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Female koalas prefer bellows in which lower formants indicate larger males
Animal Behaviour 84 (2012) 1565e1571
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Female koalas prefer bellows in which lower formants indicate larger males Benjamin D. Charlton a, *, William A. H. Ellis b, Jacqui Brumm c, Karen Nilsson c, W. Tecumseh Fitch a a
Department of Cognitive Biology, University of Vienna, Vienna, Austria Koala Research Centre of Central Queensland, Central Queensland University, Rockhampton, Queensland, Australia c Lone Pine Koala Sanctuary, Brisbane, Queensland, Australia b
a r t i c l e i n f o Article history: Received 20 July 2012 Initial acceptance 11 September 2012 Final acceptance 28 September 2012 Available online 26 October 2012 MS. number: 12-00559 Keywords: acoustic cues to body size female mating preference formant frequency koala Phascolarctos cinereus vocal communication
Despite an extensive literature on the role of acoustic cues in mate choice little is known about the specific vocal traits that female mammals prefer. We used resynthesis techniques and playback experiments to examine the behavioural responses of oestrous female koalas, Phascolarctos cinereus, to male bellows in which a specific acoustic cue to body size, the formants, were modified to simulate callers of different body size. Oestrous females looked longer towards, and spent more time in close proximity to, loudspeakers broadcasting bellows simulating larger male koalas. These findings suggest that female koalas use formants (key components of human speech) to select larger males as mating partners, and represent the first evidence of a marsupial mating preference based on a vocal signal. More generally, these results indicate that intersexual selection pressures to lower formants and exaggerate size are present in a marsupial species, raising interesting questions about the evolutionary origins of formant perception. Ó 2012 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Studies of frog and bird vocal communication systems have generated considerable empirical support for sexual selection through female mating preferences (Andersson 1994). The role of mammal vocal signals in mate choice contexts, however, has received comparatively less attention. Indeed, whereas sexual selection through female mating preferences is likely to have a significant influence on the acoustic structure of many nonhuman mammal vocal signals (McElligott et al. 1999; Croll et al. 2002; Craul et al. 2004), few studies have used systematic playback experiments that control for other aspects of the male phenotype to conclusively demonstrate a female preference for a vocal characteristic (but see: McComb 1991; Charlton et al. 2007a; Reby et al. 2010). As a result, the extent to which vocal signals contribute to mate choice in mammals remains unclear. Nevertheless, progress in bioacoustics and digital signal processing now allows us to examine, extract and manipulate specific acoustic components of mammal vocalizations. Acoustic features of mammal calls can then be linked to their production mechanisms (Fitch & Hauser 2002) and their functional relevance determined by observing behavioural responses to resynthesized calls in the context of playback experiments. In the current study, we used this approach to
* Correspondence and present address: B. D. Charlton, School of Psychology, University of Sussex, Brighton BN1 9QH, U.K. E-mail address: [email protected] (B. D. Charlton).
examine the behavioural responses of oestrous female koalas, Phascolarctos cinereus, to male bellows in which a specific acoustic cue to body size, the formants, were modified to simulate callers of different body size. Formants are the natural resonance frequencies of air in the vocal tract that appear as broadband frequency peaks in the spectral acoustic structure of vocalizations and contribute perceptually to vocal ‘quality’ or ‘timbre’ (Titze 1994). It is well established that formants are the key acoustic parameters underlying phonemic variation and vowel identity in human speech (Fant 1960; Titze 1994); however, recent studies have shown that formant variation in species-typical calls is perceived and attended to by nonhuman animals (Fitch & Kelley 2000; Reby et al. 2005; Fitch & Fritz 2006; Charlton et al. 2007a, b, 2008a, b, 2010; Ghazanfar et al. 2007; Ohms et al. 2010; Taylor et al. 2010), suggesting that formants have functional significance in many nonhuman animal vocal communication systems. In particular, formants have the potential to provide reliable information on the callers’ body size in several mammals (Fitch 1997; Riede & Fitch 1999; Reby & McComb 2003; Harris et al. 2006; Sanvito et al. 2007; Vannoni & McElligott 2008; Charlton et al. 2009) owing to a close relationship between the frequency spacing of the formants, the caller’s vocal tract length and overall body size. The highly seasonal nature of male koala bellowing indicates an exclusive association with reproductive activity (Smith 1980a; Mitchell 1990). Recent work has shown that formants are reliable
0003-3472/$38.00 Ó 2012 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.anbehav.2012.09.034
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cues to male body size in this species (Charlton et al. 2011a) and also revealed a close correspondence between the occurrence of male bellowing and female travel distances during the breeding season (Ellis et al. 2011), suggesting that male bellows might be used to attract females directly. In addition, koala bellows have a very low fundamental frequency and often include broadband noise, making them particularly well suited for highlighting formants (Fitch & Hauser 1995; Fitch & Kelley 2000), and potentially increasing the salience of size-related information. Male koalas can perceive formant frequency variation in male bellows corresponding to the natural variation in body size between a large and small adult male (Charlton et al. 2012). If, as we may expect, female koalas can also perceive relevant variation in formant frequency spacing (i.e. corresponding to the difference between a large and small adult male) they could use this information to select larger males as mating partners (Charlton et al. 2007a) and, in doing so, obtain the indirect genetic benefits of larger, more competitive offspring. Indeed, body size is a key determinant of male reproductive success in koalas (Ellis & Bercovitch 2011) and this species’ low resting larynx position (Charlton et al. 2011a) suggests particularly strong selection pressures to broadcast sizerelated formant information (Fitch & Reby 2001). In this study we manipulated male koala bellows using resynthesis techniques and played these in a two-speaker choice playback paradigm to investigate directly whether female koalas use formants as acoustic cues to body size during mate assessment. Since female preferences may only emerge during oestrus when males often maximize their copulation attempts and the implications for any potential mating decision are large (reviewed in Charlton 2008), we used behavioural observations to verify that females were in peak oestrus during the experimental period. We hypothesized that when presented with resynthesized bellows originating from four male koalas, and corresponding to both large and small adult size variants representative of the natural population, oestrous female koalas will prefer bellows in which lower formants indicate larger males. METHODS Experimental Site and Animals This research was carried out during the 2011 breeding season (NovembereDecember) at Lone Pine Koala Sanctuary (LPKS), Brisbane, Australia. The playback experiments were conducted on 16 adult female koalas aged 4e10 years (mean ¼ 6.3). The subjects were housed with three or four other female koalas in enclosures measuring approximately 5 3 m and fed a diet of Eucalyptus leaves twice daily. For the playback experiments, subjects were taken to an experimental enclosure by the head koala keeper K.N. or the head curator J.B. The female koalas that served as subjects in our experiments are fully habituated to being handled by the keepers at LPKS, and are often carried from their enclosures to the main enclosure (located around 50 m away) to be photographed with members of the general public. This study was approved by the University of Queensland Animal Ethics Committee (approval number SAS/227/10). Assessment of Female Reproductive Stage Female koalas display overt behavioural signs during oestrus (Smith 1980b; Johnston et al. 2000). These include bellowing, pseudomale behaviour (mounting other females and being mounted), jerking or convulsive behaviour, and increased activity levels. In the current study, females were only considered as potential subjects when they showed all of the above signs of
oestrus on the day of the experiment. In addition, since nonoestrous female koalas reject male advances, with aggression and loud snarl, squark or wail vocalizations (Mitchell 1990; Johnston et al. 2000), all potential subjects were presented/teased with a mature male to determine whether they were receptive or not and thus to confirm their oestrous status (sensu Johnston et al. 2003). Selection of Male Bellows for Resynthesis To construct the playback sequences we selected five different bellow recordings with high signal-to-noise ratios (i.e. low background noise) from each of four adult male koalas (20 bellows in total). In addition, to ensure that our playback stimuli were representative of male koala bellows in general (Wiley 2003), we selected calls characterized by a range of fundamental frequencies (mean SD for the four male exemplars: 24.4 9.3 Hz; 45.3 13.7 Hz; 25.0 2.5 Hz; 17.7 2.4 Hz). The male bellows were originally recorded at LPKS by B.C. in 2010 using a Sennheiser ME67 directional microphone and a Zoom H4N digital recorder (amplitude resolution: 16 bits; sampling rate: 44.1 kHz) at distances of 2e10 m. The recordings were transferred onto an Apple Macintosh Macbook Pro computer and saved in WAV format as separate sound files (16 bits amplitude resolution, 44.1 kHz sampling rate). Acoustic Analyses and Estimation of Formant Spacing Male koala bellows consist of a series of inhalation and exhalation sections, often preceded by an onset phase of abrupt exhalations (Fig. 1). The later inhalation phases have a clear pulse train structure and stable formants (Charlton et al. 2011a, b). In addition, the formant spacing of the later inhalation phases of male bellows is more tightly correlated with male body size than that of the exhalation and initial inhalation phases (Charlton et al. 2011a). Consequently, we measured the formant frequencies of the later inhalation phases of bellows to determine the appropriate readjustment factors by which to resynthesize the playback stimuli. For each bellow the formants were measured using Praat 5.1.32 DSP package (www.praat.org). The Praat settings and exact methods used to do this are described in Charlton et al. (2011a). The average formant frequency values were then used to estimate the formant spacing (DF) achieved during the inhalation sections of each bellow using the linear regression-based method of Reby & McComb (2003). Resynthesis of Playback Stimuli Previous data generated from 20 adult male koalas showed that
DF for the later inhalation sections of male bellows ranged from 297.5 to 406.7 Hz (Charlton et al. 2011a). When these values are substituted for DF in the equation VTL ¼ c/2 DF, where c ¼ the approximate speed of sound in a mammal vocal tract (350 m/s; Titze 1994), this predicts an apparent vocal tract length (VTL) of 58.8 cm and 43.0 cm if the vocal tract is modelled as a straight uniform tube closed at one end, the glottis, and open at the other end, the mouth (Titze 1994). VTL values of 58.8 cm and 43.0 cm correspond to a large and small adult male koala, respectively (Charlton et al. 2011a). Therefore, to realistically simulate the natural variation in male VTL between the largest and smallest adults representative of a population, we created a large adult size variant with a DF of 300 Hz and an apparent VTL of 58.3 cm, and a small adult size variant with a DF of 400 Hz and an apparent VTL of 43.8 cm (Fig. 2). It is worth noting that the extremely low DF of the inhalation sections of male koala bellows predicts VTLs that are
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Relative amplitude (dB)
0.82 (a)
0
Introductory phase
Exhalation phase
Inhalation phase
(b)
Frequency (Hz)
- 0.99 4000
0 0
29.7 Time (s)
Figure 1. (a) Waveform and (b) spectrogram of a male koala bellow (spectrogram settings: FFT method; window length ¼ 0.09 s; time step ¼ 0.002 s; frequency step ¼ 10 Hz; Gaussian window shape; dynamic range ¼ 50 dB).
exceptionally long for an animal the size of a koala, and that these very low formant values are likely to be produced by a combination of the oral and nasal vocal tract (for a more detailed discussion about this see Charlton et al. 2011a). Bellow modification was achieved using a PSOLA-based algorithm that shifts DF by a factor (k) while leaving all other acoustic parameters unchanged (for more details see Charlton et al. 2008b, 2010). The resynthesis factors required to change the DF of our male exemplars were simply deduced by dividing either 300 or 400 by the original measured apparent DF for each bellow. The mean intensity values for all the resynthesized bellow stimuli were then scaled to 65 dB using the ‘SCALE Intensity’ command in Praat.
Playback Sequences The playback sequences were designed to mimic a natural vocal exchange between a large and a small male (sensu Charlton et al. 2007a). To do this we created playback sequences that broadcast five different bellows from a given male exemplar that had been resynthesized to simulate a large or a small male from one speaker, and the same five bellows that had been resynthesized to simulate the alternative size variant condition (large or small male) from the other speaker (Fig. 2). In this way we controlled for all other acoustic differences between size variants across a given playback trial. In addition, we controlled for any pre-existing preferences
(a) Left speaker
Large male size variant
Right speaker
Small male size variant
Large male size variant
Small male size variant
0
2800 F6 F5 F4 F3 F2 F1
0 0
0.5039 Time (s)
Original bellow
2800 F6 F5 F4 F3 F2 F1
0 0
0.5039 Time (s)
Small male size variant F6
Frequency (Hz)
Large male size variant
Frequency (Hz)
2800
Frequency (Hz)
(b)
607
Time (s)
F5 F4 F3 F2 F1
0 0
0.5039 Time (s)
Figure 2. The structure of the playback sequences was designed to simulate a vocal exchange between two different size male koalas while controlling for all other acoustic differences. (a) The composition and ordering of the bellows within a sequence. (b) The formant modification: the spectrograms (spectrogram settings: FFT method; window length ¼ 0.05 s; time step ¼ 0.004 s; frequency step ¼ 20 Hz; Gaussian window shape; dynamic range ¼ 50 dB) show an inhalation section of a male bellow resynthesized to simulate a large and small male size variant. Note that the formants (labelled F1e6) in the original inhalation section have been lowered to create the large male size variant and raised to create the small male size variant.
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that females might have had for particular male exemplars by simultaneously presenting stimuli resynthesized from the same male exemplar to each female. The bellows within a sequence were of comparable duration (mean SD ¼ 22.1 3.9 s) and broadcast in matched pairs so that a bellow representing a large or small male was followed 5 s later by the same bellow resynthesized to simulate the alternative size variant condition (Fig. 2). The next matched pair followed 1 min later, but this time the size variant used to initiate the vocal exchange was alternated (Fig. 2). Females were presented with a total of five matched choices over the duration of each playback experiment (ca. 10 min). The size variant played from each speaker and that used to initiate the sequence were also alternated across playback trials, to control for any preferences for areas of the experimental site and/or initiators of vocal exchanges. In addition, the use of a given male exemplar was balanced across subjects so that each exemplar was used four times in the experiment. This gave us 16 different playback sequences for the playback experiments (4 2 2) to present to our 16 female subjects. By balancing the presentation of each of the four original male exemplars across subjects, we could also assess whether the origin of the resynthesized playback stimuli affected female response (Kroodsma et al. 2001).
source, determined using a Radio Shack Sound Level Meter, set for C-weighted fast response). The subject’s behaviour was videotaped during the experimental period using a Canon LEGRIA FS20 digital video camcorder mounted on a tripod, and pointing directly down on the enclosure from a central position. Behavioural Analysis The videotapes were analysed using Gamebreaker version 7.4.5 (SportsTec, Sydney, Australia) with the audio switched off and the observer (B.D.C.) blind to condition. Female behaviour was quantified throughout the entire bellow sequence (i.e. for ca. 10 min). Specifically, we measured the number of looks and the duration of each look given towards either of the speaker positions while on the tree-fork, and the amount of time spent in a 7.5 m2 proximity zone directly adjacent to either speaker position. The boundary for each of the proximity zones started 1.5 m from the enclosure wall in front of each speaker position (Fig. 3). Looking was defined as starting when the female raised or turned her head to face the speaker, having previously faced away, before maintaining a fixed head position, and ending when the head moved away from this first fixed position. Looking responses were no longer quantified once the subject had left the centrally positioned tree-fork. Entering a proximity zone was defined as starting when the female’s entire body had crossed the outer plane of the zone boundary, and ending when the first leg passed out of the zone boundary.
Playback Experiments The playback experiments were conducted in the early evenings between 1700 and 1800 hours. Females were taken into a 25 m2 enclosure (5 5 m) and placed on a tree-fork 1.5 m high that was located exactly in the centre of the enclosure. This ensured that each female would be in a standardized context and position at the onset of playback. The playback stimuli were presented using two Chaiyo Focus 505 speakers (Taipei, Taiwan) that were elevated to a height of 1.5 m from the ground and connected by coaxial cable to an Apple Macintosh MacBook Pro laptop computer. Presenting the playback stimuli at a height of 1.5 m allowed us to limit the effect of ground reflections (Nelson 2003). The speakers were placed outside the experimental enclosure 5 m away from the subject’s position at playback onset and centred on two opposite sides of the enclosure (Fig. 3). Bellows were broadcast from the speakers at equal sound pressure levels sounding equivalent to that of naturally bellowing males (75 dB mean sound pressure level at 1 m from the
1.5 m
Statistical Analysis Repeated measures general linear models were used to examine female looking responses to the two size variant conditions. We conducted two models, one in which the number of looks given to the large versus small male was entered as a within-subjects factor, and another with the duration of looks given to the large versus small male entered as a within-subjects factor. The male exemplar used to create the playback stimuli was entered as a betweensubjects factor (comprising four levels) in both models to test whether females responded differently to the four different male exemplars used to create the playback sequences (Kroodsma et al. 2001). Additionally, we entered another between-subjects factor in the models termed ‘presentation order’ to test whether the order of
2m
2.5 m
1.5 m
2.5 m
= Proximity zone Figure 3. The two-speaker choice experimental set-up. At playback onset females were placed on a tree-fork so that they were oriented directly towards the front of the enclosure and at a 90 angle to both speaker positions. The size variant condition played from each speaker (large or small male) was alternated across playback trials.
B. D. Charlton et al. / Animal Behaviour 84 (2012) 1565e1571
the bellow sequences affected female looking response. Nonparametric statistics were used to examine the proximity zone data because the amount of time spent in each proximity zone was not normally distributed (ShapiroeWilk: P < 0.05) and could not be normalized. We used Wilcoxon signed-ranks tests to detect significant differences between the time spent in each proximity zone (large versus small male), a KruskaleWallis test to test whether the origin of our resynthesized playback stimuli affected the time spent in each proximity zone, and a ManneWhitney U test to determine whether the presentation order of the playback stimuli affected this response. Since our hypothesis was that the manipulation of the formants in our four male exemplars would affect female response we used an N in inferential statistics equal to the number of subjects in our experiment (McGregor 2000). Mauchly’s test (P > 0.05) indicated that sphericity could be assumed for the repeated measures general linear model. IBM SPSS statistics version 19 (SPSS Inc., Chicago, IL, U.S.A.) for Mac OS 10.7 was used for all the analyses, significance levels were set at 0.05, and two-tailed probability values are quoted.
20
DISCUSSION
No. of looks
15 10 5 0
Large male
Small male
Looking duration (s)
160
Time in proximity zone (s)
(b)
* 120 80 40 0
Large male
Small male
100
(c) *
75 50 25 0
Large male
Small male
Figure 4. Error bar charts showing means þ SE of female behavioural responses to the two playback conditions (N ¼ 16, *P < 0.05). (a) Number of looks, (b) looking duration (s) and (c) time in proximity zone (s).
highly likely to lead to conception. Consequently, the results of the current study are indicative of a mate choice decision and, to our knowledge, represent the first evidence of a marsupial mating preference based on a vocal signal.
Table 1 Raw data for each of the female koalas in the analysis Subject
Using resynthesized bellow stimuli originating from four male koalas, we found that oestrous female koalas looked significantly more towards playback speakers broadcasting bellows with lower formant values simulating larger male callers. Females also spent longer in close proximity to playback speakers broadcasting bellows with lower formants simulating larger males. On the basis of these results, male koalas producing bellows with lower formant values and spacing would not only elicit greater attention from females, but would also be more likely to attract oestrous females towards them during the breeding season than those producing bellows with higher formant values and spacing. Furthermore, this type of behavioural response is likely to reflect an actual mating preference because oestrous females would typically be mated if they approached a male koala at this time. Female koalas are only receptive to male copulation attempts when they are in oestrus (Johnston et al. 2003) and once mated rarely continue to display oestrous behaviour. In addition, the physical act of mating triggers ovulation in this species (Johnston et al. 2000), making copulation
(a)
*
RESULTS The number (F1, 8 ¼ 6.27, P ¼ 0.037) and duration (F1, 8 ¼ 5.71, P ¼ 0.044) of looks given by females towards the speaker playing the large male size variant condition were significantly greater than those given towards the speaker playing the small male size variant condition (Fig. 4). In addition, females spent significantly longer in the large male size variant proximity zone than they did in the small male size variant proximity zone (Z ¼ 2.31, N ¼ 16, P ¼ 0.021; Fig. 4). Table 1 provides the raw data for each of the female koalas in the analysis. The male exemplar used to create the playback stimuli did not affect female looking response (number of looks: F3, 8 ¼ 0.38, P ¼ 0.769; looking duration: F3, 8 ¼ 0.04, P ¼ 0.990). In addition, the amount of time females spent in the large (c23 ¼ 0:98, P ¼ 0.807) and small (c23 ¼ 1:07, P ¼ 0.785) male proximity zones was not affected by the male exemplar used to create the playback stimuli. Taken together, these results indicate that the origin of our resynthesized playback stimuli did not affect female behavioural response. Similarly, the presentation order of the stimuli did not affect female looking response (number of looks: F3, 8 ¼ 1.52, P ¼ 0.253; looking duration: F3, 8 ¼ 2.44, P ¼ 0.157) or the time spent in either proximity zone (large male: U ¼ 18, N1 ¼ N2 ¼ 8, P ¼ 0.136; small male: U ¼ 17, N1 ¼ N2 ¼ 8, P ¼ 0.110).
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Sprite Nivea Miniata Trace Donatella Snorkle Theresa Crumpet Yulara Kat Crumble Chassis Eeyore Violet Paddle Mel Mean
Number of looks Looking duration (s) Time in proximity zone (s) Large male
Small male
Large male
Small male
Large male
Small male
12 11 24 26 26 6 19 15 21 25 12 12 25 13 9 6 16.4
14 9 18 17 13 4 14 8 19 31 8 9 23 7 15 4 13.3
99.7 50.8 244.1 135.7 109.5 43.1 239.3 245.3 119.0 98.1 195.7 82.2 192.4 56.8 169.0 40.9 132.6
94.5 64.5 122.1 99.5 61.6 40.6 93.4 103.3 97.0 144.7 80.7 30.2 190.8 44.9 162.1 48.1 92.4
61.1 122.2 0.0 15.4 85.9 248.4 0.0 0.0 70.5 55.4 86.7 124.6 0.0 109.2 0.0 121.4 68.8
18.8 91.4 0.0 29.4 42.7 100.7 0.0 0.0 31.3 34.7 19.1 116.1 0.0 111.3 0.0 127.1 45.2
‘Large male’ and ‘Small male’ refer to the large and small male size variant conditions, respectively.
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A mating preference such as this could evolve and be maintained because female koalas mating with larger males would gain the genetic benefits of larger, more competitive offspring and may, at least partly, explain the correlation that exists between male reproductive success and body size in this species (Ellis & Bercovitch 2011). In addition, this type of female mate choice behaviour would result in the differential mating success of male phenotypes able to produce lower formants in their bellows, and consequently intersexual selection will favour these individuals. Indeed, the male koala’s permanently descended larynx clearly suggests strong selection pressures for callers to elongate their vocal tracts and lower formants (Charlton et al. 2011a), to maximize the acoustic impression of their body size conveyed to receivers. Since formants fulfil important functions as acoustic cues to body size both in mate choice contexts and during intrasexual assessment in humans (Feinberg et al. 2006; Puts et al. 2007) and red deer, Cervus elaphus (Reby et al. 2005; Charlton et al. 2007a), they may have similar dual utility in koalas. Future studies should investigate whether male koalas also use formants as cues to body size during agonistic interactions with rivals, to determine whether maleemale competition has provided an additional selection pressure alongside female mating preferences for broadcasting size-related formant information in this species. In addition, further studies on koalas that compare the effect of a wider range of stimulus contrasts on female preferences based on size-related formant information are also warranted. It would be particularly interesting to see whether preferences for bellows simulating larger callers persist when the size variants used for the bellow stimuli fall outside the typical adult range, and also when bellows representing small versus medium and medium versus large male size variants are presented. Such preferences would indicate the presence of directional sexual selection on this feature of male bellows, and help to explain the existence of the extremely low formant frequencies present in male bellows. Furthermore, as acoustic signals are typically composed of several components, possible interactions between them can mean that female responses to variation in single acoustic parameters may not accurately predict how they respond to variation in different combinations of acoustic components (Ryan & Rand 2003; Olvido & Wagner 2004; Scheuber et al. 2004). Indeed, recent work has shown that male rock hyrax, Procavia capensis, ‘songs’ simultaneously encode information on body weight, size, current condition, social status and hormonal state (Koren & Geffen 2009), and it seems likely that multiple levels of information are present in male koala bellows. Future studies should determine whether other cues to male quality exist in male koala bellows, and use a playback approach to show how this information interacts with formants to influence mate choice decisions. In conclusion, our findings show that formants play a key role (as acoustic cues to body size) in affecting female koala mating behaviour and, hence, that intersexual selection pressures constitute an important evolutionary force acting on the vocal displays of male koalas. More generally, by showing that female koalas use size-related formant information in a mate choice context, our results reinforce the hypothesis that the perceptual relevance of formant frequencies (which are critical for phonemic variation in human speech: Fant 1960; Titze 1994) may have initially evolved as a result of sexual selection pressures linked to the expression of information on body size (Fitch 1997). Ovulating women find male speakers with low formants particularly attractive (Feinberg et al. 2006) and oestrous female red deer prefer male roars with lower formants indicating larger callers (Charlton et al. 2007a). In the current study we have shown that intersexual selection pressures for males to broadcast size-related formant information are also present in a marsupial species. Comparative data such as this suggest that female use of formants in a mate choice context could
be prevalent throughout mammals, and may have important implications for understanding the evolution of spoken language. Acknowledgments We thank the staff at LPKS for building the experimental enclosure and allowing us to conduct the experiments. Special thanks go to Mai Ueda for her help conducting some of the experiments. B.D.C. was supported by a European Research Council Advanced Grant SOMACCA (No. 230604) awarded to W.T.F. References Andersson, M. 1994. Sexual Selection. Princeton, New Jersey: Princeton University Press. Charlton, B. D. 2008. Female mate choice in nonhuman mammals. In: Animal Behavior: New Research (Ed. by E. A. Weber & L. H. Krause), pp. 35e56. New York: Nova Science Publishers. Charlton, B. D., Reby, D. & McComb, K. 2007a. Female red deer prefer the roars of larger males. Biology Letters, 3, 382e385. Charlton, B. D., Reby, D. & McComb, K. 2007b. Female perception of size-related formants in red deer, Cervus elaphus. Animal Behaviour, 74, 707e714. Charlton, B. D., McComb, K. & Reby, D. 2008a. Free-ranging red deer hinds show greater attentiveness to roars with formant frequencies typical of young males. Ethology, 114, 1023e1031. Charlton, B. D., Reby, D. & McComb, K. 2008b. Effect of combined source (F0) and filter (formant) variation on red deer hind responses to male roars. Journal of the Acoustical Society of America, 123, 2936e2943. Charlton, B. D., Zhihe, Z. & Snyder, R. J. 2009. The information content of giant panda, Ailuropoda melanoleuca, bleats: acoustic cues to sex, age and size. Animal Behaviour, 78, 893e898. Charlton, B. D., Zhihe, Z. & Snyder, R. J. 2010. Giant pandas perceive and attend to formant frequency variation in male bleats. Animal Behaviour, 79, 1221e1227. Charlton, B. D., Ellis, W. A. H., McKinnon, A. J., Cowin, G. J., Brumm, J., Nilsson, K. & Fitch, W. T. 2011a. Cues to body size in the formant spacing of male koala (Phascolarctos cinereus) bellows: honesty in an exaggerated trait. Journal of Experimental Biology, 214, 3414e3422. Charlton, B. D., Ellis, W. A. H., McKinnon, A. J., Brumm, J., Nilsson, K. & Fitch, W. T. 2011b. Perception of male caller identity in koalas (Phascolarctos cinereus): acoustic analysis and playback experiments. PLoS ONE, 6, e20329. Charlton, B. D., Ellis, W. A. H., Larkin, R. & Fitch, W. T. 2012. Perception of sizerelated formant information in male koalas (Phascolarctos cinereus). Animal Cognition, 15, 999e1006. Craul, M., Zimmermann, E. & Radespiel, U. 2004. First experimental evidence for female mate choice in a nocturnal primate. Primates, 45, 271e274. Croll, D. A., Clark, C. W., Acevedo, A., Tershy, B., Flores, S., Gedamke, J. & Urban, J. 2002. Bioacoustics: only male fin whales sing loud songs: these mammals need to call long-distance when it comes to attracting females. Nature, 417, 809. Ellis, W. A. H. & Bercovitch, F. B. 2011. Body size and sexual selection in the koala. Behavioral Ecology and Sociobiology, 65, 1229e1235. Ellis, W. A. H., Bercovitch, F. B., FitzGibbon, S., Roe, P., Wimmer, J., Melzer, A. & Wilson, R. 2011. Koala bellows and their association with the spatial dynamics of free-ranging koalas. Behavioral Ecology, 22, 372e377. Fant, G. 1960. Acoustic Theory of Speech Production. The Hague: Mouton. Feinberg, D. R., Jones, B. C., Law-Smith, M. J., Moore, F. R., DeBruine, L. M., Cornwell, R. E., Hillier, S. G. & Perrett, D. I. 2006. Menstrual cycle, trait estrogen level, and masculinity preferences in the human voice. Hormones and Behavior, 49, 215e222. Fitch, W. T. 1997. Vocal tract length and formant frequency dispersion correlate with body size in rhesus macaques. Journal of the Acoustical Society of America, 102, 1213e1222. Fitch, W. T. & Fritz, J. B. 2006. Rhesus macaques spontaneously perceive formants in conspecific vocalisations. Journal of the Acoustical Society of America, 120, 2132e2141. Fitch, W. T. & Hauser, M. D. 1995. Vocal production in nonhuman-primates e acoustics, physiology, and functional constraints on honest advertisement. American Journal of Primatology, 37, 191e219. Fitch, W. T. & Hauser, M. D. 2002. Unpacking ‘honesty’: generating and extracting information from acoustic signals. In: Animal Communication (Ed. by A. MegalaSimmons & A. Popper), pp. 65e137. Berlin: Springer-Verlag. Fitch, W. T. & Kelley, J. P. 2000. Perception of vocal tract resonances by whooping cranes Grus americana. Ethology, 106, 559e574. Fitch, W. T. & Reby, D. 2001. The descended larynx is not uniquely human. Proceedings of the Royal Society B, 268, 1669e1675. Ghazanfar, A., Turesson, H., Maier, J., Vandinther, R., Patterson, R. D. & Logothetis, N. 2007. Vocal-tract resonances as indexical cues in rhesus monkeys. Current Biology, 17, 425e430. Harris, T. R., Fitch, W. T., Goldstein, L. M. & Fashing, P. J. 2006. Black and white colobus monkey (Colobus guereza) roars as a source of both honest and exaggerated information about body mass. Ethology, 112, 911e920.
B. D. Charlton et al. / Animal Behaviour 84 (2012) 1565e1571 Johnston, S. D., McGowan, M. R., O’Callaghan, P., Cox, R. & Nicolson, V. 2000. Natural and artificial methods for inducing the luteal phase in the koala (Phascolarctos cinereus). Reproduction, 120, 59e64. Johnston, S. D., McGowan, M. R., O’Callaghan, P., Cox, P., Houlden, B., Haig, S. & Taddeo, G. 2003. Birth of koalas Phascolarctos cinereus at Lone Pine Koala Sanctuary following artificial insemination. International Zoo Yearbook, 38, 160e172. Koren, L. & Geffen, E. 2009. Complex call in male rock hyrax (Procavia capensis): a multi-information distributing channel. Behavioral Ecology and Sociobiology, 63, 581e590. Kroodsma, D. E., Byers, B. E., Goodale, E., Johnson, S. & Liu, W. C. 2001. Pseudoreplication in playback experiments, revisited a decade later. Animal Behaviour, 61, 1029e1033. McComb, K. E. 1991. Female choice for high roaring rates in red deer, Cervus elaphus. Animal Behaviour, 41, 79e88. McElligott, A. G., O’Neill, K. P. & Hayden, T. J. 1999. Cumulative long-term investment in vocalization and mating success of fallow bucks, Dama dama. Animal Behaviour, 57, 1159e1167. McGregor, P. K. 2000. Playback experiments: design and analysis. Acta Ethologica, 3, 3e8. Mitchell, P. 1990. Social behaviour and communication of koalas. In: Biology of the Koala (Ed. by A. K. Lee, K. A. Handasyde & G. D. Sanson), pp. 151e170. Chipping Norton: Surrey Beatty. Nelson, B. 2003. Reliability of sound attenuation in Florida scrub habitat and behavioral implications. Journal of the Acoustical Society of America, 113, 2901e 2911. Ohms, V., Gill, A., Van Heijningen, C., Beckers, G. & ten Cate, C. 2010. Zebra finches exhibit speaker-independent phonetic perception of human speech. Proceedings of the Royal Society B, 277, 1003e1009. Olvido, A. E. & Wagner, W. E. 2004. Signal components, acoustic preference functions and sexual selection in a cricket. Biological Journal of the Linnean Society, 83, 461e472. Puts, D. A., Hodges, C. R., Cardenas, R. A. & Gaulin, S. J. C. 2007. Men’s voices as dominance signals: vocal fundamental and formant frequencies influence
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Animal Behaviour journal homepage: www.elsevier.com/locate/anbehav
Female koalas prefer bellows in which lower formants indicate larger males Benjamin D. Charlton a, *, William A. H. Ellis b, Jacqui Brumm c, Karen Nilsson c, W. Tecumseh Fitch a a
Department of Cognitive Biology, University of Vienna, Vienna, Austria Koala Research Centre of Central Queensland, Central Queensland University, Rockhampton, Queensland, Australia c Lone Pine Koala Sanctuary, Brisbane, Queensland, Australia b
a r t i c l e i n f o Article history: Received 20 July 2012 Initial acceptance 11 September 2012 Final acceptance 28 September 2012 Available online 26 October 2012 MS. number: 12-00559 Keywords: acoustic cues to body size female mating preference formant frequency koala Phascolarctos cinereus vocal communication
Despite an extensive literature on the role of acoustic cues in mate choice little is known about the specific vocal traits that female mammals prefer. We used resynthesis techniques and playback experiments to examine the behavioural responses of oestrous female koalas, Phascolarctos cinereus, to male bellows in which a specific acoustic cue to body size, the formants, were modified to simulate callers of different body size. Oestrous females looked longer towards, and spent more time in close proximity to, loudspeakers broadcasting bellows simulating larger male koalas. These findings suggest that female koalas use formants (key components of human speech) to select larger males as mating partners, and represent the first evidence of a marsupial mating preference based on a vocal signal. More generally, these results indicate that intersexual selection pressures to lower formants and exaggerate size are present in a marsupial species, raising interesting questions about the evolutionary origins of formant perception. Ó 2012 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Studies of frog and bird vocal communication systems have generated considerable empirical support for sexual selection through female mating preferences (Andersson 1994). The role of mammal vocal signals in mate choice contexts, however, has received comparatively less attention. Indeed, whereas sexual selection through female mating preferences is likely to have a significant influence on the acoustic structure of many nonhuman mammal vocal signals (McElligott et al. 1999; Croll et al. 2002; Craul et al. 2004), few studies have used systematic playback experiments that control for other aspects of the male phenotype to conclusively demonstrate a female preference for a vocal characteristic (but see: McComb 1991; Charlton et al. 2007a; Reby et al. 2010). As a result, the extent to which vocal signals contribute to mate choice in mammals remains unclear. Nevertheless, progress in bioacoustics and digital signal processing now allows us to examine, extract and manipulate specific acoustic components of mammal vocalizations. Acoustic features of mammal calls can then be linked to their production mechanisms (Fitch & Hauser 2002) and their functional relevance determined by observing behavioural responses to resynthesized calls in the context of playback experiments. In the current study, we used this approach to
* Correspondence and present address: B. D. Charlton, School of Psychology, University of Sussex, Brighton BN1 9QH, U.K. E-mail address: [email protected] (B. D. Charlton).
examine the behavioural responses of oestrous female koalas, Phascolarctos cinereus, to male bellows in which a specific acoustic cue to body size, the formants, were modified to simulate callers of different body size. Formants are the natural resonance frequencies of air in the vocal tract that appear as broadband frequency peaks in the spectral acoustic structure of vocalizations and contribute perceptually to vocal ‘quality’ or ‘timbre’ (Titze 1994). It is well established that formants are the key acoustic parameters underlying phonemic variation and vowel identity in human speech (Fant 1960; Titze 1994); however, recent studies have shown that formant variation in species-typical calls is perceived and attended to by nonhuman animals (Fitch & Kelley 2000; Reby et al. 2005; Fitch & Fritz 2006; Charlton et al. 2007a, b, 2008a, b, 2010; Ghazanfar et al. 2007; Ohms et al. 2010; Taylor et al. 2010), suggesting that formants have functional significance in many nonhuman animal vocal communication systems. In particular, formants have the potential to provide reliable information on the callers’ body size in several mammals (Fitch 1997; Riede & Fitch 1999; Reby & McComb 2003; Harris et al. 2006; Sanvito et al. 2007; Vannoni & McElligott 2008; Charlton et al. 2009) owing to a close relationship between the frequency spacing of the formants, the caller’s vocal tract length and overall body size. The highly seasonal nature of male koala bellowing indicates an exclusive association with reproductive activity (Smith 1980a; Mitchell 1990). Recent work has shown that formants are reliable
0003-3472/$38.00 Ó 2012 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.anbehav.2012.09.034
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cues to male body size in this species (Charlton et al. 2011a) and also revealed a close correspondence between the occurrence of male bellowing and female travel distances during the breeding season (Ellis et al. 2011), suggesting that male bellows might be used to attract females directly. In addition, koala bellows have a very low fundamental frequency and often include broadband noise, making them particularly well suited for highlighting formants (Fitch & Hauser 1995; Fitch & Kelley 2000), and potentially increasing the salience of size-related information. Male koalas can perceive formant frequency variation in male bellows corresponding to the natural variation in body size between a large and small adult male (Charlton et al. 2012). If, as we may expect, female koalas can also perceive relevant variation in formant frequency spacing (i.e. corresponding to the difference between a large and small adult male) they could use this information to select larger males as mating partners (Charlton et al. 2007a) and, in doing so, obtain the indirect genetic benefits of larger, more competitive offspring. Indeed, body size is a key determinant of male reproductive success in koalas (Ellis & Bercovitch 2011) and this species’ low resting larynx position (Charlton et al. 2011a) suggests particularly strong selection pressures to broadcast sizerelated formant information (Fitch & Reby 2001). In this study we manipulated male koala bellows using resynthesis techniques and played these in a two-speaker choice playback paradigm to investigate directly whether female koalas use formants as acoustic cues to body size during mate assessment. Since female preferences may only emerge during oestrus when males often maximize their copulation attempts and the implications for any potential mating decision are large (reviewed in Charlton 2008), we used behavioural observations to verify that females were in peak oestrus during the experimental period. We hypothesized that when presented with resynthesized bellows originating from four male koalas, and corresponding to both large and small adult size variants representative of the natural population, oestrous female koalas will prefer bellows in which lower formants indicate larger males. METHODS Experimental Site and Animals This research was carried out during the 2011 breeding season (NovembereDecember) at Lone Pine Koala Sanctuary (LPKS), Brisbane, Australia. The playback experiments were conducted on 16 adult female koalas aged 4e10 years (mean ¼ 6.3). The subjects were housed with three or four other female koalas in enclosures measuring approximately 5 3 m and fed a diet of Eucalyptus leaves twice daily. For the playback experiments, subjects were taken to an experimental enclosure by the head koala keeper K.N. or the head curator J.B. The female koalas that served as subjects in our experiments are fully habituated to being handled by the keepers at LPKS, and are often carried from their enclosures to the main enclosure (located around 50 m away) to be photographed with members of the general public. This study was approved by the University of Queensland Animal Ethics Committee (approval number SAS/227/10). Assessment of Female Reproductive Stage Female koalas display overt behavioural signs during oestrus (Smith 1980b; Johnston et al. 2000). These include bellowing, pseudomale behaviour (mounting other females and being mounted), jerking or convulsive behaviour, and increased activity levels. In the current study, females were only considered as potential subjects when they showed all of the above signs of
oestrus on the day of the experiment. In addition, since nonoestrous female koalas reject male advances, with aggression and loud snarl, squark or wail vocalizations (Mitchell 1990; Johnston et al. 2000), all potential subjects were presented/teased with a mature male to determine whether they were receptive or not and thus to confirm their oestrous status (sensu Johnston et al. 2003). Selection of Male Bellows for Resynthesis To construct the playback sequences we selected five different bellow recordings with high signal-to-noise ratios (i.e. low background noise) from each of four adult male koalas (20 bellows in total). In addition, to ensure that our playback stimuli were representative of male koala bellows in general (Wiley 2003), we selected calls characterized by a range of fundamental frequencies (mean SD for the four male exemplars: 24.4 9.3 Hz; 45.3 13.7 Hz; 25.0 2.5 Hz; 17.7 2.4 Hz). The male bellows were originally recorded at LPKS by B.C. in 2010 using a Sennheiser ME67 directional microphone and a Zoom H4N digital recorder (amplitude resolution: 16 bits; sampling rate: 44.1 kHz) at distances of 2e10 m. The recordings were transferred onto an Apple Macintosh Macbook Pro computer and saved in WAV format as separate sound files (16 bits amplitude resolution, 44.1 kHz sampling rate). Acoustic Analyses and Estimation of Formant Spacing Male koala bellows consist of a series of inhalation and exhalation sections, often preceded by an onset phase of abrupt exhalations (Fig. 1). The later inhalation phases have a clear pulse train structure and stable formants (Charlton et al. 2011a, b). In addition, the formant spacing of the later inhalation phases of male bellows is more tightly correlated with male body size than that of the exhalation and initial inhalation phases (Charlton et al. 2011a). Consequently, we measured the formant frequencies of the later inhalation phases of bellows to determine the appropriate readjustment factors by which to resynthesize the playback stimuli. For each bellow the formants were measured using Praat 5.1.32 DSP package (www.praat.org). The Praat settings and exact methods used to do this are described in Charlton et al. (2011a). The average formant frequency values were then used to estimate the formant spacing (DF) achieved during the inhalation sections of each bellow using the linear regression-based method of Reby & McComb (2003). Resynthesis of Playback Stimuli Previous data generated from 20 adult male koalas showed that
DF for the later inhalation sections of male bellows ranged from 297.5 to 406.7 Hz (Charlton et al. 2011a). When these values are substituted for DF in the equation VTL ¼ c/2 DF, where c ¼ the approximate speed of sound in a mammal vocal tract (350 m/s; Titze 1994), this predicts an apparent vocal tract length (VTL) of 58.8 cm and 43.0 cm if the vocal tract is modelled as a straight uniform tube closed at one end, the glottis, and open at the other end, the mouth (Titze 1994). VTL values of 58.8 cm and 43.0 cm correspond to a large and small adult male koala, respectively (Charlton et al. 2011a). Therefore, to realistically simulate the natural variation in male VTL between the largest and smallest adults representative of a population, we created a large adult size variant with a DF of 300 Hz and an apparent VTL of 58.3 cm, and a small adult size variant with a DF of 400 Hz and an apparent VTL of 43.8 cm (Fig. 2). It is worth noting that the extremely low DF of the inhalation sections of male koala bellows predicts VTLs that are
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Relative amplitude (dB)
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Figure 1. (a) Waveform and (b) spectrogram of a male koala bellow (spectrogram settings: FFT method; window length ¼ 0.09 s; time step ¼ 0.002 s; frequency step ¼ 10 Hz; Gaussian window shape; dynamic range ¼ 50 dB).
exceptionally long for an animal the size of a koala, and that these very low formant values are likely to be produced by a combination of the oral and nasal vocal tract (for a more detailed discussion about this see Charlton et al. 2011a). Bellow modification was achieved using a PSOLA-based algorithm that shifts DF by a factor (k) while leaving all other acoustic parameters unchanged (for more details see Charlton et al. 2008b, 2010). The resynthesis factors required to change the DF of our male exemplars were simply deduced by dividing either 300 or 400 by the original measured apparent DF for each bellow. The mean intensity values for all the resynthesized bellow stimuli were then scaled to 65 dB using the ‘SCALE Intensity’ command in Praat.
Playback Sequences The playback sequences were designed to mimic a natural vocal exchange between a large and a small male (sensu Charlton et al. 2007a). To do this we created playback sequences that broadcast five different bellows from a given male exemplar that had been resynthesized to simulate a large or a small male from one speaker, and the same five bellows that had been resynthesized to simulate the alternative size variant condition (large or small male) from the other speaker (Fig. 2). In this way we controlled for all other acoustic differences between size variants across a given playback trial. In addition, we controlled for any pre-existing preferences
(a) Left speaker
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Figure 2. The structure of the playback sequences was designed to simulate a vocal exchange between two different size male koalas while controlling for all other acoustic differences. (a) The composition and ordering of the bellows within a sequence. (b) The formant modification: the spectrograms (spectrogram settings: FFT method; window length ¼ 0.05 s; time step ¼ 0.004 s; frequency step ¼ 20 Hz; Gaussian window shape; dynamic range ¼ 50 dB) show an inhalation section of a male bellow resynthesized to simulate a large and small male size variant. Note that the formants (labelled F1e6) in the original inhalation section have been lowered to create the large male size variant and raised to create the small male size variant.
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that females might have had for particular male exemplars by simultaneously presenting stimuli resynthesized from the same male exemplar to each female. The bellows within a sequence were of comparable duration (mean SD ¼ 22.1 3.9 s) and broadcast in matched pairs so that a bellow representing a large or small male was followed 5 s later by the same bellow resynthesized to simulate the alternative size variant condition (Fig. 2). The next matched pair followed 1 min later, but this time the size variant used to initiate the vocal exchange was alternated (Fig. 2). Females were presented with a total of five matched choices over the duration of each playback experiment (ca. 10 min). The size variant played from each speaker and that used to initiate the sequence were also alternated across playback trials, to control for any preferences for areas of the experimental site and/or initiators of vocal exchanges. In addition, the use of a given male exemplar was balanced across subjects so that each exemplar was used four times in the experiment. This gave us 16 different playback sequences for the playback experiments (4 2 2) to present to our 16 female subjects. By balancing the presentation of each of the four original male exemplars across subjects, we could also assess whether the origin of the resynthesized playback stimuli affected female response (Kroodsma et al. 2001).
source, determined using a Radio Shack Sound Level Meter, set for C-weighted fast response). The subject’s behaviour was videotaped during the experimental period using a Canon LEGRIA FS20 digital video camcorder mounted on a tripod, and pointing directly down on the enclosure from a central position. Behavioural Analysis The videotapes were analysed using Gamebreaker version 7.4.5 (SportsTec, Sydney, Australia) with the audio switched off and the observer (B.D.C.) blind to condition. Female behaviour was quantified throughout the entire bellow sequence (i.e. for ca. 10 min). Specifically, we measured the number of looks and the duration of each look given towards either of the speaker positions while on the tree-fork, and the amount of time spent in a 7.5 m2 proximity zone directly adjacent to either speaker position. The boundary for each of the proximity zones started 1.5 m from the enclosure wall in front of each speaker position (Fig. 3). Looking was defined as starting when the female raised or turned her head to face the speaker, having previously faced away, before maintaining a fixed head position, and ending when the head moved away from this first fixed position. Looking responses were no longer quantified once the subject had left the centrally positioned tree-fork. Entering a proximity zone was defined as starting when the female’s entire body had crossed the outer plane of the zone boundary, and ending when the first leg passed out of the zone boundary.
Playback Experiments The playback experiments were conducted in the early evenings between 1700 and 1800 hours. Females were taken into a 25 m2 enclosure (5 5 m) and placed on a tree-fork 1.5 m high that was located exactly in the centre of the enclosure. This ensured that each female would be in a standardized context and position at the onset of playback. The playback stimuli were presented using two Chaiyo Focus 505 speakers (Taipei, Taiwan) that were elevated to a height of 1.5 m from the ground and connected by coaxial cable to an Apple Macintosh MacBook Pro laptop computer. Presenting the playback stimuli at a height of 1.5 m allowed us to limit the effect of ground reflections (Nelson 2003). The speakers were placed outside the experimental enclosure 5 m away from the subject’s position at playback onset and centred on two opposite sides of the enclosure (Fig. 3). Bellows were broadcast from the speakers at equal sound pressure levels sounding equivalent to that of naturally bellowing males (75 dB mean sound pressure level at 1 m from the
1.5 m
Statistical Analysis Repeated measures general linear models were used to examine female looking responses to the two size variant conditions. We conducted two models, one in which the number of looks given to the large versus small male was entered as a within-subjects factor, and another with the duration of looks given to the large versus small male entered as a within-subjects factor. The male exemplar used to create the playback stimuli was entered as a betweensubjects factor (comprising four levels) in both models to test whether females responded differently to the four different male exemplars used to create the playback sequences (Kroodsma et al. 2001). Additionally, we entered another between-subjects factor in the models termed ‘presentation order’ to test whether the order of
2m
2.5 m
1.5 m
2.5 m
= Proximity zone Figure 3. The two-speaker choice experimental set-up. At playback onset females were placed on a tree-fork so that they were oriented directly towards the front of the enclosure and at a 90 angle to both speaker positions. The size variant condition played from each speaker (large or small male) was alternated across playback trials.
B. D. Charlton et al. / Animal Behaviour 84 (2012) 1565e1571
the bellow sequences affected female looking response. Nonparametric statistics were used to examine the proximity zone data because the amount of time spent in each proximity zone was not normally distributed (ShapiroeWilk: P < 0.05) and could not be normalized. We used Wilcoxon signed-ranks tests to detect significant differences between the time spent in each proximity zone (large versus small male), a KruskaleWallis test to test whether the origin of our resynthesized playback stimuli affected the time spent in each proximity zone, and a ManneWhitney U test to determine whether the presentation order of the playback stimuli affected this response. Since our hypothesis was that the manipulation of the formants in our four male exemplars would affect female response we used an N in inferential statistics equal to the number of subjects in our experiment (McGregor 2000). Mauchly’s test (P > 0.05) indicated that sphericity could be assumed for the repeated measures general linear model. IBM SPSS statistics version 19 (SPSS Inc., Chicago, IL, U.S.A.) for Mac OS 10.7 was used for all the analyses, significance levels were set at 0.05, and two-tailed probability values are quoted.
20
DISCUSSION
No. of looks
15 10 5 0
Large male
Small male
Looking duration (s)
160
Time in proximity zone (s)
(b)
* 120 80 40 0
Large male
Small male
100
(c) *
75 50 25 0
Large male
Small male
Figure 4. Error bar charts showing means þ SE of female behavioural responses to the two playback conditions (N ¼ 16, *P < 0.05). (a) Number of looks, (b) looking duration (s) and (c) time in proximity zone (s).
highly likely to lead to conception. Consequently, the results of the current study are indicative of a mate choice decision and, to our knowledge, represent the first evidence of a marsupial mating preference based on a vocal signal.
Table 1 Raw data for each of the female koalas in the analysis Subject
Using resynthesized bellow stimuli originating from four male koalas, we found that oestrous female koalas looked significantly more towards playback speakers broadcasting bellows with lower formant values simulating larger male callers. Females also spent longer in close proximity to playback speakers broadcasting bellows with lower formants simulating larger males. On the basis of these results, male koalas producing bellows with lower formant values and spacing would not only elicit greater attention from females, but would also be more likely to attract oestrous females towards them during the breeding season than those producing bellows with higher formant values and spacing. Furthermore, this type of behavioural response is likely to reflect an actual mating preference because oestrous females would typically be mated if they approached a male koala at this time. Female koalas are only receptive to male copulation attempts when they are in oestrus (Johnston et al. 2003) and once mated rarely continue to display oestrous behaviour. In addition, the physical act of mating triggers ovulation in this species (Johnston et al. 2000), making copulation
(a)
*
RESULTS The number (F1, 8 ¼ 6.27, P ¼ 0.037) and duration (F1, 8 ¼ 5.71, P ¼ 0.044) of looks given by females towards the speaker playing the large male size variant condition were significantly greater than those given towards the speaker playing the small male size variant condition (Fig. 4). In addition, females spent significantly longer in the large male size variant proximity zone than they did in the small male size variant proximity zone (Z ¼ 2.31, N ¼ 16, P ¼ 0.021; Fig. 4). Table 1 provides the raw data for each of the female koalas in the analysis. The male exemplar used to create the playback stimuli did not affect female looking response (number of looks: F3, 8 ¼ 0.38, P ¼ 0.769; looking duration: F3, 8 ¼ 0.04, P ¼ 0.990). In addition, the amount of time females spent in the large (c23 ¼ 0:98, P ¼ 0.807) and small (c23 ¼ 1:07, P ¼ 0.785) male proximity zones was not affected by the male exemplar used to create the playback stimuli. Taken together, these results indicate that the origin of our resynthesized playback stimuli did not affect female behavioural response. Similarly, the presentation order of the stimuli did not affect female looking response (number of looks: F3, 8 ¼ 1.52, P ¼ 0.253; looking duration: F3, 8 ¼ 2.44, P ¼ 0.157) or the time spent in either proximity zone (large male: U ¼ 18, N1 ¼ N2 ¼ 8, P ¼ 0.136; small male: U ¼ 17, N1 ¼ N2 ¼ 8, P ¼ 0.110).
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Sprite Nivea Miniata Trace Donatella Snorkle Theresa Crumpet Yulara Kat Crumble Chassis Eeyore Violet Paddle Mel Mean
Number of looks Looking duration (s) Time in proximity zone (s) Large male
Small male
Large male
Small male
Large male
Small male
12 11 24 26 26 6 19 15 21 25 12 12 25 13 9 6 16.4
14 9 18 17 13 4 14 8 19 31 8 9 23 7 15 4 13.3
99.7 50.8 244.1 135.7 109.5 43.1 239.3 245.3 119.0 98.1 195.7 82.2 192.4 56.8 169.0 40.9 132.6
94.5 64.5 122.1 99.5 61.6 40.6 93.4 103.3 97.0 144.7 80.7 30.2 190.8 44.9 162.1 48.1 92.4
61.1 122.2 0.0 15.4 85.9 248.4 0.0 0.0 70.5 55.4 86.7 124.6 0.0 109.2 0.0 121.4 68.8
18.8 91.4 0.0 29.4 42.7 100.7 0.0 0.0 31.3 34.7 19.1 116.1 0.0 111.3 0.0 127.1 45.2
‘Large male’ and ‘Small male’ refer to the large and small male size variant conditions, respectively.
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A mating preference such as this could evolve and be maintained because female koalas mating with larger males would gain the genetic benefits of larger, more competitive offspring and may, at least partly, explain the correlation that exists between male reproductive success and body size in this species (Ellis & Bercovitch 2011). In addition, this type of female mate choice behaviour would result in the differential mating success of male phenotypes able to produce lower formants in their bellows, and consequently intersexual selection will favour these individuals. Indeed, the male koala’s permanently descended larynx clearly suggests strong selection pressures for callers to elongate their vocal tracts and lower formants (Charlton et al. 2011a), to maximize the acoustic impression of their body size conveyed to receivers. Since formants fulfil important functions as acoustic cues to body size both in mate choice contexts and during intrasexual assessment in humans (Feinberg et al. 2006; Puts et al. 2007) and red deer, Cervus elaphus (Reby et al. 2005; Charlton et al. 2007a), they may have similar dual utility in koalas. Future studies should investigate whether male koalas also use formants as cues to body size during agonistic interactions with rivals, to determine whether maleemale competition has provided an additional selection pressure alongside female mating preferences for broadcasting size-related formant information in this species. In addition, further studies on koalas that compare the effect of a wider range of stimulus contrasts on female preferences based on size-related formant information are also warranted. It would be particularly interesting to see whether preferences for bellows simulating larger callers persist when the size variants used for the bellow stimuli fall outside the typical adult range, and also when bellows representing small versus medium and medium versus large male size variants are presented. Such preferences would indicate the presence of directional sexual selection on this feature of male bellows, and help to explain the existence of the extremely low formant frequencies present in male bellows. Furthermore, as acoustic signals are typically composed of several components, possible interactions between them can mean that female responses to variation in single acoustic parameters may not accurately predict how they respond to variation in different combinations of acoustic components (Ryan & Rand 2003; Olvido & Wagner 2004; Scheuber et al. 2004). Indeed, recent work has shown that male rock hyrax, Procavia capensis, ‘songs’ simultaneously encode information on body weight, size, current condition, social status and hormonal state (Koren & Geffen 2009), and it seems likely that multiple levels of information are present in male koala bellows. Future studies should determine whether other cues to male quality exist in male koala bellows, and use a playback approach to show how this information interacts with formants to influence mate choice decisions. In conclusion, our findings show that formants play a key role (as acoustic cues to body size) in affecting female koala mating behaviour and, hence, that intersexual selection pressures constitute an important evolutionary force acting on the vocal displays of male koalas. More generally, by showing that female koalas use size-related formant information in a mate choice context, our results reinforce the hypothesis that the perceptual relevance of formant frequencies (which are critical for phonemic variation in human speech: Fant 1960; Titze 1994) may have initially evolved as a result of sexual selection pressures linked to the expression of information on body size (Fitch 1997). Ovulating women find male speakers with low formants particularly attractive (Feinberg et al. 2006) and oestrous female red deer prefer male roars with lower formants indicating larger callers (Charlton et al. 2007a). In the current study we have shown that intersexual selection pressures for males to broadcast size-related formant information are also present in a marsupial species. Comparative data such as this suggest that female use of formants in a mate choice context could
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