Beyond sound: bimodal acoustic calls used in mate-choice and aggression by red-eyed treefrogs.

Airborne sound signals function as key mediators of mate-choice, aggression and other social interactions in a wide range of vertebrate and invertebrate animals. Calling animals produce more than sound, however. When displaying on or near a solid substrate, such as vegetation or soil, they also unavoidably excite substrate vibrations because of the physics of sound production and of acoustic propagation, and these vibrations can propagate to receivers. Despite their near ubiquity, these vibrational signal components have received very little research attention and in vertebrates it is unknown whether they are relevant to mate-choice, an important driver of evolutionary divergence. Here, we show that female red-eyed treefrogs are more than twice as likely to choose a male mating call when airborne sound is paired with its corresponding substrate vibrations. Furthermore, males of the same species are more aggressive towards and display a greater range of aggressive behaviors in response to bimodal (sound and vibration) versus unimodal (sound or vibration alone) calls. In aggressive contexts, at least, air- and substrate-borne signal components function non-redundantly. These results are a clear demonstration that vibrations produced by a calling animal can function together with airborne sound to markedly enhance the function of a signal. If this phenomenon proves widespread, this finding has the potential to substantially influence our understanding of the function and evolution of acoustic signals.

Collateral damage or a shadow of safety? The effects of signalling heterospecific neighbours on the risks of parasitism and predation.

Although males often display from mixed-species aggregations, the influence of nearby heterospecifics on risks associated with sexual signalling has not been previously examined. We tested whether predation and parasitism risks depend on proximity to heterospecific signallers. Using field playback experiments with calls of two species that often display from the same ponds, túngara frogs and hourglass treefrogs, we tested two hypotheses: (1) calling near heterospecific signallers attractive to eavesdroppers results in increased attention from predatory bats and parasitic midges (collateral damage hypothesis) or (2) calling near heterospecific signallers reduces an individual's predation and parasitism risks, as eavesdroppers are drawn to the heterospecifics (shadow of safety hypothesis). Bat visitation was not affected by calling neighbours. The number of frog-biting midges attracted to hourglass treefrog calls, however, rose threefold when played near túngara calls, supporting the collateral damage hypothesis. We thus show that proximity to heterospecific signallers can drastically alter both the absolute risks of signalling and the relative strengths of pressures from predation and parasitism. Through these mechanisms, interactions between heterospecific guild members are likely to influence the evolution of signalling strategies and the distribution of species at both local and larger scales.

Inherent Directionality Determines Spatial Release from Masking at the Tympanum in a Vertebrate with Internally Coupled Ears.

In contrast to humans and other mammals, many animals have internally coupled ears that function as inherently directional pressure-gradient receivers. Two important but unanswered questions are to what extent and how do animals with such ears exploit spatial cues in the perceptual analysis of noisy and complex acoustic scenes? This study of Cope's gray treefrog (Hyla chrysoscelis) investigated how the inherent directionality of internally coupled ears contributes to spatial release from masking. We used laser vibrometry and signal detection theory to determine the threshold signal-to-noise ratio at which the tympanum's response to vocalizations could be reliably detected in noise. Thresholds were determined as a function of signal location, noise location, and signal-noise separation. Vocalizations were broadcast from one of three azimuthal locations: frontal (0 °), to the right (+90 °), and to the left (-90 °). Masking noise was broadcast from each of 12 azimuthal angles around the frog (0 to 330 °, 30 ° separation). Variation in the position of the noise source resulted in, on average, 4 dB of spatial release from masking relative to co-located conditions. However, detection thresholds could be up to 9 dB lower in the "best ear for listening" compared to the other ear. The pattern and magnitude of spatial release from masking were well predicted by the tympanum's inherent directionality. We discuss how the magnitude of masking release observed in the tympanum's response to spatially separated signals and noise relates to that observed in previous behavioral and neurophysiological studies of frog hearing and communication.

Spatial hearing in Cope's gray treefrog: I. Open and closed loop experiments on sound localization in the presence and absence of noise.

The ability to reliably locate sound sources is critical to anurans, which navigate acoustically complex breeding choruses when choosing mates. Yet, the factors influencing sound localization performance in frogs remain largely unexplored. We applied two complementary methodologies, open and closed loop playback trials, to identify influences on localization abilities in Cope's gray treefrog, Hyla chrysoscelis. We examined localization acuity and phonotaxis behavior of females in response to advertisement calls presented from 12 azimuthal angles, at two signal levels, in the presence and absence of noise, and at two noise levels. Orientation responses were consistent with precise localization of sound sources, rather than binary discrimination between sources on either side of the body (lateralization). Frogs were unable to discriminate between sounds arriving from forward and rearward directions, and accurate localization was limited to forward sound presentation angles. Within this region, sound presentation angle had little effect on localization acuity. The presence of noise and low signal-to-noise ratios also did not strongly impair localization ability in open loop trials, but females exhibited reduced phonotaxis performance consistent with impaired localization during closed loop trials. We discuss these results in light of previous work on spatial hearing in anurans.

Spatial hearing in Cope's gray treefrog: II. Frequency-dependent directionality in the amplitude and phase of tympanum vibrations.

Anuran ears function as pressure difference receivers, and the amplitude and phase of tympanum vibrations are inherently directional, varying with sound incident angle. We quantified the nature of this directionality for Cope's gray treefrog, Hyla chrysoscelis. We presented subjects with pure tones, advertisement calls, and frequency-modulated sweeps to examine the influence of frequency, signal level, lung inflation, and sex on ear directionality. Interaural differences in the amplitude of tympanum vibrations were 1-4 dB greater than sound pressure differences adjacent to the two tympana, while interaural differences in the phase of tympanum vibration were similar to or smaller than those in sound phase. Directionality in the amplitude and phase of tympanum vibration were highly dependent on sound frequency, and directionality in amplitude varied slightly with signal level. Directionality in the amplitude and phase of tone- and call-evoked responses did not differ between sexes. Lung inflation strongly affected tympanum directionality over a narrow frequency range that, in females, included call frequencies. This study provides a foundation for further work on the biomechanics and neural mechanisms of spatial hearing in H. chrysoscelis, and lends valuable perspective to behavioral studies on the use of spatial information by this species and other frogs.

The Response of Gray Treefrogs to Anesthesia by Tricaine Methanesulfonate (TMS or MS-222).

The design of anesthetic protocols for frogs is commonly hindered by lack of information. Results from fishes and rodents do not always apply to frogs, and the literature in anurans is concentrated on a few species. We report on the response of treefrogs (Hyla chrysoscelis and H. versicolor) to tricaine methanesulfonate. Body mass did not differ significantly between the species or between sexes. In the first exposure of a frog to TMS, variation in induction time was best explained by species (H. chrysoscelis resisted longer) and body mass (larger animals resisted longer). Multiple exposures revealed a strong effect of individual variation on induction time and a significant increase of induction time with number of previous anesthesia events within the same day. Recovery time was mostly explained by individual variation, but it increased with total time in anesthetic and decreased with induction time. It also increased with number of days since the last series of anesthesias and decreased with number of previous uses of the anesthetic bath. This is one of the first studies of anesthesia in hylids and also one of the first assessments of the factors that influence the variability of the response to anesthesia within a species.

Vibrational signaling in the agonistic interactions of red-eyed treefrogs.

Sensitivity to substrate-borne vibrations is widespread in animals and evolutionarily precedes hearing but, compared with other sensory modalities, we know little about vibrational communication, particularly in vertebrates. For plant-dwelling arthropods, vibrations are likely as important as sound. Arboreal vertebrates excite plant vibrations with most movements, but the behavioral relevance of these vibrations has not been tested experimentally. In playback experiments using a robotic model frog and an electrodynamic shaker, we demonstrate that plant-borne vibrations generated by the shaking (tremulation) display of male red-eyed treefrogs (Agalychnis callidryas) are a vibrational signal, necessary and sufficient to elicit tremulations in response. A trend toward increased aggression during visual playbacks suggests that the visual component of tremulations may also convey information. In male-male contests, tremulations were the most frequent aggressive display, and their use and vibrational characteristics varied with male size and conflict context. Nearly all of A. callidryas' signaling behaviors, including tremulations and acoustic calls, excite strong, stereotyped vibrations that travel through plants and could be informative to receivers. Our results demonstrate that vibrational signals serve a key role in the biology of one well-known arboreal frog and suggest that consideration of the vibrational modality may significantly broaden our appreciation of the behavior and evolution of arboreal vertebrates.

Frequency information in the vibration-cued escape hatching of red-eyed treefrogs.

Incidental acoustic and vibrational cues generated by predators are a potential source of information for prey assessing risk. Substrate vibrations should be excited by most predators, and frequency, amplitude or temporal properties could allow prey to distinguish predator from benign-source vibrations. Red-eyed treefrog embryos detect egg predators using vibrations excited during attacks, hatching rapidly and prematurely to escape. We recorded vibrations in egg clutches during attacks by five species of predators and three common types of benign physical disturbance. We analyzed their frequency distributions to assess if and how frequency properties could be used to discriminate between vibration sources and used vibration playbacks to examine the effects of frequency properties on the escape hatching response. Vibrations produced by predators and benign disturbances generally have broad and overlapping frequency distributions, and all frequencies excited by attacks are also excited by benign disturbances. Decision rules based on the frequency distribution of vibrations alone would therefore result in either high levels of hatching in response to benign vibrations (false alarms) or common failures to hatch in response to predators (missed cues). Nevertheless, embryos hatch in response to predator and not benign disturbances in nature, and our playback results show that vibration frequency information is an important component of their hatching decision. Embryos combine frequency with temporal information to refine their hatching response. Moreover, comparing frequency spectra of predator and benign vibrations suggests that the presence of energy in frequencies outside the range characteristic of attacks might serve as an indicator of benign disturbance.

Flexible information sampling in vibrational assessment of predation risk by red-eyed treefrog embryos.

Prey assessing risk may miss cues and fail to defend themselves, or respond unnecessarily to false alarms. Error rates can be ameliorated with more information, but sampling predator cues entails risk. Red-eyed treefrogs have arboreal eggs and aquatic tadpoles. The embryos use vibrations in snake attacks to cue behaviorally mediated premature hatching, and escape, but vibrations from benign sources rarely induce hatching. Missed cues and false alarms are costly; embryos that fail to hatch are eaten and hatching prematurely increases predation by aquatic predators. Embryos use vibration duration and spacing to inform their hatching decision. This information accrues with cycles of vibration, while risk accrues over time as snakes feed. We used vibration playback experiments to test if embryos adjust sampling of information based on its cost, and measured latency to initiate hatching in videotaped snake attacks. Embryos did not initiate hatching immediately in attacks or playbacks, and the delay varied with the rate at which information accrued. Embryos started hatching sooner in response to stimuli with shorter cycles but sampled fewer cycles (less information) of longer-cycle stimuli before hatching. This flexible sampling is consistent with embryos balancing a trade-off between the value and cost of information.

Temporal pattern cues in vibrational risk assessment by embryos of the red-eyed treefrog, Agalychnis callidryas.

The embryos of red-eyed treefrogs, Agalychnis callidryas, use vibrations transmitted through their arboreal egg clutch to cue escape hatching behavior when attacked by egg-eating snakes. Hatching early increases the risk of predation in the water, so embryos should avoid it unless they are in danger. We exposed egg clutches to intermittent vibrations with different combinations of vibration duration and spacing to examine the role of simple temporal pattern cues in the escape hatching response. Stimuli were bursts of synthetic white noise from 0 to 100 Hz, including the range of frequencies with substantial energy in snake attacks, and had approximately rectangular amplitude envelopes. Embryos hatched in response to a small range of temporal patterns and not in response to many others, rather than hatching to most vibrations except for certain patterns perceived as safe. Neither cycle length nor duty cycle predicted hatching response, except at extreme values where no hatching occurred; the highest energy stimuli elicited little or no hatching. Both vibration duration and inter-vibration interval strongly affected the hatching response. The highest levels of hatching were to durations of 0.5 s combined with intervals of 1.5-2.5 s, and hatching decreased gradually with increasing difference of either duration or interval from these most effective stimuli. Vibration duration and interval appear to function as two necessary elements of a composite cue, rather than as redundant cues. This increases response specificity and reduces the range of stimuli that elicit hatching, likely reducing the chance of hatching unnecessarily in a benign disturbance. Vibration-cued hatching in A. callidryas embryos offers an opportunity to experimentally assess the behavioral decision rules underlying an effective and costly anti-predator defense.