterospecific. This possibility seems reasonable because interspecific matings rarely result in viable offspring (Gerhardt et al. 1994) and although the costs of aggressive interactions in these species are unknown,

9 58 Behavioral Ecology they are probably minimal given the lack of weaponry and potential for injury (Reichert and Gerhardt 011). Furthermore, as hypothesized by Cody (1969, 1973), similarity in aggressive signals may in fact be selected for in some cases when heterospecific aggression is a particularly important component of a species life history. Our results cannot directly address hypotheses of character convergence or divergence in aggressive signals because we did not sample from a large range of populations in both sympatry and allopatry for each species. Nevertheless, the limited information on geographical variation in aggressive signals from this study and that of a previous study of these species (Pierce and Ralin 197) suggests that the presence of heterospecifics may not have any effects on aggressive-call structure. There were no obvious differences between the aggressive calls of male H. chrysoscelis from a sympatric population in Missouri and an allopatric population in Florida. In addition, characteristics of the aggressive calls of male H. chrysoscelis measured by Pierce and Ralin (197) from populations in Texas that were likely sympatric with H. versicolor are within the range of variation in aggressive calls for the populations of H. chrysoscelis measured in this study. Pierce and Ralin (197) also presented measurements of males from populations of H. versicolor in Texas that were largely similar to the data we present for H. versicolor from an allopatric population in Missouri. The only major difference between the studies is that Pierce and Ralin (197) found that the aggressive calls of both H. chrysoscelis and H. versicolor were amplitude modulated, whereas in the present study, H. versicolor was found to be much less likely to have amplitude-modulated calls than H. chrysoscelis. However, the conclusions of Pierce and Ralin (197) are apparently based on measurements of only male H. versicolor, and as we noted above, our study found some overlap in the extent to which the calls of each species showed amplitude modulation. Thus, there is some evidence for differences at the species level in aggressive-call structure, although we caution that sampling over a much larger geographic range using uniform recording techniques and accounting for variation in mass is necessary before any firm conclusions can be reached. A related question is whether the differences in aggressive-call structure described here are meaningful to receivers. The staged interactions showed that individuals responded to aggressive signals of heterospecifics, and we can conclude that the differences between the species calls are insufficient to eliminate heterospecific aggression. Thus, selection for character displacement of aggressive signals is probably weak at best in this system and several alternative hypotheses can be posed to explain species divergence in aggressive calls. First, there may be differences in the signal characteristics that are important for intraspecific aggressive interactions. For example, the amplitude modulation of aggressive calls may be a relevant signal in interactions between male H. chrysoscelis but not between male H. versicolor. Second, the differences between the species aggressive calls may be driven by divergence in advertisement calls that, due to a shared vocal production system, resulted in correlated changes in aggressive-call characteristics. Evidence consistent with such a process was found in the tree frog Dendropsophus ebraccatus, in which there were strong positive between-individual correlations between advertisement and aggressive-call characteristics even though these characteristics tend to vary in opposite directions within individuals in response to competition (Reichert 013b). Third, the differences between the aggressive calls of the species could be an incidental consequence of drift since the lineages diverged. These hypotheses can be addressed by playbacks testing the effects of variation in aggressive-call characteristics on the responses of receivers of each species, measurements of correlated selection on advertisement and aggressive-call characteristics, and a more thorough sampling of geographic variation in aggressive calls. Likewise, comparative studies of aggressive calls across a range of species could reveal broader patterns in the diversification of aggressive-signal characteristics. There has been little work to date on this subject (Owen PC, unpublished data). Differences in receiver responses and contest structure Perhaps the most significant results of this study were the differences in behavior of males in interactions that were staged either between conspecific H. versicolor or interspecific interactions between a male H. versicolor and a male H. chrysoscelis. Interspecific interactions tended to be longer and more escalated than intraspecific interactions. Predictions for interaction duration and likelihood of aggression are unclear for heterospecific interactions and depend on not only the expected degree of resource overlap but also on the response function of individuals to variation in signals characteristic of conspecifics and heterospecifics (Ord and Stamps 009). Although the resource under dispute, the calling space, is presumably identical in intraspecific and interspecific interactions of these species, the negative consequences of a nearby competitor may differ, leading to differences in the predicted behaviors of individuals in these situations. Specifically, a major negative effect of nearby competitors is that overlap from their calls may reduce an individual s likelihood of attracting a mate because females are more attracted to calls that are free from overlap or spatially separated from the source of overlap (Schwartz and Wells 1983b; Schwartz 1987; Schwartz and Gerhardt 1995). Interestingly, the effects of heterospecific call overlap are asymmetric in these species. Marshall et al. (006) presented females of each species with playbacks simulating call overlap between a conspecific and a heterospecific. Surprisingly, female H. versicolor almost always chose the heterospecific call in this situation, likely because these calls happened to contain more leading pulses, which are strongly attractive to females (Marshall et al. 006; Marshall and Gerhardt 010). A caveat in the context of this study is that such masking was contingent on complete overlap of all of the advertisement calls presented to the females. Although complete overlap is probably very rare in natural vocal interactions, we note that the incidence of call overlap increases substantially when males interact at close range (Reichert and Gerhardt 013b). In contrast, females of H. chrysoscelis generally chose the speaker broadcasting conspecific calls. Thus, a male H. versicolor may have more to lose by the presence of a heterospecific competitor than would a male H. chrysoscelis. Because of this, male H. versicolor may be expected to be more likely to initiate aggressive behaviors in this situation, and in fact because of the asymmetry in costs, perhaps be more likely to win interactions against H. chrysoscelis (Maynard Smith and Parker 1976; Hammerstein and Parker 198). Although the latter prediction was not fulfilled in this study, and for the most part the contestant s behaviors were relatively evenly matched, it is interesting in this context that when interactions escalated to the most intense level, physical fighting, physical contact was nearly always instigated by H. versicolor. A similar asymmetry was apparent in the aggressivethreshold measurements. Although H. versicolor males were equally likely to respond with aggressive calls to both conspecific and heterospecific stimuli, H. chrysoscelis males responded less to the heterospecific than to the conspecific stimulus. Asymmetries in the aggressive responses of members of the species are a typical feature of interspecific aggressive interactions (Schwartz and Wells 1985; Robinson