Propagation-induced song modifications

We first examined the environmental constraints impinging on the white-browed warbler's acoustic communication by analyzing the modifications affecting the song during its transmission through the forest using propagation experiments. The striking result is that the first half of the signal corresponding to the higher frequencies is embedded in the background noise after 100 m of propagation (Fig. 1b, audio S2)[18]. In addition to this propagation-induced shortening of the emitted song, amplitude and frequency modulations are also modified (correlations between control and propagated signals for amplitude envelope: at 25 m, r = 0.84; at 50 m, r = 0.56; at 100 m, r = 0.36; spectrographic cross-correlation between control and propagated signals: at 25 m, r = 0.93; at 50 m, r = 0.91; at 100 m, r = 0.68; all p<0.01, n = 6).

Species-specific information

To understand how the territorial song supports species-specific recognition, we performed playback experiments with acoustic lures. Preliminary tests (n = 4) showed that the emission of a control song - recorded from a remote individual - from inside a male's territory always elicits a strong reaction from the owner, consisting of songs in reply and approach toward the sound source. To identify which acoustic parameter(s) support(s) the species-specific message, we synthesized experimental signals with acoustic features of the original song selectively modified and performed playback tests. We found that species-specific recognition relies on the slope of the frequency modulation of the carrier frequency. A synthetic continuous pure tone mimicking the natural decreasing frequency slope of the carrier of the natural song (Fig. 1c, audio S3) induces a strong territorial behavior from challenged males (Fig. 2; post-hoc multiple comparisons revealed no significant difference between the control song and a synthetic pure tone: latency of response, Z = 0.446, p = 1.000, n = 11; approach to the speaker, Z = 0.266, p = 1.000, n = 11; calls emission rate, Z = 0.802, p = 1.000, n = 11; songs emission rate, Z = 0.294, p = 1.000, n = 11). Conversely, when all the acoustic features of the song are kept at their natural values except the frequency slope, the behavioral response decreases. Males showed a weak tolerance towards modifications of this parameter since modifications of the slope (multiplied by 1.5: audio S4, or divided by 2: audio S5, Fig. 3a,b) impair the species-specific recognition process (Fig. 2; control song compared with experimental song with FM slope multiplied by 1.5, post-hoc multiple comparisons: latency of response, Z = 3.47, p<0.01, n = 10; approach to the speaker, Z = 3,49, p<0.01, n = 10; calls emission rate, Z = 1.82, p = 1.000, n = 10; songs emission rate, Z = 0.417, p = 1.000, n = 10; control song compared with experimental song with FM slope divided by 2, post-hoc multiple comparisons: latency of response, Z = 2.95, p<0.05, n = 10; approach to the speaker, Z = 2.13, p = 0.503, n = 10; calls emission rate, Z = 3.43, p<0.01, n = 10; songs emission rate, Z = 1.76, p = 1.000, n = 10). We also found that the whole song is not necessary. If only the second half of the song was played-back (audio S6, Fig. 3c), males showed a territorial response (Fig. 2; post-hoc multiple comparisons revealed no significant difference between control compared with experimental song: latency of response, Z = 0.64, p = 1.000, n = 6; approach to the speaker, Z = 2.57, p = 0.154, n = 6; calls emission rate, Z = 0.54, p = 1.000, n = 6; songs emission rate, Z = 0.24, p = 1.000, n = 6). Thus, white-browed warbler males are highly tolerant towards song simplification in species-specific recognition and seem to disregard parameters that are substantially altered during transmission: the duration of notes and silences, the fine intra-note frequency modulation and the amplitude modulation. Moreover, they do not need to hear the first half of the song, which is likely to disappear in the background noise. We conclude that the relevant parameter is the average slope of the slow frequency modulation of the carrier frequency, a propagation-resistant parameter, enabling species identity information to be transmitted at long range.

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Figure 2. Behavioral responses to experimental signals.

(control = control song, n = 19 birds; ptone = synthetic continuous pure tone, n = 11; mul1.5 = slope multiplied by 1.5, n = 10; div2 = slope divided by 2, n = 10; 2half = second half of the song, n = 6; noAM = without AM song, n = 6). Results of Krukal-Wallis tests: latency, H (5, n = 62) = 34.38, P = 0.000; approach, H (5, n = 62) = 22.24, P = 0.000; calls, H (5, n = 62) = 15.94, P = 0.007; songs, H (5, n = 62) = 4.55, P = 0.473. *: significant difference with control (post-hoc multiple comparison tests; see text for details).

https://doi.org/10.1371/journal.pone.0001580.g002

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Figure 3. Experimental songs with modified FM slope or modified duration.

a, FM slope multiplied by 1.5. b, FM slope divided by 2. c, second half of the song.

https://doi.org/10.1371/journal.pone.0001580.g003

Individual identity information

Within the first half of the song, the frequency slope is disrupted by gaps in frequency between notes (arrows in Fig. 1a). The temporal position as well as the frequency interval of these gaps are remarkably similar within the songs of an individual while the number of gaps and their characteristics differ between individuals [18]. We deciphered the individual coding-decoding process by playback experiments. Males respond less aggressively to a neighbors' song than to an unfamiliar song (sign test, p<0.01, n = 9). As a result of playback experiments, a modification of the position of the main gap impaired the recognition of a neighbor's song (control neighbor's song compared with experimental neighbor's song with gap shifted in time, sign test, p<0.05, n = 6; control neighbor's song compared with experimental neighbor's song with gap shifted in frequency, sign test, p<0.05, n = 5; audios S7 and S8; Fig. 4). After long range transmission, the first half of the signal, and consequently the individual vocal signature, disappears into the background noise. Individuality is thus likely to be a privatized information with an “active space” restricted to the immediate surroundings of the emitting bird.

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Figure 4. Experimental songs with the position of the main frequency “gap” modified.

a, in the time domain. a, in the frequency domain.

https://doi.org/10.1371/journal.pone.0001580.g004

Localization of the singer

Study site

The study was performed in the Atlantic forest of Southeastern Brazil (Morro Grande State Reserve), during November 2000 and 2001 (temperature: 16–22°C, relative humidity: 85–100%). The white-browed warbler is a small Parulidae, feeding on insects and living in the understory [11]. The species was chosen for its high abundance and regular singing activity. In the Morro Grande reserve, the white-browed warbler show a very high IPA- Index of Point Abundance and an estimated density is about 15–20 pairs per km² [30, Vielliard, pers.obs.].

Recording and playback procedures

Recordings of wild birds were performed using an ultra-directional Sennheiser MKH 816 microphone connected to a Sony TCD-D10 DAT recorder (recording distance: less than 6 meters from the bird; sampling frequency: 48 kHz; frequency response: flat within the range 20–20000 Hz) and subsequently digitised at a sampling frequency of 22 kHz. For both propagation and playback experiments, the emission chain was made of a Sony TCD-D7 DAT tape recorder connected to a 10W customized amplifier and an Audax loudspeaker (frequency response: 100–8600±2.5 dB; sounds emitted at 90 dB_SPL measured at 1 m from the loudspeaker with a Bruel & Kjaer sound level meter type 2235 equipped with a 1/2 inch 4126 microphone, slow setting, linear scale). For propagation experiments, the reception chain was made of an omnidirectional Beyer dynamic M69 TG microphone (frequency response 150–15000 Hz±2dB), connected to a Sony TCD-D10 DAT tape recorder (sampling frequency 48 kHz, flat response within the range 20–20000 Hz).

During playbacks, two observers were hidden at a few meters from the loudspeaker. One of the observer was in charge of emitting sounds; the other was blind to condition. In the case of localization experiments (two-loudspeakers set up), there was one observer hidden close to each loudspeaker. The reaction of the bird was assessed by both observers and the distance at which the bird approached the loudspeaker was visually assessed and categorized as being more than 10 m or less than 10 m.

Sound stimuli

We built synthetic control by using the Synthesizer of Avisoft SASLab Pro software [31]. Its principle is to first use the spectrograms of natural songs as models, then scan the frequency contour and amplitude envelope at regular time intervals, and finally save the corresponding values into a WAV file. Experimental signals were made from these synthetic copies by selectively modifying acoustic values in the time and frequency domains. The synthetic continuous pure tone was built using Syntana software [32]. The 50m-propagated songs were obtained by performing transmission experiments as described below.

Propagation experiments

A synthetic copy of a representative song of the white-browed warbler was broadcast and re-recorded at 6 m high (typical song post height) over four distances (control signal: 1.5 m; propagated signals: 25, 50, and 100 m which represents the extreme diameter of a territory; propagation transect chosen to be well representative of the forest environment). We analyzed the re-recorded sounds by comparing the propagated signals with the control. To assess modifications of the main amplitude fluctuations, signals envelopes were digitally filtered (150 Hz high-pass) and averaged (n = 6) for each propagation distances. Correlations (r values) between control and propagated signals envelopes and their significance were calculated using Bravais-Pearson product-moment. To assess modifications of the frequency modulation, signals spectrograms were averaged (n = 6) for each propagation distance, and compared using the digital spectrographic cross-correlation method [33], [34].

Playback experiments 1: species-specific information

Experiments were performed between 06h15 and 10h30 (series of five consecutive experimental songs played at natural song rate), situated in the approximate center of the bird territory, at 2 meters high). Only one treatment per tested individual was done to avoid habituation (a total of 56 birds was tested). To limit pseudo-replication, synthetic copies of three songs from different individuals were used as original control songs. Modified experimental signals were made from these copies by digital synthesis using Avisoft SasLab Pro [31] and Syntana software [32]. Each of the control copies and their corresponding experimental signals were used in a balanced manner between the tested birds (i.e., one third of the birds were tested with signals built from one of the three original songs, another third of the birds were tested with signals built from another of the three original songs, the remaining birds were tested with signals built from the third original song). The behavioral responses observed during the playback trial and the three minutes following it, were compared with the bird behavior during the three minutes before the playback. Four response measures were scored:

• latency of response –in terms of approach, song and/or calls emitted in reply- (score 0: no response within 3 minutes after the end of the playback session; score 1: response between 1 and 3 minutes after the playback session; score 2: response during playback or within 1 minute after the playback session);
• approach to the speaker (score 0: none; score 1: >10 m; score 2: <10 m);
• variation of call emission rate (score 0: no variation; score 1: weak variation, i.e. up to 3 times the initial calling rate in terms of number of calls/minute; score 2: strong variation, i.e. more than 3 times the initial calling rate);
• variation of song emission rate (score 0: no variation; score 1: weak variation, i.e. until 3 times the initial singing rate in terms of number of songs/minute; score 2: strong variation, i.e. more than 3 times the initial singing rate).

Responses to control song and experimental signals were compared using Kruskal-Wallis tests followed by post-hoc non parametric multiple comparisons [35].

Playback experiments 2: individual information

All the birds tested here were different from the ones used in the previous experiments. Each experiment consisted of two consecutive trials (paired test, one experiment per tested bird, n = 20), one presenting an original neighbor's song (control) and one presenting either an unfamiliar song or a modified neighbor's song (experimental). Each trial was composed of a series of five consecutive songs played at natural song rate; the loudspeaker being situated at the border between the tested bird territory and its neighbor's one, at 2 meters high. The order of trials presentation was balanced between the tests (time interval: at least 10 minutes). Behavioral responses to playback were assessed independently for each trial using the following behavioral scale: 1: the tested bird answered vocally to playback signals and approached at less than 10 meters from the loudspeaker, 2: the tested bird answered vocally but did not approach, 3: the tested bird showed no reaction to playback. A sign test was used to compare responses between neighbor's song and unfamiliar or modified song. If contiguous neighbors were tested with this protocol, tests were performed on different days and using different stimuli to avoid habituation.

Playback experiments 3: ranging

All the birds tested here were different from those in the other experiment and each individual was challenged only once (n = 18). Experimental songs were recorded from remote individuals (not neighbors). Behavioral response to the experimental signal was assessed in term of latency of departure from the first loudspeaker (‘immediate departure’, i.e., departure during the emission of the experimental signal, or ‘delayed departure’, i.e., after the end of the experimental signal playback), and in term of approaching distance to the second loudspeaker (‘close approach’, i.e., at less than 10 m, or ‘distant approach’, i.e. more than 10 m). Fisher's exact test was used for comparisons.

Playback experiments 4: azimuth assessment

The procedure was identical to that of experiment 3, except that the experimental signal emitted by the second loudspeaker was either a control song, either a song without AM (n = 18 birds tested, different than in previous experiments). Besides latency of departure and distance of approach, behavioral response was also assessed in term of directionality (‘correct direction’: the birds flew directly, with a precision of ± 20°, towards the second loudspeaker in reaction to the experimental signal; ‘incorrect direction’: the birds flew in a direction different of more than 20° to the second loudspeaker). Directionality was assessed by the observer situated close to the first loudspeaker. Fisher's exact test was used for comparisons.

All statistical tests have been performed using STATISTICA 6.1 software.