© 2015. Published by The Company of Biologists Ltd | The Journal of Experimental Biology (2015) 218, 2442-2447 doi:10.1242/jeb.119552 RESEARCH ARTICLE A Chinese alligator in heliox: formant frequencies in a crocodilian Stephan A. Reber1,*, Takeshi Nishimura2, Judith Janisch1, Mark Robertson3 and W. Tecumseh Fitch1,* ABSTRACT perceived as honest signals of body size (Budka and Osiejuk, 2013; Crocodilians are among the most vocal non-avian reptiles. Adults of Fitch and Kelley, 2000). However, there is little evidence for formants both sexes produce loud vocalizations known as ‘bellows’ year round, in anurans (Rand and Dudley, 1993), and to date formants have not with the highest rate during the mating season. Although the specific been shown to play a role in the vocalizations of non-avian reptiles. function of these vocalizations remains unclear, they may advertise Crocodilians are among the most vocal non-avian reptiles the caller’s body size, because relative size differences strongly affect (Britton, 2001; Campbell, 1973), and adults of either sex produce ‘ ’ courtship and territorial behaviour in crocodilians. In mammals and loud vocalizations, called bellows , year round with the highest birds, a common mechanism for producing honest acoustic signals of occurrence during the mating season (Vergne et al., 2009). It has body size is via formant frequencies (vocal tract resonances). To our been suggested that bellows serve as advertisement calls, carrying knowledge, formants have to date never been documented in any cues to sex and size (Garrick and Lang, 1977). In all extant non-avian reptile, and formants do not seem to play a role in the crocodilian species, relative size differences strongly affect both vocalizations of anurans. We tested for formants in crocodilian courtship and agonistic behaviour. Females only accept males larger vocalizations by using playbacks to induce a female Chinese alligator than themselves as mates (Brazaitis and Watanabe, 2011), and size (Alligator sinensis) to bellow in an airtight chamber. During reliably predicts the outcome of agonistic interactions (Tucker et al., vocalizations, the animal inhaled either normal air or a helium/ 1998; Webb et al., 1983). Accurate acoustic cues to body size would ’ oxygen mixture (heliox) in which the velocity of sound is increased. facilitate several aspect of crocodilians social behaviour, because Although heliox allows normal respiration, it alters the formant both sexes compete over breeding grounds and mates (Garrick and distribution of the sound spectrum. An acoustic analysis of the calls Lang, 1977). In some crocodilian species, bellow displays differ showed that the source signal components remained constant under between sexes; for example, in American alligators (Alligator both conditions, but an upward shift of high-energy frequency bands mississippiensis), only males appear to exhibit sub-audible ‘ ’ was observed in heliox. We conclude that these frequency bands vibrations (visible by the water dance ) before the audible bellow represent formants. We suggest that crocodilian vocalizations could (Vliet, 1989). Additionally, bellowing seems to stimulate equivalent thus provide an acoustic indication of body size via formants. Because vocal responses in other conspecifics and to attract individuals birds and crocodilians share a common ancestor with all dinosaurs, a towards the sound source (Wang et al., 2009b). Finally, bellows are better understanding of their vocal production systems may also rather noisy vocalizations with a broad energy spectrum (Garrick provide insight into the communication of extinct Archosaurians. and Lang, 1977), which would make these calls well suited to indicate size with formants. KEY WORDS: Bioacoustics, Source-filter theory, Alligator sinensis, No study to date has demonstrated that crocodilian bellows Vocal tract resonance, Bellow, Archosauria exhibit formants; indeed, the existence of formants has not been demonstrated in any non-avian reptile. A powerful method to INTRODUCTION identify the presence of formants is to compare an animal’s In many vertebrates, long-distance vocalizations convey cues to a vocalizations produced in the atmosphere conditions of ambient air caller’s physical attributes, such as body size (Hardouin et al., 2007, and heliox, a helium/oxygen mixture (Herrsch, 1966; Madsen et al., 2014; Harris et al., 2006; Vannoni and McElligott, 2008). In 2012; Nowicki, 1987; Rand and Dudley, 1993; Roberts, 1975). mammals, formant frequencies (vocal tract resonances) represent a Because helium is lighter than air, the speed of sound is higher in widespread mechanism for producing such honest acoustic signals heliox, shifting resonances in a gas-filled cavity upward in (Charlton et al., 2012, 2011; Fitch and Hauser, 2002; Reby et al., frequency (Koda et al., 2012). Although heliox allows normal 2005). Formants are often negatively correlated with a caller’s vocal respiration, it sharply alters the distribution of formants in the sound tract length and hence its size (Fitch, 1997; Reby and McComb, 2003; spectrum (Beil, 1962). In contrast, frequency bands produced by the Riede and Fitch, 1999). Bird vocalizations possess formants sound source (e.g. vocal fold or other tissue vibrations) will remain (Nowicki, 1987) and although they have several functions, it is unchanged in the two conditions. The heliox method can thus thought that in at least some speciesthey can serve (Fitch, 1999; Hinds clearly disentangle the independent contributions of the source and and Calder, 1971; Jones and Witt, 2014; Miller et al., 2007) and be the filter to the acoustic output (Fant, 1960; Holywell and Harvey, 1964). The two are currently not known to be coupled in vertebrate 1Department of Cognitive Biology, University of Vienna, Vienna 1150, Austria. vocalizations. By means of the heliox method it was shown, for 2Primate Research Institute, Kyoto University, Inuyama, Aichi 484-8506, Japan. 3St Augustine Alligator Farm Zoological Park, St Augustine, FL 32080, USA. example, that the vocal sac of four species of anurans does not act as a cavity resonator (Rand and Dudley, 1993) and that source *Authors for correspondence ([email protected]; characteristics do not shift frequency appreciably in heliox in [email protected]) humans (Beil, 1962), gibbons (Koda et al., 2012), bats (Hartley and This is an Open Access article distributed under the terms of the Creative Commons Attribution Suthers, 1988; Roberts, 1973) or birds (Brittan-Powell et al., 1997; License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. Gaunt et al., 1987; Nowicki, 1987). In this study, we sought evidence for formants in crocodilians by Received 17 January 2015; Accepted 19 May 2015 comparing bellows produced in air and in heliox by a female The Journal of Experimental Biology 2442 RESEARCH ARTICLE The Journal of Experimental Biology (2015) 218, 2442-2447 doi:10.1242/jeb.119552 Chinese alligator (Alligator sinensis Fauvel 1879). As relatively Table 1. Acoustic parameters of the bellows emitted in ambient air small but highly vocal crocodilians, Chinese alligators are well and heliox suited for such experiments (Garrick, 1975; Wang et al., 2009a, Air Heliox 2007). We induced our subject (total length 125 cm) to bellow in an Day 1 Day 2 Day 1 Day 2 airproof chamber using playbacks of its own vocalizations. The alligator breathed either ambient air or heliox (88% helium, 12% N (PF) 27 16 9 38 oxygen). We calculated the speed of sound for these two gas PF (Hz) 36.7±7.5 37.6±6.5 32.7±9 38.4±7 ’ N (others) 34 24 21 47 mixtures and estimated the subject s vocal tract length using the DF (Hz) 138.8±36.3 155±98.4 121.2±8.7 131.6±8.7 externally measured head length (16 cm). Based on these values, Intensity (dB) 66.6±1.8 69.1±2.5 65.2±3 68.7±1.2 we performed an automated acoustic analysis of the recordings. Our Duration (s) 0.43±0.05 0.43±0.03 0.4±0.05 0.47±0.07 aims were (1) to identify a source signal, which should remain N, number of bellows analysed; PF, pulse frequency; DF, dominant frequency. unchanged in the two conditions (Taylor and Reby, 2010), and (2) PF, DF, relative intensity (sound pressure level, SPL) and duration data are to determine whether the calls contained formant frequencies, means±s.d. PF could not be measured in all recordings, hence the reduced which should be significantly altered in the two different sample size. atmospheres. (Z=−11.32, P<0.001) were consistently and significantly higher in RESULTS the heliox than in the normal air condition (Table 2). Playbacks were highly effective in inducing bellowing in our Next, a subsample of call recordings with a high signal-to- subject: typically a bout of two call playbacks elicited a bellowing noise ratio was selected from both treatments for a more detailed response. The animal bellowed in every section of the experiment analysis. The first two formants were measured (Query: Formant on two testing days. For all analysis steps, the recordings from these listing) by manually adjusting the settings to create a good visual 2 days were pooled and compared by atmosphere treatment. overlap between the formant frequencies as indicated in Praat’s editing window and the frequency bands of high energy visible in Source signal, intensity and duration the spectrogram. The first formant was identified on average Bellows of Chinese alligators are relatively chaotic vocalizations at 425.7±46.0 Hz (mean±s.d.) in air and at 825.1±26.1 Hz in with very little harmonic signal, and the fundamental frequency heliox. For the second formant, the average frequencies were at could not be measured with confidence. However, the bellows do 1618.4±112.7 Hz in air and at 3155.4±119.5 Hz in heliox. typically show a pulsed structure around 35 Hz, which was visible in Both formants were significantly higher in heliox than in air the spectrogram.