Territorial Olive Frogs Display Lower Aggression Towards Neighbours Than Strangers Based on Individual Vocal Signatures

Animal Behaviour 123 (2017) 217e228

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Animal Behaviour

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Territorial olive frogs display lower aggression towards neighbours than strangers based on individual vocal signatures

* * Ming-Feng Chuang a, Yeong-Choy Kam a, , Mark A. Bee b, c, a Department of Life Science, Tunghai University, Taichung, Taiwan b Department of Ecology, Evolution, and Behavior, University of Minnesota e Twin Cities, St Paul, MN, U.S.A. c Graduate Program in Neuroscience, University of Minnesota e Twin Cities, St Paul, MN, U.S.A. article info Some territorial animals display a form of social recognition in which they direct low levels of aggression Article history: towards established neighbours, but maintain greater readiness to respond aggressively towards unfa- Received 25 April 2016 miliar individuals. In many taxa, such as songbirds, this so-called ‘dear enemy’ effect involves discrim- Initial acceptance 26 August 2016 ination between neighbours and strangers based on individually distinctive vocal signatures. Although Final acceptance 12 September 2016 most anuran amphibians (frogs and toads) are highly vocal, and many are also territorial, we know very little about neighbourestranger discrimination in this group. In the present study of the olive frog, MS. number: A16-00364R Babina adenopleura (Ranidae), we show that the vocal signals of males are individually distinct, and that territory holders use this information to direct lower levels of aggression towards their nearby neigh- Keywords: bours. Analyses of individual variation in advertisement calls revealed many individually distinctive acoustic communication spectral and temporal acoustic properties, with spectral properties contributing most towards statistical aggression discrimination among individuals. In a ﬁeld playback experiment that simulated territorial intrusions, contest individual discrimination territorial males had higher thresholds for producing aggressive calls in response to the advertisement individual recognition calls of their nearby neighbours compared with those of strangers. A simple model based on sound maleemale competition attenuation due to spherical spreading estimated that males responded aggressively to strangers at Rana adenopleura distances that were approximately twice as far away as for neighbours and that were similar to intermale social category distances recorded in the ﬁeld. Together, results from this study indicate that territorial male olive frogs territoriality develop vocally mediated dear enemy relationships with their nearby neighbours. These results highlight the potential for convergence in social recognition systems across diverse taxa. © 2016 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Animals discriminate among learned categories of conspecifics territories, which likely pose less of a threat to territory ownership in a diversity of social contexts (Bee, 2006; Beer, 1970; Colgan, 1983; than less familiar, nonterritorial individuals (Temeles, 1994). The Sherman, Reeve, & Pfennig, 1997; Starks, 2004; Wiley, 2013). These ability to discriminate between neighbours and strangers has been categories may be broad and inclusive (e.g. familiar versus unfa- studied most thoroughly in the acoustic modality in songbirds miliar, kin versus nonkin), or they may correspond to specific in- (Falls, 1982; Lambrechts & Dhondt, 1995; Stoddard, 1996), although dividuals. A form of learned category discrimination common it occurs in other sensory modalities and is taxonomically wide- among territorial animals occurs when territory holders display spread, occurring in mammals (delBarco-Trillo, McPhee, & relatively lower levels of aggression towards their established Johnston, 2009; Palphramand & White, 2007), lizards (Husak & neighbours compared with unfamiliar strangers (Temeles, 1994). Fox, 2003a, 2003b), amphibians (Davis, 1987; Jaeger, 1981), fish This so-called ‘dear enemy’ effect (Fisher, 1954) is hypothesized to (Myrberg & Riggio, 1985; Sogawa, Ota, & Kohda, 2016), crustaceans function in allowing territory holders to avoid the costs of repeated (Booksmythe, Jennions, & Backwell, 2010) and insects (Langen, aggressive interactions with the familiar occupants of nearby Tripet, & Nonacs, 2000). Anuran amphibians (frogs and toads) represent a promising taxonomic group for elucidating social and ecological factors that * Correspondence: Y. -C. Kam, Department of Life Science, Tunghai University, promote the evolution of vocally mediated neighbourestranger Taichung 40704, Taiwan; M. A. Bee, Department of Ecology, Evolution, and Behavior, discrimination and its underlying causal mechanisms (Bee, in e University of Minnesota Twin Cities, St Paul, MN 55108, U.S.A. press; Bee, Reichert, & Tumulty, 2016). In addition to being highly E-mail addresses: [email protected] (Y. -C. Kam), [email protected] (M. A. Bee). http://dx.doi.org/10.1016/j.anbehav.2016.11.001 0003-3472/© 2016 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. 218 M.-F. Chuang et al. / Animal Behaviour 123 (2017) 217e228 vocal (Gerhardt & Huber, 2002), anurans exhibit remarkable di- METHODS versity in their social and reproductive behaviours (Wells, 1977, 2007). Even within the same genus, different species exhibit this Study Sites diversity, which can range from nonterritorial scramble competition, to lekking, to the aggressive defence of long-term territories This study was conducted at the Lien-Hua-Chih Research Centre, that contain breeding resources (Wells, 1977, 2007). Territorial Taiwan. We made acoustic recordings of actively calling males be- behaviour is common among male anurans and involves a suite of tween June and September in 2007, 2008 and 2009. These re- vocal and physical behaviours used in contests with intruders. The cordings were made either in a natural forest (12052059.500E, costs of contests in anurans can include reduced mate attraction, 235508.900N) or in a nearby palm farm where areca nuts were physical injury, and increased energy expenditure and predation grown (Areca catechu; also known as betel nuts; 1205301200E, risk (Dyson, Reichert, & Halliday, 2013). Hence, we might expect 235500100N). The two sites were located approximately 600 m territorial individuals to direct reduced levels of aggression towards apart. In the natural forest, males established territories around the their established neighbours, and indeed they do in some species perimeter of a permanent pond (5 10 m, maximum depth of (Davis, 1987). However, only a few previous studies have tested the approximately 1 m; see Figure 1 in Chuang et al., 2013). The site at hypothesis that anurans exhibit dear enemy behaviour. Moreover, the palm farm consisted of numerous shallow (<30 cm depth), results from some of these studies are equivocal because of poor flooded areas where males established territories. This site was also experimental designs, insufficient reporting of data, or inadequate used for conducting playback experiments in 2009. Territorial male consideration of the study species' natural history (reviewed in Bee, olive frogs were abundant at both sites, where they invariably in press; Bee et al., 2016). Answering questions about the mecha- called while floating on the surface of the water, similar to some nisms and evolution of dear enemy behaviour in anurans demands North American ranids (e.g. Howard, 1978; Wells, 1978). additional investigation. We investigated vocally mediated neighbourestranger Sound Recordings discrimination in the olive frog, Babina adenopleura (Ranidae; formerly Rana adenopleura). The olive frog is a common species Advertisement calls were used for acoustic analyses of individ- throughout Southeast Asia and is abundant in Taiwan, where this ual distinctiveness and as stimuli in playback tests. Calls were only study was conducted. The olive frog mating system is resource recorded from males that were not interacting with other con- defence polygyny (Chuang, Bee, & Kam, 2013). Males use a com- specifics at the time recordings were made. Upon selecting a focal bination of distinct vocalizations and physical combat to defend male for recording, we placed a directional microphone (Sennhe- small territories that function as oviposition sites. Males occupy iser ME67) mounted on a tripod approximately 0.5 m away from and defend their territories for an average of 8.4 consecutive days, the male. We recorded calls (44.1 kHz sampling rate, 16-bit reso- although some individuals have been observed to defend the same lution) onto a digital recorder (Marantz PMD670). After completing territory for up to 43 days (Chuang et al., 2013). The vocal reper- a recording, we captured the focal male, measured its body length toire of the male olive frog consists of several different call types (snoutevent length (SVL) to the nearest 0.01 mm) using callipers (Chuang, Kam, & Bee, 2016). Advertisement calls are by far the (Mitutoyo 505-666 dial calliper and Mitutoyo 500-672 digital most common call produced by males (Fig. 1a). As in other frogs, calliper) and its mass (to the nearest 0.05 g) using a portable this call type almost certainly functions both in attracting mates electronic balance (Hiroda MT-300). Measurements of SVL and and repelling rival males (Wells, 1977, 2007). Males also produce mass were used to compute an index of physical condition (i.e. two acoustically distinct types of calls in aggressive contexts that length-independent mass) following Baker (1992). We also recor- involve close-range vocal and physical interactions between ded the water temperature to the nearest 0.1 C at the male's calling competing males (Chuang et al., 2013, 2016). Following convention site using a Tecpel DTM-3108 digital thermometer. (e.g. Toledo et al., 2014; Wells, 2007), these have been termed We used a Tecpel DSL-332 sound level meter to record the territorial calls (Fig. 1b) and encounter calls (Fig. 1c) (Chuang et al., sound pressure level (SPL re 20 mPa, fast RMS, C-weighted) of the 2016). Territorial calls appear to function early in contest escala- calls of 11 males. We positioned the microphone of the sound level tion, whereas encounter calls are used in more highly escalated meter at a distance of 1 m from the frog and recorded the contests. maximum SPL of between 4 and 10 calls for each individual. We The present study had two aims. First, we tested the hypothesis computed the minimum, median and maximum SPLs of the sample that advertisement calls are individually distinct and could function of 11 males based on using individual median values computed as identity signals. Our predictions were that acoustic properties of over the sample of calls recorded from each individual. this signal would be more variable among individuals than within individuals, and that this pattern of individual variation would Individual Vocal Distinctiveness allow calls to be assigned to the correct individual above chance levels in multivariate statistical analyses. Second, we tested the We tested the hypothesis that advertisement calls are individ- hypothesis that territorial males behaviourally discriminate be- ually distinct by analysing patterns of individual variation in their tween the advertisement calls of neighbours and strangers. We acoustic properties. A quantitative description of the call is pro- measured ‘aggressive thresholds’ in response to calls representing vided elsewhere (Chuang et al., 2016). We used Raven Pro 1.4 these two social categories in a field playback experiment. The (Charif, Waack, & Strickman, 2010) to analyse 70 acoustic proper- aggressive threshold was operationally defined as the lowest ties of 899 advertisement calls recorded from 45 males in 2008 (20 stimulus amplitude that elicited aggressive vocalizations calls for each of 44 males and 19 calls for one male). Supplementary (Brenowitz & Rose, 1994; Marshall, Humfeld, & Bee, 2003; Table S1 provides a full description of all 70 acoustic properties, Robertson, 1984; Rose & Brenowitz, 1991). According to a dear which we summarize briefly here. We analysed four temporal enemy hypothesis, we predicted aggressive thresholds would be properties using the entire call as our unit of measurement (Fig. 2), relatively lower (i.e. males would be more aggressive) in response including call period (Fig. 2b), call duration (Fig. 2c), the number of to the calls of a stranger. notes per call (Fig. 2c) and note rate. Twenty-two additional M.-F. Chuang et al. / Animal Behaviour 123 (2017) 217e228 219

(a) Advertisement call

7 6 5 4 3 2

1 kHz 0.5

100 ms (c) Encounter call (b) Territorial call

7 7 6 6 5 5 4 4 3 3 2 2

1 1 kHz 0.5 kHz 0.5

100 ms 100 ms

Figure 1. Vocal repertoire of male olive frogs, Babina adenopleura. Shown here are waveforms (top) and spectrograms (bottom) for (a) an advertisement call, (b) a territorial call and (c) an encounter call. properties were separately measured for the first, middle and last 25 ms windows. All frequency measurements were based on 1024- note of each call. If there was an odd of number of notes, we point fast Fourier transforms and Hann windows and were made randomly selected one of the two middle notes for analysis. Seven from the average power spectrum computed over the duration of of these 22 properties were temporal properties that described the each 25 ms window. amplitude envelope of notes (Fig. 2d) and included note duration, Descriptive statistics for all 70 call properties analysed in this the rise and fall times, the duration of a central plateau of near study are reported in Supplementary Table S2. Temperature vari- steady-state amplitude between two peaks in the amplitude en- ation can have large effects on the measured acoustic properties of velope, and three measures of relative amplitude between the first frog vocalizations (Gerhardt & Huber, 2002). However, water peak, the plateau and the second peak. The remaining 15 of these temperature varied over a narrow range of just 2 C across our 22 properties quantified features of each note's bimodal frequency sample of recordings (range 22.8e24.8 C; X ± SD ¼ 23.9 ± 0.7 C). spectrum (Fig. 2e). We determined the low peak frequency and the In separate linear regression models examining the relationship high peak frequency in the bimodal spectrum, and their relative between water temperature and each acoustic property (see amplitude, in four separate 25 ms windows. These four analysis Table S2), only 2 of 70 properties were significantly related to windows were centred on the first and second peaks of the temperature (relative amplitude in the second 25 ms window of amplitude envelope, the envelope's central plateau and the last the first call note and frequency modulation between the first and portion of each note following the second peak (see Fig. 2d). The second 25 ms windows in the middle call note). We statistically low peak frequency invariably corresponds to the second harmonic adjusted values of these two properties to the mean water tem- of a missing fundamental frequency, whereas the high peak fre- perature following Platz and Forester (1988) prior to all statistical quency typically corresponds to the eighth or ninth harmonic analyses. (Chuang et al., 2016). To describe the frequency modulation present We evaluated the individual distinctiveness of advertisement in advertisement call notes (see Fig. 1a), we also computed the calls as follows. First, we compared the magnitudes of among- differences between the measured values of the high peak fre- individual and within-individual variation in each measured quency in the first 25 ms window and each of the subsequent three acoustic property. As descriptive measures of individual variation, 220 M.-F. Chuang et al. / Animal Behaviour 123 (2017) 217e228

(a)

10 s Call period

(b)

2 s

Call duration Note duration

(c)

50 ms

1st Peak 2nd Peak Rise Fall Plateau time time

(d) 2 3 25 ms 1

Four 25 ms windows for sampling spectral properties

(e) 110 Relative amplitude 105 100 95 90 High peak frequency

Amplitude (db) 85 80 Low peak frequency 75 0.05 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Frequency (kHz)

Figure 2. Schematic illustration of analyses of spectral and temporal properties of advertisement calls. See Table S1 for a complete description of measured call properties. (a) A series of advertisement calls produced by a territorial male. (b) A subset of two calls from panel (a) illustrating the measurement of call period. (c) A three-note call from panel (b) illustrating the measurement of call duration and note duration. (d) A single note from the call in panel (c) illustrating the measurements of several features of the amplitude envelope. Also shown in panel (d) is the relative timing of the four 25 ms windows over which some properties were measured. Note the different timescales for panels (a)e(d). (e) The power spectrum of a single advertisement call illustrating the measurement of several spectral properties, including low peak frequency, high peak frequency, and their relative amplitude.

we report coefficients of variation (CV ¼ 100% jSD/Xj). Within- CVw values computed over 45 individuals. Among-individual vari- individual variability (CVw) was computed separately for each in- ability (CVa) was calculated using the mean and standard deviation dividual using the mean and standard deviation calculated over the of the 45 individual means. The ratio of among-individual to 19 or 20 calls recorded from that individual. We report the mean of within-individual variability (CVa/CVw) was computed to compare M.-F. Chuang et al. / Animal Behaviour 123 (2017) 217e228 221 the relative variability among and within individuals. We also used (areca palm farm), the playback experiment was conducted; model II ANOVAs to determine whether among-individual varia- therefore, we assume that these stranger stimulus-donor males tion was significantly greater than within-individual variation. We were unfamiliar to our subjects. Each stimulus consisted of two caution that these ANOVAs should not be regarded as independent advertisement calls, each composed of three or four notes, sepa- due to intercorrelations among call properties (Beecher, 1989b). rated by a 20 s intercall interval, which is typical for this species Second, we used a discriminant function analysis (DFA) both to (Table S2). All stimuli used for neighbour and stranger playbacks estimate the success rate for classifying calls to the correct in- were created and edited as .wav files (44.1 kHz sampling rate,16-bit dividuals and to evaluate the contribution of different acoustic resolution) using Adobe Audition v.1.5. Stimuli were broadcast us- properties towards successful classification. Values for each ing Adobe Audition v.1.5 running on an ASUS Eee PC 900 computer. acoustic property were first standardized by converting them to z The output of the computer's sound card was broadcast through a scores and then subjected to a principal component analysis (PCA) Mineroff SME-AFS portable field speaker. Prior to commencing to create a new set of orthogonal variables (see Supplementary each playback test, we calibrated the SPL of the selected acoustic Table S3). We used principal component scores from all factors stimulus to be 94 dB at a distance of 1 m. These calibrations were having eigenvalues greater than 1.0 as the input variables for the performed at the field site in habitat similar to that from which the DFA (see Supplementary Table S4). The use of PCA in this context chosen subject called, but well away from the subject to avoid served as a data-reduction tool to ensure subsequent analyses were eliciting aggressive behaviour. focused on a subset of uncorrelated variables. Results from the PCA Playback tests for both treatment groups were conducted as and DFA are summarized in Tables S3 and S4, respectively. Classi- follows. We first identified an actively calling subject with at least fication success was computed using a cross-validation procedure one nearby, actively calling neighbour within approximately 3 m. and prior probabilities that were proportional to the number of Males that were more isolated from other calling males were not calls recorded per male. The methods outlined by Titus, Mosher, used as subjects; based on previous work in this system, males and Williams (1984) were used to determine whether our probably do not interact with other males that establish territories observed classification success significantly exceeded that expected greater than 3 m away (Chuang et al., 2013). Next, we removed the by chance alone. We also used linear regressions, with sequential neighbour, marked and measured it as described above, and Bonferroni corrections (Rice, 1989) for multiple comparisons, to temporarily restrained it in a plastic container with a small amount evaluate the relationships between principal component scores and of water for the short duration of the playback test to prevent it SVL, mass and condition. from calling. We also marked the neighbour's exact calling site with Finally, we followed procedures outlined by Beecher (1989b) to a piece of flagging tape. The playback speaker was positioned on a compute the total information capacity (Hs, in bits) of advertise- floating Styrofoam platform and placed approximately 1 m from a ment calls with respect to individual identity. These computations subject along a straight line connecting the subject and the were based on the results of all significant model II ANOVAs con- neighbour's marked calling site. We also placed the Sennheiser ducted on scores obtained for all 70 principal components resulting microphone 50e100 cm from the subject so that its vocal responses from the PCA. Given the large number of call properties analysed, could be monitored over headphones and recorded using the we also conducted this analysis using only those principal com- Marantz recorder. ponents with an eigenvalue greater than 1.0 to derive a more Once the subject resumed what appeared to be normal calling conservative estimate of information capacity. behaviour following placement of the recording microphone (usually within 5e10 min), we monitored its vocalizations during a NeighboureStranger Discrimination 2 min prestimulus period to ensure that it was not interacting aggressively with other frogs. Immediately following this presti- We tested the hypothesis that territorial males behaviourally mulus period, we began repeated playbacks of the selected stim- discriminate between the advertisement calls of neighbours and ulus on a 1 min loop. The initial broadcast of the stimulus was strangers in a field playback experiment. Playback trials were attenuated in software by 24 dB to achieve a broadcast SPL of conducted at night (between 2000 and 0300 hours) during August approximately 70 dB at 1 m. Preliminary trials had indicated that and September of 2009. A between-subjects design was used to this SPL would be well below subjects' aggressive thresholds. measure aggressive thresholds in response to broadcasts of the During the silent interval following each stimulus presentation on calls of a subject's nearest neighbour or a stranger. Both types of the 1 min loop, we increased the SPL of the next stimulus by 2 dB. calls were broadcast from the direction of the neighbour's territory. So, for example, at the start of the eighth minute of playbacks, the Subjects were assigned randomly to the neighbour or stranger stimulus SPL had increased from approximately 70 dBe84 dB. We treatment groups with the caveat that there be an equal number of continued increasing the amplitude of the 1 min loop stimulus in subjects in each group (total N ¼ 20). As reported in Supplementary 2 dB steps until the frog responded with one or more territorial Table S5, there were no differences between subjects assigned to calls, encounter calls, or both. the neighbour and stranger treatment groups in terms of the water Subjects typically gave territorial calls at lower playback SPLs temperature at the time of testing, SVL, mass, condition, speaker than those required to elicit encounter calls. We attempted to distance or nearest neighbour distance. continue playbacks in the manner just described until each subject Each subject was tested with a unique neighbour or stranger had responded with both territorial calls and encounter calls, but stimulus to avoid potential problems of pseudoreplication and this was not always possible due to the technical limitations of our external validity associated with other playback designs playback set-up. (We were unable to reproduce amplitudes in (Kroodsma, 1989a, 1989b, 1990; Searcy, 1989). The stimuli used in excess of about 103 dB without introducing considerable harmonic neighbour playback trials were generated using calls recorded as distortion.) We ended a trial once the animal responded aggres- described above from each subject's nearest neighbour on the night sively with encounter calls or once we reached the technical limit of of the playback test prior to commencing the test. The stimuli used our playback system. We noted the attenuation levels at which in stranger playback trials were generated using calls recorded in subjects gave territorial calls and encounter calls. If the subject was 2007 or 2008 from marked and measured individuals calling in the not already marked, we captured, marked and measured it as permanent pond. These males were neither recaptured nor described above. We used a tape measure to determine the distance observed calling during the year (2009), or at the place where between the subject's original calling site and both the playback 222 M.-F. Chuang et al. / Animal Behaviour 123 (2017) 217e228 speaker and the calling site of its nearest neighbour. The Tecpel marked. Marked individuals typically resumed normal activities sound level meter was used to measure the actual SPL of the (e.g. calling) within 5 min of being released. playback stimulus from the location of the subject's calling site using the attenuation settings that elicited territorial calls and RESULTS encounter calls. These SPL measurements (in dB) represent our response variables. Similar SPL measurements have been used Individual Vocal Distinctiveness previously to assess aggressive thresholds in other frogs (Brenowitz & Rose, 1994; Marshall et al., 2003; Robertson, 1984; Rose & Values of the CVa/CVw ratio were typically greater than 1.0, and Brenowitz, 1991). each acoustic property varied significantly more among individuals We report both median and mean thresholds below. Because the than within individuals (all Ps < 0.001; Table 1). Values of partial h2, data did not meet the requisite assumptions for using parametric which represent the size of the effect of individual identity in these tests, ManneWhitney U tests were used to separately compare analyses, ranged between 0.10 and 0.85. The spectral properties of median thresholds for eliciting territorial calls and encounter calls high peak frequency and low peak frequency had the highest values between the neighbour and stranger treatment groups. We used of partial h2 (means of 0.69 and 0.59, respectively) and the greatest Spearman rank correlations, with sequential Bonferroni corrections CVa/CVw ratios (means of 2.0 and 2.6, respectively) (Table 1). These (Rice, 1989) for multiple comparisons, to evaluate the relationships two spectral properties also exhibited the lowest variation within between thresholds and SVL, mass, condition, distance to the individuals, with mean CVw values of 5.6% and 3.3%, respectively, speaker and distance to the nearest neighbour's calling site. We also compared to a mean CVw of 38.9% averaged over all other proper- compared the acoustic properties listed in Table S1 between the ties combined. The spectral properties of relative amplitude and neighbour and stranger stimuli to evaluate the possibility that frequency modulation also had high values of partial h2 (means of acoustic differences unrelated to individual identity might be 0.59 and 0.37, respectively) and CVa/CVw ratios (median of 2.0 for correlated with differences in aggressive thresholds elicited by relative amplitude, to reduce the influence of one outlier, and a these two types of calls. We subjected standardized values (z mean of 1.1 for frequency modulation) (Table 1). With mean CVw scores) to a PCA to create a set of orthogonal variables. We used values of 44.0% and 88.4%, respectively, relative amplitude and ManneWhitney U tests to compare principal component scores frequency modulation were much more variable within individuals between the neighbour and stranger stimuli for each PCA factor compared with other spectral properties. Among temporal prop- having an eigenvalue greater than 1.0. None of these analyses erties, those of calls (call duration, note number and note rate; 2 returned significant results (see Supplementary Table S6). Hence, Table 1) had the highest mean partial h (0.52) and CVa/CVw ratio any observed differences in aggressive thresholds in response to (1.6), followed by the temporal properties of individual notes (note neighbour and stranger stimuli could not be attributed to inherent duration, rise time, fall time, plateau duration, envelope amplitude 2 acoustic differences between these stimuli aside from those arising ratios; mean partial h ¼ 0.48; mean CVa/CVw ratio ¼ 1.5; Table 1). from differences in the individual identity and social category The PCA returned 19 factors with eigenvalues greater than 1.0, (neighbour versus stranger) of the stimulus donors. accounting for 74.6% of the variation in the original call variables (Table S3). In the DFA, the first 10 functions had eigenvalues greater Ethical Note than 1.0 and accounted for 94.0% of the variation in PCA scores (Table S4). Examination of the confusion matrix from the DFA This study was conducted under permit (no. revealed that 96.4% of advertisement calls were correctly classified TFRILHC0950001568) from the Lien-Hua-Chih Research Centre of as belonging to the individual that produced them (N ¼ 45). This the Taiwan Forestry Research Institute. No other permits were level of correct classification was significantly greater than ex- required because these field studies were not conducted in a pro- pected by chance (1/45 or 2.2%; Cohen's Kappa ¼ 0.963; 95% con- tected area nor were our subjects an endangered or protected fidence interval ¼ 0.951e0.976; Z ¼ 191.6, P < 0.001). species. All methodological procedures complied with legal re- An inspection of the factor loadings from the PCA and DFA quirements in Taiwan and were reviewed and approved by the (Tables S3 and S4, Fig. 3) indicated that the spectral properties of Experimental Animal Care and Use Committee of Tunghai Univer- high peak frequency and low peak frequency contributed most sity (no. 100e19) as well as the Institutional Animal Care and Use towards the classification of calls to the correct individuals. The first Committee of the University of Minnesota (no. 0809A46721). In canonical root from the DFA, which explained 24.3% of the variation total, 72 male frogs were used as subjects in this study. Individual in PCA scores, loaded most heavily on PCA factor 1, which itself identification of subjects was desirable to avoid pseudoreplication explained 10.7% of the variation in the original call variables and arising from unknowingly recording or testing the same individual loaded heavily on high peak frequency (Fig. 3). The second ca- repeatedly. Unfortunately, physical markings, such as dorsal colour nonical root from the DFA explained 20.6% of the variation in PCA patterns, proved inadequately distinct to allow accurate individual scores and loaded most heavily on PCA factor 2, which explained identification of subjects. Therefore, each subject was toe-clipped 10.0% of the variation in the original call variables and loaded most and temporarily marked using a numbered waistband. Our toe- heavily on low peak frequency (Fig. 3). PCA factor 2, which was clipping procedures followed the Guidelines for use of live amphib- positively correlated with low peak frequency (Table S3), was ians and reptiles in field and laboratory research (HACC, 2004; see significantly negatively correlated with SVL, mass and condition also; Perry, Wallace, Perry, Curzer, & Muhlberger, 2011). Temporary (Table 2). This result indicates that both larger males and males in waistbands were made from small (1.5 cm diameter) circular pieces better condition had lower values of low peak frequency. Other PCA of waterproof paper tied to a cotton thread. These waistbands did factors were not related to body size or condition (Table 2). The not appear to impede the animal's normal behaviour and typically third canonical root, which explained an additional 14% of the came loose within 2 weeks. Temporary waistbands allowed us to variation in PCA scores, loaded almost equally on PCA factors 3 and assess subject identification at a distance so as to avoid disturbing 5, both of which explained about 5% of the variation in the original the animal while also reducing the risk of wounds caused by long- call variables (Fig. 3). PCA factor 3 loaded most heavily on temporal lasting waistbands. We removed waistbands from individuals that properties related to the note structure within calls, including note could be relocated at the end of the study. All animals were released rate and note duration, whereas PCA factor 5 loaded most heavily at their site of capture within 10e30 min of being captured and on two temporal features of the amplitude envelope (amplitude M.-F. Chuang et al. / Animal Behaviour 123 (2017) 217e228 223

Table 1

Coefﬁcients of variation (CV) for 70 acoustic properties of advertisement calls measured within individuals (CVw) and among individuals (CVa), the ratio of among-individual to within-individual variation (CVa/CVw), and results from model II ANOVAs (all Ps < 0.001). Data are shown separately for calls and ﬁrst, middle and last notes

2 Property CVw CVa CVa/CVw F44,854 Partial h Call or Middle Last Call or Middle Last Call or Middle Last Call or Middle Last Call or Middle Last first note note note first note note note first note note note first note note note first note note note

Call period 25.7 ee27.0 ee1.1 ee3.2 ee0.14 ee Call duration 14.5 ee18.5 ee1.3 ee28.4 ee0.59 ee Note per call 12.8 ee20.8 ee1.6 ee40.1 ee0.67 ee Note rate 3.5 ee 7.9 ee2.2 ee41.1 ee0.68 ee Note duration 6.5 5.9 5.7 9.4 10.7 10.5 1.5 1.8 1.8 30.0 29.8 20.7 0.61 0.61 0.52 Note rise time 36.2 16.6 19.3 50.7 28.2 30.6 1.4 1.7 1.6 17.6 5.6 5.8 0.48 0.22 0.23 Plateau duration 9.3 16.3 16.6 19.9 27.0 29.4 2.1 1.7 1.8 34.0 25.7 33.5 0.64 0.57 0.63 Note fall time 42.3 53.3 46.3 19.6 16.7 18.8 0.5 0.3 0.4 4.2 2.5 2.3 0.18 0.12 0.11 Amplitude ratio 1 14.1 14.1 18.9 25.1 23.7 17.7 1.8 1.7 0.9 39.2 30.6 10.7 0.67 0.61 0.36 Amplitude ratio 2 13.1 13.2 18.7 35.3 22.2 22.3 2.7 1.7 1.2 90.3 32.7 13.5 0.82 0.63 0.41 Amplitude ratio 3 16.1 11.8 16.8 34.7 15.5 15.2 2.2 1.3 0.9 62.4 22.8 15.1 0.76 0.54 0.44 Low peak frequency 1 6.8 3.5 3.5 14.0 7.4 7.7 2.1 2.1 2.2 18.7 53.4 39.9 0.49 0.73 0.67 Low peak frequency 2 2.7 2.4 2.7 7.3 7.9 7.7 2.7 3.3 2.8 58.7 2.2 74.3 0.75 0.10 0.79 Low peak frequency 3 2.7 2.4 2.8 7.9 7.4 7.8 2.9 3.1 2.8 65.8 2.2 14.1 0.77 0.10 0.42 Low peak frequency 4 2.7 2.8 4.3 8.0 7.8 8.1 3.0 2.8 1.9 75.7 59.6 48.7 0.80 0.75 0.72 High peak frequency 1 6.8 3.9 4.7 7.7 8.3 7.8 1.1 2.1 1.7 15.8 61.0 43.5 0.45 0.76 0.69 High peak frequency 2 4.9 5.0 5.5 9.7 9.2 9.2 2.0 1.8 1.7 50.2 54.4 37.8 0.72 0.74 0.66 High peak frequency 3 2.9 3.3 3.7 10.1 10.5 9.8 3.4 3.2 2.7 108.2 58.3 63.0 0.85 0.75 0.76 High peak frequency 4 9.0 7.0 10.4 13.6 12.7 14.9 1.5 1.8 1.4 25.3 41.1 31.1 0.57 0.68 0.62 Relative amplitude 1 64.9 28.4 46.5 105.3 42.1 49.9 1.6 1.5 1.1 16.6 24.5 16.7 0.46 0.56 0.46 Relative amplitude 2 48.0 34.5 54.9 131.9 93.5 89.3 2.7 2.7 1.6 34.1 42.3 19.8 0.64 0.69 0.50 Relative amplitude 3 29.9 32.0 48.3 57.4 76.3 83.5 1.9 2.4 1.7 34.2 54.3 27.7 0.64 0.74 0.59 Relative amplitude 4 59.6 13.2 67.8 199.1 335.7 145.1 3.3 25.5 2.1 28.6 36.6 24.1 0.60 0.65 0.55 Frequency modulation 1 86.7 113.3 144.9 112.5 123.6 105.9 1.3 1.0 0.7 13.2 9.0 7.6 0.41 0.32 0.28 Frequency modulation 2 73.5 92.1 103.4 74.6 81.3 103.7 1.0 0.9 1.0 13.2 5.6 9.8 0.40 0.22 0.34 Frequency modulation 3 68.9 54.6 58.2 68.2 86.7 79.3 1.0 1.6 1.4 11.6 21.9 16.4 0.37 0.53 0.46

ratio 1 and 2), and a feature of the spectral envelope (relative mean thresholds was 4.2 dB (Fig. 4a). The lowest stimulus level that amplitude 2). Each of the remaining canonical roots from the DFA elicited territorial calls (see lower-bound whiskers in Fig. 4a) in explained less than 10% of the variation in PCA scores. response to strangers (83.9 dB) was 7.2 dB lower than the lowest The total information capacity (HS) of advertisement calls was stimulus level eliciting territorial calls in response to neighbours 7.3 bits, suggesting these signals could uniquely identify an upper (91.1 dB). In contrast to thresholds for eliciting territorial calls, there limit of 158 (27.3) different individuals, assuming an ideal receiver was no significant difference in the median threshold for eliciting (Beecher, 1989b). In our more conservative analysis of information encounter calls in response to neighbours (98.2 dB; X ¼ 97.4 dB) capacity based on principal components with eigenvalues greater and strangers (97.3 dB; X ¼ 96.0 dB) (ManneWhitney U test: than 1.0, the information capacity was estimated to be 3.9 bits, Z ¼0.39, P ¼ 0.6982, N1 ¼8, N2 ¼ 6,; Fig. 4a). The differences in which would correspond to an upper limit of 15 individuals, again median and mean thresholds were less than 1 dB and 1.5 dB, assuming an ideal receiver. respectively. However, the minimum threshold (see lower-bound whiskers in Fig. 4a) for responding to strangers with encounter NeighboureStranger Discrimination calls (86.2 dB) was 4.9 dB lower than the minimum threshold for responding to neighbours (91.1 dB). All 20 subjects responded to playbacks by producing territorial The significant difference in thresholds for responding to calls, and 14 of 20 subjects also produced encounter calls before we neighbours and strangers with territorial calls can be placed in a reached the technical limits of our playback system. The median biological context using a simple visual model that jointly considers stimulus levels required to elicit territorial calls and encounter calls the SPLs of natural advertisement calls, sound attenuation in the were 94.5 dB and 97.4 dB, respectively. Aggressive thresholds for frog's habitat and the distances that typically separate adjacent territorial calls ranged between 83.9 dB and 100.7 dB (X ¼ 94.8 dB; territorial males. The median SPL of advertisement calls recorded at N ¼ 20), and those for encounter calls ranged between 86.2 dB and 1 m was 87.0 dB and ranged between 84.7 dB and 90.4 dB (N ¼ 11 101.6 dB (X ¼ 96.8 dB; N ¼ 14). Thresholds for responding with individuals). This range is depicted as the two black circles in encounter calls were negatively correlated with nearest-neighbour Fig. 4b. This figure also illustrates the expected SPLs of calls over a distance (Table 3), such that males with closer neighbours also had range of behaviourally relevant distances from the source assuming higher thresholds for responding aggressively with encounter calls, signal attenuation due only to spherical spreading (Fig. 4b). This as reported in some other frogs (Marshall et al., 2003; Rose & assumption is justified based on field measurements of frequency- Brenowitz, 1991). No other correlations between aggressive dependent attenuation in a study of North American bullfrogs thresholds and measures of body size, speaker distance or nearest- (Rana catesbeiana, Ranidae). Like olive frogs, bullfrogs call while neighbour distance were statistically significant. floating on the surface of the water. In a sound transmission study, The median threshold for eliciting territorial calls was signifi- the SPL of bullfrog calls, as well as pure tones with frequencies cantly lower in response to the calls of strangers compared with the spanning the range of those contained in olive frogs calls, decreased calls of neighbours (ManneWhitney U test: Z ¼2.12, P ¼ 0.0343, by 6 dB with each doubling of distance in the animal's natural N1 ¼ N2 ¼ 10; Fig. 4a). The median threshold in response to habitat, consistent with attenuation due to spherical spreading strangers (94.3 dB; X ¼ 92.7 dB) was 3.7 dB lower than that in (Boatright-Horowitz, Cheney, & Simmons, 1999). Estimates of response to neighbours (98.0 dB; X ¼ 96.9 dB); the difference in signal attenuation due to spherical spreading loss can be combined 224 M.-F. Chuang et al. / Animal Behaviour 123 (2017) 217e228

0.76 First note: High peak frequency 2

0.90 First note: High peak frequency 3

0.87 Middle note: High peak frequency 1

PCA 0.81 Middle note: High peak frequency 2 DFA –0.89 High peak CR 1 Factor 1 (10.7%) frequency (24.3%) 0.87 Middle note: High peak frequency 3

0.82 Last note: High peak frequency 1

0.81 Last note: High peak frequency 2

0.88 Last note: High peak frequency 3

0.82 First note: Low peak frequency 2

0.83 First note: Low peak frequency 3

0.85 First note: Low peak frequency 4

0.74 PCA Middle note: Low peak frequency 1 DFA –0.79 Low peak CR 2 Factor 2 Discriminant function analysis 0.85 frequency (20.6%) (10.0%) Middle note: Low peak frequency 4

0.79 Last note: Low peak frequency 1

0.85 Last note: Low peak frequency 2

0.76 Last note: Low peak frequency 4

0.73 Call: Note rate

PCA –0.76 First note: Note duration Factor 3 Note rate/ (4.9%) –0.84 Note duration Middle note: Note duration

0.75 –0.74 DFA Last note: Note duration CR 3 (14.0%) –0.76 –0.85 Middle note: Amplitude ratio 1 PCA –0.78 Amplitude ratio/ Factor 5 Middle note: Amplitude ratio 2 (4.9%) Relative amplitude 0.72 Middle note: Relative amplitude 2

Figure 3. The combined results of a principal component analysis (PCA) of analysed call properties and a subsequent discriminant function analysis (DFA) of principal component scores. The tree structure illustrated here is a visual representation of standardized factor loadings of canonical roots (CR1eCR3) from the DFA onto factors from the PCA, and of the PCA factors onto the original acoustic properties measured. Estimates of the percentage of variation explained are provided in parentheses. The complete results from the PCA and DFA are included in Supplementary Tables S3 and S4, respectively. with measures of aggressive thresholds for territorial calls to model for each treatment and the maximum individual SPL from our the distances over which subjects respond aggressively towards sample of 11 recorded males. The resulting estimated distances, neighbours and strangers. For both the neighbour and stranger thus, simulate interactions between a highly aggressive territorial ‘response zones’ depicted in Fig. 4b, the zone's upper bound was resident and an intruder that produces high-amplitude calls. Lower determined as the intersection of the lowest aggressive threshold bounds for each response zone were determined as the intersection M.-F. Chuang et al. / Animal Behaviour 123 (2017) 217e228 225

Table 2 Results from linear regressions of principal component scores on snout-to-vent length (SVL), mass and condition

Factor SVL Mass Condition

2 2 2 b R F1,43 P b R F1,43 P b R F1,43 P 1 0.02 <0.01 <0.1 0.89 0.00 <0.01 <0.1 0.99 0.02 <0.01 <0.1 0.88 2 0.42 0.18 9.1 <0.01* 0.62 0.39 27.2 <0.01* 0.48 0.23 12.6 <0.01* 3 0.01 <0.01 <0.1 0.96 0.04 <0.01 <0.1 0.78 0.06 <0.01 0.2 0.69 4 0.17 0.03 1.3 0.26 0.10 0.01 0.4 0.52 0.06 <0.01 0.2 0.68 5 0.35 0.12 5.9 0.02 0.16 0.03 1.2 0.29 0.18 0.03 1.5 0.23 6 0.11 0.01 0.6 0.46 0.20 0.04 1.7 0.20 0.18 0.03 1.4 0.25 7 0.13 0.02 0.8 0.39 0.20 0.04 1.8 0.19 0.16 0.02 1.1 0.30 8 0.01 <0.01 <0.1 0.96 0.12 0.01 0.6 0.43 0.19 0.04 1.6 0.21 9 0.21 0.04 2.0 0.16 0.27 0.07 3.4 0.07 0.17 0.03 1.3 0.26 10 0.05 <0.01 0.1 0.74 0.07 <0.01 0.2 0.65 0.18 0.03 1.4 0.24 11 0.20 0.04 1.8 0.19 0.13 0.02 0.7 0.40 0.05 <0.01 0.1 0.76 12 0.20 0.04 1.7 0.20 0.20 0.04 1.9 0.18 0.08 0.01 0.3 0.60 13 0.17 0.03 1.3 0.26 0.22 0.05 2.1 0.15 0.13 0.02 0.7 0.40 14 0.07 <0.01 0.2 0.65 0.14 0.02 0.9 0.35 0.14 0.02 0.9 0.35 15 0.11 0.01 0.6 0.45 0.09 0.01 0.3 0.57 0.01 <0.01 <0.1 0.96 16 0.15 0.02 0.9 0.34 0.09 0.01 0.4 0.55 0.04 <0.01 <0.1 0.80 17 0.06 <0.01 0.2 0.70 0.01 <0.01 <0.1 0.97 0.07 <0.01 0.2 0.66 18 0.05 <0.01 0.1 0.72 0.05 <0.01 0.1 0.75 0.15 0.02 1.0 0.33 19 0.10 0.01 0.5 0.50 0.09 0.01 0.4 0.54 0.28 0.08 3.8 0.06

P values marked with an asterisk (*) were signiﬁcant following a sequential Bonferroni correction for multiple comparisons.

Table 3 Results of Spearman rank correlations between aggressive thresholds for territorial present study extends this body of work by describing the patterns calls (N ¼ 20) and encounter calls (N ¼ 14) and snout-to-vent length (SVL), mass, of individual variation in the advertisement calls of a territorial condition and distances between the subject's calling site and the speaker and the species that also behaviourally discriminates among individuals calling site of the nearest neighbour based on learning about this variation. The success of multivariate Territorial calls Encounter calls statistics in assigning calls to the correct individuals was quite high rPrP(96.1% correct classification). The spectral properties of low peak frequency and high peak frequency contributed most towards suc- SVL 0.03 0.890 0.13 0.658 fi Mass 0.05 0.845 0.57 0.034 cessful classi cation. Low peak frequency, which invariably corre- Condition 0.03 0.895 0.55 0.044 sponds to the second harmonic of the fundamental frequency Speaker distance 0.17 0.487 0.06 0.241 (Chuang et al., 2016), was negatively related to body size and con- Neighbour distance 0.25 0.298 0.68 0.008* dition. In frogs, fundamental frequency is highly dependent on the P values marked with an asterisk (*) were significant following a sequential Bon- length and mass of the vocal folds (Martin, 1971), which in turn are ferroni correction for multiple comparisons. usually related to overall body size. Thus, individual differences in size are a potentially important source of individual differences in of the highest aggressive threshold for each treatment and the spectral properties correlated with fundamental frequency (Bee & minimum individual SPL from our sample of 11 recorded males, Gerhardt, 2001b; Bee, Kozich, Blackwell, & Gerhardt, 2001). thereby simulating interactions between a tolerant territory resi- The total information capacity of advertisement calls (7.3 bits and dent and an intruder producing low-amplitude calls. Based on this 158 individuals, or more conservatively, 3.9 bits and 15 individuals), model, the response zone over which strangers are able to elicit a measure that assumes an ideal receiver, far exceeds the typical aggressive calling from territory holders extends out to about 2.1 m number of neighbours a territorial male might have at any one time. and is approximately twice the size of the corresponding response Based on previous observations (Chuang et al., 2013), territory res- zone for neighbours (Fig. 4b). As illustrated in Fig. 4b, the modelled idents typically have between zero and six neighbours (median ¼ 2 upper bound of the stranger response zone corresponds almost neighbours; see Figure 1 in Chuang et al., 2013). Hence, the infor- fi exactly to the nearest-neighbour distances measured in the eld mation content of advertisement calls is nearly one to two orders of ± ¼ (2.12 0.67 m; N 20 individuals; Table S5). magnitude higher than would seem, on the surface, to be necessary to meet the much more modest demands of recognizing the voices DISCUSSION of a small number of neighbours. This apparent ‘over identification’ may be necessary, however, to enable robust discrimination under This study yielded two main findings. First, advertisement calls natural conditions. Signals become degraded as a result of propa- are individually distinct. Second, territorial males have higher gation in the environment (Richards & Wiley, 1980; Wiley & thresholds for responding aggressively towards neighbours Richards, 1978), and ambient noise can mask important signal compared with strangers. Together, these findings confirm that two components (Brumm & Slabbekoorn, 2005). Moreover, receivers are key elements of a functional social recognition system for neigh- not ideal, as the measure of total information capacity assumes, bourestranger discrimination are present in a territorial frog (Bee, in because they must decode identity information using perceptual press; Bee et al., 2016): individually distinctive signals (or ‘signa- systems with limited resolution (e.g. just-noticeable differences; tures’) and the ability to perceptually and behaviourally discriminate Akre & Johnsen, in press; Nelson & Marler, 1990). Placing our esti- among signals produced by different categories of individuals. In the mate of total information capacity into a broader, comparative context of territorial aggression, the social recognition system iden- context, as in studies of parenteoffspring recognition in swallows tified here provides a functional basis for the dear enemy effect. (Beecher, 1982, 1989a), is currently impossible with frogs because A general consensus is emerging that anuran advertisement calls we lack estimates of the individual identity information for other can function as identity signals (reviewed in Bee et al., 2016). The species. 226 M.-F. Chuang et al. / Animal Behaviour 123 (2017) 217e228

104 chorus settings (Brenowitz & Rose, 1994; Marshall et al., 2003). (a) P = 0.0343 P = 0.6982 Results from our visual model using sound propagation, measured 102 call amplitudes and empirically determined aggressive thresholds to 100 estimate the size of response zones for neighbours and strangers 98 indicated that strangers elicit aggressive responses over greater distances. Interestingly, the upper bound of the stranger response 96 3.7 dB zone corresponded closely with the typical distance separating two 94 neighbouring males. Our interpretation of this finding is that 92 established territory holders use aggressive signals and other be- ‘ ’ 90 haviours to prevent new neighbours , which are initially unfamiliar, 4.9 dB from calling too closely. Eventually, they become more tolerant as 88 7.2 dB the neighbour becomes more familiar and is perceived as posing 86 Response threshold (dB SPL) little threat to territory ownership. Our results on territorial calls are, 84 therefore, consistent with the prediction that neighbouring territorial male olive frogs can behave as dear enemies. 82 Neighbour Stranger Neighbour Stranger In contrast to territorial calls, median aggressive thresholds for fi Territorial call Encounter call encounter calls did not differ signi cantly in response to neighbours versus strangers. However, the minimum threshold for evoking encounter calls was nearly 5 dB lower in response to (b) strangers compared with neighbours, a result consistent with dear enemy recognition. At present, we can only speculate as to why 106 dear enemy relationships might be more pronounced in the context NNeighboureighbour resresponseponse zozonene of territorial calling versus encounter calling. Recall that territorial 100 StrangerStranger responseresponse zone calls appear to function early in contest escalation, for example to maintain minimum intermale spacing, whereas encounter calls are 94 used in more highly escalated contests that may also involve 90.490.4 dB physical aggression (Chuang et al., 2013, 2016). This functional 88 distinction is supported by the relatively higher aggressive 84.7 dB thresholds for eliciting encounter calls measured in the present 82 study. Therefore, regardless of a male's status as a familiar neighbour or a stranger, individuals that intrude too closely appear to Sound pressure level (dB) evoke the same, more escalated aggressive response. This pattern 76 of results indicates that the cognitive mechanisms underlying dear enemy relationships in olive frogs are sufficiently sophisticated to 70 0 0.5 1 1.5 2 2.5 3 provide for some degree of functional context dependence. Only a few previous experimental studies of frogs have investi- Distance from source (m) gated a vocally mediated dear enemy effect (reviewed in Bee, in Figure 4. Neighbourestranger discrimination by territorial males of the olive frog, press; Bee et al., 2016). Territorial male bullfrogs (R. catesbeiana), Babina adenopleura. (a) Aggressive thresholds (measured in dB sound pressure level exhibit dear enemy behaviours similar to those reported in songbirds (SPL)) for territorial calls (left) and encounter calls (right). Box plots depict the median (Falls, 1982; Lambrechts & Dhondt, 1995; Stoddard, 1996). They (line, square), interquartile range (box) and range (whiskers) of the lowest stimulus SPLs that were required to elicit territorial calls and encounter calls in response to the respond less aggressively to playbacks of the calls of an adjacent calls of neighbours (N ¼ 10) and strangers (N ¼ 10). P values are from ManneWhitney territorial neighbour coming from the direction of the neighbour's U tests. (b) A visual model translating aggressive thresholds (dB SPL) into response usual territory compared with playbacks of both the calls of strangers zones (in metres). This model relates the range of SPLs of olive frog advertisement calls from the neighbour's location and the calls of the neighbour from as a function of spherical spreading loss (grey region) to the size of response zones outside its usual territory (Davis, 1987). Bullfrog advertisement calls computed for neighbours and strangers (see text for computations) and to distances separating adjacently territorial males. Black circles depict the upper and lower SPLs are individually distinct, and, as we found with olive frogs, size- measured from a sample of 11 males recorded at a distance of 1 m. Blue and red circles related variation in fundamental frequency and correlated spectral depict the intersections of either the highest signal SPL with the lowest aggressive properties contribute most towards the statistical discrimination threshold, or the lowest signal SPL with the highest aggressive threshold, which were among individuals (Bee, 2004; Bee & Gerhardt, 2001b). Field play- used to map out the upper and lower bounds of the corresponding neighbour and back experiments using the habituationediscrimination paradigm stranger response zones (striped bars indicate overlapping zones). Black and white square and horizontal error bars depict the mean ± SD nearest-neighbour distance have shown that male bullfrogs can learn about individually (N ¼ 20). distinctive acoustic properties of a neighbour's advertisement calls, such as fundamental frequency, as well as the neighbour's usual territorial location, as a result of repeatedly hearing its calls coming In our playback experiment, thresholds for eliciting territorial from a fixed location across a newly established boundary (Bee, fi calls were signi cantly higher in responses to neighbours' calls than 2003a; Bee & Gerhardt, 2001a, 2001b, 2002; Bee et al., 2001). Par- in responses to strangers' calls. This result unequivocally demon- allel studies should investigate the perceptual and cognitive bases of strates the ability of territorial male olive frogs to perceptually and neighbourestranger discrimination in olive frogs, including its behaviourally discriminate between the calls of neighbours and location specificity. strangers. Because received sound amplitude is inversely related to Studies of the dear enemy effect in other anuran species have source distance (Boatright-Horowitz et al., 1999), lower thresholds been more preliminary in nature and have been plagued by insuf- in responses to strangers' calls are also indicative of a lower will- ficient natural history data or a variety of methodological issues ingness on the part of territory holders to tolerate encroachment by that complicate interpretations of their results (reviewed in Bee, in an unfamiliar male. This interpretation is based on earlier work press; Bee et al., 2016). Bourne, Collins, Holder, and McCarthy demonstrating robust plasticity in aggressive thresholds in frogs in (2001) reported that territorial males of the golden rocket frog, M.-F. Chuang et al. / Animal Behaviour 123 (2017) 217e228 227

Anomaloglossus (formerly Colostethus) beebei (Dendrobatidae), Council (NSC 101-2621-B-029-002-MY3). M.A.B. was funded by a called antiphonally in response to the calls of a neighbour (a grant-in-aid of research from the University of Minnesota Graduate nonaggressive response), but responded aggressively to the calls of School and by a Land-Grant Professorship from the McKnight strangers. Unfortunately, quantitative data were not provided to Foundation. illustrate these reported behavioural differences. Lesbarreres and Lode (2002) reported that males of the agile frog, Rana dalmatina Supplementary Material (Ranidae), produce individually distinctive calls and behaviourally discriminate between the calls of familiar neighbours and unfa- Supplementary material associated with this article can be miliar individuals. However, their behavioural assay (changes in found, in the online version, at http://dx.doi.org/10.1016/j.anbehav. call duration) was not clearly associated with overt aggression 2016.11.001. directed towards opponent males. In addition, the males that served as the donors of neighbour call stimuli were not necessarily References a subject's adjacent neighbour, but instead were males calling from the same pond. Feng, Arch, et al. (2009) and Feng, Riede, et al. Akre, K. L., & Johnsen, S. (in press). Communication through a window of error: (2009) showed that males of the concave-eared torrent frog, Proportional processing and signal categorization. In M. A. Bee, & C. T. Miller (Eds.), Psychological mechanisms in animal communication. New York, NY: Odorrana tormota (Ranidae, formerly Amolops tormotus), produce Springer. individually distinctive vocalizations and respond more aggres- Baker, J. M. R. (1992). Body condition and tail height in great crested newts, Triturus sively towards the calls of unfamiliar males compared with familiar cristatus. Animal Behaviour, 43,157e159. delBarco-Trillo, J., McPhee, M. E., & Johnston, R. E. (2009). Nonagonistic familiarity males. Interpretation of these results, however, is complicated by decreases aggression in male Turkish hamsters, Mesocricetus brandti. Animal the fact that the animals were tested in the laboratory, making it Behaviour, 77, 389e393. unclear what precisely the aggressive males were defending. In Bee, M. A. (2003a). Experience-based plasticity of acoustically evoked aggression in a territorial frog. Journal of Comparative Physiology A, 189(6), 485e496. addition, and as in the study by Lesbarreres and Lode (2002), the Bee, M. A. (2003b). A test of the ‘dear enemy effect’ in the strawberry dart-poison males serving as donors of neighbour call stimuli were not neces- frog (Dendrobates pumilio). Behavioral Ecology and Sociobiology, 54(6), 601e610. sarily the subject's adjacent neighbour in the field, but instead were Bee, M. A. (2004). Within-individual variation in bullfrog vocalizations: Implica- males that called in the same general area. tions for a vocally mediated social recognition system. Journal of the Acoustical Society of America, 116(6), 3770e3781. Not all territorial anurans discriminate behaviourally between Bee, M. A. (2006). Individual recognition in animal species. In M. Naguib (Ed.), neighbours and strangers. Males of the strawberry poison frog, Encyclopedia of language and linguistics (2nd ed., pp. 617e626). New York, NY: Elsevier. Oophaga pumilio (Dendrobatidae; formerly Dendrobates pumilio) & € Bee, M. A. (in press). Social recognition in anurans. In M. A. Bee, C. T. Miller (Eds.), aggressively defend long-term territories (Prohl, 1997, 2005). They Psychological mechanisms in animal communication. New York, NY: Springer. also produce individually distinct vocalizations (Prohl,€ 2003). In Bee, M. A., & Gerhardt, H. C. (2001a). Habituation as a mechanism of reduced two field playback experiments, however, Bee (2003b) failed to find aggression between neighboring territorial male bullfrogs (Rana catesbeiana). Journal of Comparative Psychology, 115(1), 68e82. evidence that territorial males behaviourally discriminated be- Bee, M. A., & Gerhardt, H. C. (2001b). Neighbourestranger discrimination by terri- tween the calls of neighbours and strangers. When stimuli were torial male bullfrogs (Rana catesbeiana): I. Acoustic basis. Animal Behaviour, 62, broadcast from midway between the subject's and neighbour's 1129e1140. Bee, M. A., & Gerhardt, H. C. (2002). Individual voice recognition in a territorial frog territories, they responded strongly but equally to the calls of both (Rana catesbeiana). Proceedings of the Royal Society of B: Biological Sciences, neighbours and strangers. When both types of calls were played 269(1499), 1443e1448. back from the centre of the neighbour's territory, responses were Bee, M. A., Kozich, C. E., Blackwell, K. J., & Gerhardt, H. C. (2001). Individual variation in advertisement calls of territorial male green frogs, Rana clamitans: Implica- weaker, but again, there was no evidence of behavioural discrimi- tions for individual discrimination. Ethology, 107(1), 65e84. nation in responses to neighbours and strangers. Bee, M. A., Reichert, M. S., & Tumulty, J. P. (2016). Assessment and recognition of Previous methodological difficulties and null results aside, the rivals in anuran contests. Advances in the Study of Behavior, 48,161e249. present study and previous work on bullfrogs (Davis, 1987) and Beecher, M. D. (1982). Signature systems and kin recognition. American Zoologist, 22(3), 477e490. golden rocket frogs (Bourne et al., 2001) suggest discrimination Beecher, M. D. (1989a). 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