Female natural call in the laboratory
Prior to ovulation at night, captive gravid females (O. graminea) in an indoor arena produce simple calls repeatedly about once every 10 minutes. The female calls immediately stimulate male vocalizations. The audio and video recordings clearly show that the female’s natural calls induce receptive males’ antiphonal responses followed by phonotactic movements, finally even resulting in amplexus. Therefore, such a call has been shown to be a highly attractive stimulus, called as ‘female courtship call’ (abbr. FCC). The FCC is a single note, or sometimes a ‘two-note’ call, of shallow frequency-modulated stacks. The fundamental harmonic sweeps from 4.47 ± 0.57 kHz downward to 2.68 ± 0.61 kHz, across the duration (26.5 ± 8.3 ms, n = 82), with an intensity 85 dB SPL measured at a distance of 50 cm above the female frog’s head, much above the ambient noise level at the calling site, that averages from 58 to more than 70 dB SPL in the frequency range between 50 and 4000 Hz. As shown in Fig. 1, a single-note FCC has several harmonics, the frequencies of the fundamental harmonic (F0 or F1) to fifth harmonic (F5) marked by the red vertical line were 5.2 kHz, 10.4 kHz, 15.6 kHz, 20.8 kHz, and 26.0 kHz, respectively, and the call duration was ca. 40 ms with energy extending into ultrasonic range (up to 36 kHz). The ‘two-note’ call has approximately the same spectrum as a single note FCC, and the second note (FCC2) has a shorter duration (the difference about 8 ms), is less intense (the difference about 5 dB), with an inter-note interval between FCC1 and FCC2 of 255 ± 47 ms (n = 14).
Male antiphonal calls in the laboratory
When a male frog in captivity in a quiet darkened room was separated by the black cotton gauze mesh with a gravid female frog in another small chamber, the male was often induced to vocalize by the female courtship call. Six examples of immediately evoked antiphonal responses (abbr. AR) are shown in Fig. 2. The latencies to the male’s first AR were 0.2 s, 0.35 s, 2.7 s, 1.5 s, 1.6 s, and 1.2 s, respectively, measured from the onset of the FCC or the first note of female call (FCC1).
The AR was generally a short upward-downward tonal call with a duration of about 60-85 ms (e.g. Fig. 2a, b), exhibiting a number of harmonics and different F0, although recorded from the same male. The mean F0 of a single AR note was 4.47 kHz, maximum F0 4.75 kHz, minimum F0 4.15 kHz, as shown in Fig. 2a, and the frequency of the harmonics marked by the red vertical line was 4.9 kHz, 9.8 kHz, 14.7 kHz and 19.6 kHz, respectively. In Fig. 2b, the mean F0 of a single AR was 5.66 kHz, maximum F0 6.26 kHz, minimum F0 4.89 kHz, and the frequency of these harmonics marked by the red vertical line was 6.3 kHz, 12.6 kHz, 18.9 kHz and 25.2 kHz, respectively.
Sometimes the ARs were short frequency-modulated (FM) calls, frequently followed by a multi-note long call with a duration of 600~800 ms, including 4-5 separate notes, as shown in Fig. 2c-f. The analysis results show that the mean F0 of an individual AR note varied. For example, the ARs were often quite complex, when evoked by a single FCC (Fig. 2c, f) or a ‘two-note’ call (FCC1 and FCC2), as in Fig. 2d, e.
The mean F0 of eight elicited AR notes in Fig. 2c was 3.0 kHz, 2.6 kHz, 0.6 kHz, 4.1 kHz, 4.7 kHz, 5.9 kHz, 4.3 kHz and 4.5 kHz, respectively. In Fig. 2d, the mean F0 for seven elicited AR notes was 2.6 kHz, 2.6 kHz, 3.6 kHz, 3.9 kHz, 5.0 kHz, 4.9 kHz and 5.8 kHz, respectively. In Fig. 2e, the mean F0 for five elicited AR notes was 2.6 kHz, 2.7 kHz, 3.1 kHz, 4.0 kHz, and 3.5 kHz, respectively. In Fig. 2f, the mean F0 for eight elicited AR notes was 2.2 kHz, 1.8 kHz, 2.0 kHz, 2.3 kHz, 2.6 kHz, 3.3 kHz, 11.6 kHz and 12.7 kHz, respectively. Moreover, the last long call in Fig. 2c-f was like a singing tenor- characterized by pronounced and varying frequency modulation patterns, and occurrence of nonlinear phenomena (i.e., frequency jumps, subharmonics, and deterministic chaos), and having energy in the ultrasonic range up to ca. 42 kHz. As far as we know, only males of O. graminea have such a complicated and varied antiphonal response. Its functional significance may be an important trait that transmits a male’s fitness to the female. These ARs were rare in other anuran species (Ryan 1985; Narins et al. 2000).
Male phonotaxis in the laboratory
We found that in a quiet, dark indoor arena, newly captured males were most responsive during the phonotaxis experiments. In response to the FCC stimuli at a playback level of 85 dB SPL, in addition to vocal responses evoked, males rapidly and accurately localized the loudspeaker. The representative phonotactic trajectories elicited from the males by the FCC playback are illustrated in Fig. 3. The FCC playback resulted in the attraction of receptive males to the speaker. Analysis of the speaker-derived phonotactic paths indicated that males of O. graminea are mainly characterized by fast response with a minimum latency of less than 1 s, a long jump with a maximum distance more than 1 meter, and extraordinary localization acuity. Upon hearing the FCC playback, a receptive male immediately turned his body towards the speaker and made a long jump (range: ca. 60-100 cm) toward the speaker. Most males would go directly to or near the loudspeaker, sometimes touching the foam on the wall above the loudspeaker, and falling. A small number of males jumped a short distance (ca. 30 cm) and then quickly crawled toward the playback speaker. When the male frog heard the second or third acoustic stimulus (FCC playback), it would immediately orient itself, adjust its trajectory, take a big leap, and make contact with the speaker, possibly leading to amplexus if the sound source were a live gravid female frog. Therefore, we believe that the FCC is a salient stimulus and has a significant attraction to males.
The precision of the long-distance jumps (more than 30 cm) of receptive male O. graminea was remarkable, with an average azimuthal error of jumps just 0.7° ± 0.8° (n = 14), even smaller than that in male concave-eared frogs (Shen et al. 2008). This accuracy suggests that large odorous frogs have the capacity to localize their mates in noisy, dark habitats.