Electronic supplementary material

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  1. Arena design and electronic equipment

The tests were conducted in an indoor 1.5×1.5-meter arena (Figure 1) to control for ambient noise, light and weather conditions. Visual stimuli were broadcast from two 20” LCD computer screens (Samsung Syncmaster 2032 BW, screen resolution: 1680 x 1050 pixels) connected to a Pavillion dv5000 notebook computer via a 4-port Abix video splitter. Both screens had a wide viewing angle (170°) to allow animals to view stimuli from a large range of positions, and were covered with matt black foam board except for a 5×3 cm rectangle where the visual stimulus was displayed and which was covered with a 40% neutral density filter to reduce light intensity. The screens were set 1 meter apart, forming a triangle with the frog such that the angle of the screens relative to the point where the animal was placed for habituation was 60°.

Directly in front of each screen, under the floor of the arena and covered with thin wire netting to allow good sound propagation, we placed a Monacor SP7 loudspeaker. These were connected to the notebook computer via a stereo Stageline 102 amplifier. Call amplitude was calibrated at the female release point in the centre of the arena using a MT-8820 Sound Level Meter (Voltcraft©, C-weighted, ‘fast’ root-mean-square (RMS) setting, tolerance ± 1.5 dB).

The acoustic stimuli were broadcast antiphonally throughout the test with a 1.5-second interval of silence between alternating call bouts, meaning there were effectively no leading or following calls (a factor we have shown affects mate choice in Hyla arborea, Richardson et al. 2008).

The frogs’ movements within the arena were recorded using a Sony HDR-SR7E infrared camera.

Figure 1: Experimental setup used to conduct audiovisual choice tests

  1. Constructing the acoustic stimuli

The aim of this study was to determine whether visual cues can affect female perception of the attractiveness of male acoustic cues. We therefore needed to construct male calls with contrasting attractiveness. In a previous study (Richardson et al. subm.), we established the distribution within the study population of several call parameters and found through two-stimuli choice tests a significant female preference for calls with (i) a high amplitude, (ii) a low peak frequency (i.e. the frequency with the highest energy content), and (iii) a high call rate.

From our digital laboratory recordings of male H. arborea calls, we selected a representative call (16-bit, 16 kHz) with mean population values for duration (55 ms). Using Avisoft SAS-lab Pro software (Avisoft Bioacoustics) we then adjusted peak frequency and amplitude of this representative call to synthesize the following calls (see figure 2 to visualize the sonograms of a natural advertisement call bout of H. arborea and an artificial call bout used in this study):

- an “unattractive call” with a peak frequency value corresponding to that of the 9th decile of the population distribution (2.87kHz), and an amplitude value corresponding to that of the 1st decile of the population distribution (85dB at 1m).

- an “attractive call” with a peak frequency value corresponding to that of the 1st decile of the population distribution (2.31kHz), and an amplitude value corresponding to that of the 9th decile of the population distribution (91dB at 1m).

Using these calls we then constructed with Avisoft SAS-lab Pro the two acoustic stimuli used in this study (call parameter values used are summarized in table 1 below):

  • an “all attractive call bout”: a 3.4s bout (mean population value for bout length) containing 25 “attractive calls” emitted at a rate of 7.35 calls/s (call value for the 9th decile of the population distribution).

  • an “all unattractive call bout”: a 3.4s containing 18 “unattractive calls” emitted at a rate of 5.29 calls/s (call value for the 1st decile of the population distribution).

Using the audio editing software Cool Edit Pro we finally mounted these two call bouts onto a stereo track so that the two call bouts were broadcast antiphonally with a 1.5-second interval of silence between alternating bouts.

Figure 2. Sonograms of (a) the first 5 calls of a natural advertisement call bout of H. arborea and (b) the first 5 calls of an artificial call bout used in this study.
Table 1: Parameter values used for the acoustic stimuli

Call parameter

Parameter value for the attractive call bout

Parameter value for the unattractive call bout

Bout length (s)



Call rate (calls/s)



Call duration (ms)



Peak frequency (kHz)



Amplitude (dB)



  1. Construction of the visual stimuli

Video screens are designed for human vision and fail to render natural colours as perceived by animals with a different visual sensitivity. Modifying video colours to create a perceptual match for the animal vision between ‘natural’ and ‘video’ stimuli is a crucial procedure when using video playbacks (Fleishman et al. 1998). This was a two-step procedure: (i) determination of the colour signals naturally encountered in males in the Ile Crémieu metapopulation. (ii) adjustment of the RGB values of each colour patch on the videos to render colours as perceived by frogs at night. We did so using Matlab and Adobe Premiere.

First, we measured the natural variation of male coloration in the local population. A total of 125 males had been captured from the Ile Crémieu metapopulation two years before the experiment. Their back, belly and vocal sac colouration had been measured in reflectance spectrometry, with a spectrometer (Avantes AvaSpec-3648-SPU2), a deuterium-halogen light source (Avantes AvaLight-DHS) and a coaxial optic fibre (Avantes FCR-7UV200-2-45-ME). Measurements were done relative to a white reference (Spectralon 99%) and black noise. Male vocal sac coloration varied from light orange to dark red; vocal sac coloration being partly based on carotenoids (Richardson et al. 2009), the darker and redder coloration likely corresponded to an increase carotenoid pigmentation. We analysed spectra using AVICOL (Gomez 2006) and obtained the spectra corresponding to the coloration of a pale (light orange, 1st quartile), the average and an intensely coloured (dark red, 4th quartile) vocal sac.

Second, we video recorded a calling male and modified video colours to render male natural coloration as it would be perceived by frogs under moonlight. Whatever the mechanism of nocturnal vision, the natural and video stimuli would be perceived as identical if they elicited a similar response for each class of photoreceptor. Colours were matched for the two rod classes identified in frogs and involved in night vision by using information about maximal rod sensitivity (peaks 435 nm and 503 nm in Hyla cinerea King et al. 1993), templates established for vertebrate rod pigments (Govardovskii et al. 2000) and lens transmission spectrum established for the Northern leopard frog Rana pipiens, the only anuran species for which this data was available (Kennedy & Milkman 1956). All details concerning these procedures can be found in Gomez et al. (2009). We finally controlled that the light intensity delivered at the female release point in the arena was similar to full moonlight conditions by using a luminancemeter (LMT L1009). Direct measurements of light intensity at 1m from a screen ranged from 8 10-3 lux for pale orange to 12 10-3 lux for the dark red stimulus. As a reference, full moon light intensity computed from Warrant (2004) was 16 10-3 lux.

We performed a preliminary experiment to test whether females were sensitive to the presence of vocal sac vibrations. Females exhibited no significant preference for a video of a male with a vibrating inflated vocal sac over a male with an inflated but static vocal sac, when presented with identical acoustic stimuli and identical average vocal sac coloration (only 16 out of 25 chose the vibrating stimulus, P=0.23 using a two-tailed binomial test). We therefore constructed all video playbacks with males showing an inflated vocal sac with regular breathing movements but no vibration.

  1. Female utilization

Because of the scarcity of females available during one breeding season, each female was used in several tests (in random order), following the subsequent rule: if a female failed to make an effective choice during a test, the trial was discarded and the female was retested later in the night in order to obtain one valid response for each of the two experiments. Thus each female never provided more than one data point for each of the 2 experiments.

There was a minimum time-out of 20 min for females between two successive tests, during which they were held individually in dark plastic boxes (10×7×7cm, L×W×H) containing a thin layer of water, and exposed to a recording of a conspecific chorus (we looped a 10-minute recording of a small chorus, ca. 15 males, made on the study site in 2005) broadcast from an Hitachi CX70 CD player at an amplitude of 50 dB.

Control experiments have demonstrated that the phonotactic behaviour of female hylids (H. cinerea and H. versicolor) is not biased by previous two-stimulus testing (Gerhardt et al. 2000), a result which can likely be generalized to H. arborea. Because we analyzed the results of each experiment independently, our using each female in several experiments did not violate requirements for independence of replicates.

Fleishman, L. J., McClintock, W. J., D'Eath, R. B., Brainard, D. H. & Endler, J. A. 1998 Colour perception and the use of video playback experiments in animal behaviour. Anim. Behav., 56, 1035-1040.

Gerhardt, H. C., Tanner, S. D., Corrigan, C. M. & Walton, H. C. 2000 Female preference functions based on call duration in the gray tree frog (Hyla versicolor). Behav. Ecol., 11, 663-669.

Gomez, D. 2006 AVICOL, a program to analyse spectrometric data. Available from the author upon request at dodogomez@yahoo.fr.

Gomez, D., Richardson, C., Lengagne, T., Plenet, S., Joly, P., Léna, J.-P. & Théry, M. 2009 The role of nocturnal vision in mate choice: females prefer conspicuous males in the European tree frog (Hyla arborea). Proc. R. Soc. Lond. B, 276, 2351-2358.

Govardovskii, V. I., Fyhrquist, N., Reuter, T., Kuzmin, D. G. & Donner, K. 2000 In search of the visual pigment template. Visual Neuroscience, 17, 509-528.

King, R. B., Douglass, J. K., Phillips, J. B. & Baube, C. L. 1993 Scotopic Spectral Sensitivity of the Optomotor Response in the Green Treefrog Hyla cinerea. J. Exp. Zool., 267, 40-46.

Kennedy, D. & Milkman, R. D. 1956 Selective light absorption by the lenses of lower vertebrates, and its influence on spectral sensitivity. Biol. Bull., 111, 375–386. (doi:10.2307/1539144)

Richardson, C., Lena, J. P., Joly, P. & Lengagne, T. 2008 Are leaders good mates? A study of call timing and male quality in a chorus situation. Anim. Behav., 76, 1487-1495.

Richardson, C., Popovici, J., Bellvert, F. & Lengagne, T. 2009 Conspicuous coloration of the vocal sac of a nocturnal chorusing treefrog: carotenoid-based? Amph.-Rept., 30, 576-580.

Richardson, C., Joly, P., Lena, J.-P., Plenet, S. & Lengagne, T. Submitted. Use of multiple acoustic cues in mate choice in a chorusing anuran, the European treefrog Hyla arborea.

Warrant, E. 2004 Vision in the dimmest habitats on Earth. J. Comp. Phys. A, 190, 765–789. (doi:10.1007/s00359-004-0546-z)

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