Convergent evolution of a blood-red nectar pigment in vertebrate-pollinated flowers.

Nearly 90% of flowering plants depend on animals for reproduction. One of the main rewards plants offer to pollinators for visitation is nectar. Nesocodon mauritianus (Campanulaceae) produces a blood-red nectar that has been proposed to serve as a visual attractant for pollinator visitation. Here, we show that the nectar's red color is derived from a previously undescribed alkaloid termed nesocodin. The first nectar produced is acidic and pale yellow in color, but slowly becomes alkaline before taking on its characteristic red color. Three enzymes secreted into the nectar are either necessary or sufficient for pigment production, including a carbonic anhydrase that increases nectar pH, an aryl-alcohol oxidase that produces a pigment precursor, and a ferritin-like catalase that protects the pigment from degradation by hydrogen peroxide. Our findings demonstrate how these three enzymatic activities allow for the condensation of sinapaldehyde and proline to form a pigment with a stable imine bond. We subsequently verified that synthetic nesocodin is indeed attractive to Phelsuma geckos, the most likely pollinators of Nesocodon We also identify nesocodin in the red nectar of the distantly related and hummingbird-visited Jaltomata herrerae and provide molecular evidence for convergent evolution of this trait. This work cumulatively identifies a convergently evolved trait in two vertebrate-pollinated species, suggesting that the red pigment is selectively favored and that only a limited number of compounds are likely to underlie this type of adaptation.

Perceptually salient differences in a species recognition cue do not promote auditory streaming in eastern grey treefrogs (Hyla versicolor).

Auditory streaming underlies a receiver's ability to organize complex mixtures of auditory input into distinct perceptual "streams" that represent different sound sources in the environment. During auditory streaming, sounds produced by the same source are integrated through time into a single, coherent auditory stream that is perceptually segregated from other concurrent sounds. Based on human psychoacoustic studies, one hypothesis regarding auditory streaming is that any sufficiently salient perceptual difference may lead to stream segregation. Here, we used the eastern grey treefrog, Hyla versicolor, to test this hypothesis in the context of vocal communication in a non-human animal. In this system, females choose their mate based on perceiving species-specific features of a male's pulsatile advertisement calls in social environments (choruses) characterized by mixtures of overlapping vocalizations. We employed an experimental paradigm from human psychoacoustics to design interleaved pulsatile sequences (ABAB…) that mimicked key features of the species' advertisement call, and in which alternating pulses differed in pulse rise time, which is a robust species recognition cue in eastern grey treefrogs. Using phonotaxis assays, we found no evidence that perceptually salient differences in pulse rise time promoted the segregation of interleaved pulse sequences into distinct auditory streams. These results do not support the hypothesis that any perceptually salient acoustic difference can be exploited as a cue for stream segregation in all species. We discuss these findings in the context of cues used for species recognition and auditory streaming.

In your CORT: Corticosterone and its receptors in the brain underlie mate choosiness in female Cope's gray treefrogs (Hyla chrysoscelis).

Selecting an attractive mate can involve trade-offs related to investment in sampling effort. Glucocorticoids like corticosterone (CORT) are involved in resolving energetic trade-offs. However, CORT is rarely studied in the context of mate choice, despite its elevated levels during reproductive readiness and the energetic transitions that characterize reproduction. Few systems are as well suited as anuran amphibians to evaluate how females resolve energetic trade-offs during mate choice. Phonotaxis tests provide a robust bioassay of mate choice that permit the precise measurement of inter-individual variation in traits such as choosiness-the willingness to pursue the most attractive mate despite costs. In Cope's gray treefrogs (Hyla chrysoscelis), females exhibit remarkable variation in circulating CORT as well as choosiness during mate choice, and a moderate dose of exogenous CORT rapidly (<1 h) and reliably induce large increases in choosiness. Here we measured the expression of glucocorticoid (GR) and mineralocorticoid (MR) receptors in the brains of females previously treated with exogenous CORT and tested for mate choosiness. We report a large decrease in GR expression in the hindbrain and midbrain of females that were treated with the moderate dosage of CORT-the same treatment group that exhibited a dramatic increase in choosiness following CORT treatment. This association, however, does not appear to be causal, as only forebrain GR levels, which are not affected by CORT injection, are positively associated with variation in choosiness. No strong effects were found for MR. We discuss these findings and suggest future studies to test the influence of glucocorticoids on mate choice.

Female preferences for the spectral content of advertisement calls in Cope's gray treefrog (Hyla chrysoscelis).

Amphibians have inner ears with two sensory papillae tuned to different frequency ranges of airborne sounds. In frogs, male advertisement calls possess distinct spectral components that match the tuning of one or both sensory papillae. Female preferences for the spectral content of advertisement calls can depend on signal amplitude and can vary among closely related lineages. In this study of Cope's gray tree frog (Hyla chrysoscelis), we investigated the amplitude dependence of female preferences for the spectral content of male advertisement calls, which have a "bimodal" spectrum with separate low-frequency (1.25 kHz) and high-frequency (2.5 kHz) components. In two-alternative choice tests, females generally preferred synthetic calls with bimodal spectra over "unimodal" calls having only one of the two spectral components. They also preferred unimodal calls with a high-frequency component over one with the low-frequency component. With few exceptions, preferences were largely independent of amplitude across both a 30 dB range of overall signal amplitude and an 11 dB range in the relative amplitudes of the two spectral components. We discuss these results in the context of evolutionary lability in female preferences for the spectral content of advertisement calls in North American tree frogs in the genus Hyla.

Neuroethology of sound localization in anurans.

Albert Feng pioneered the study of neuroethology of sound localization in anurans by combining behavioral experiments on phonotaxis with detailed investigations of neural processing of sound direction from the periphery to the central nervous system. The main advantage of these studies is that many species of female frogs readily perform phonotaxis towards loudspeakers emitting the species-specific advertisement call. Behavioral studies using synthetic calls can identify which parameters are important for phonotaxis and also quantify localization accuracy. Feng was the first to investigate binaural processing using single-unit recordings in the first two auditory nuclei in the central auditory pathway and later investigated the directional properties of auditory nerve fibers with free-field stimulation. These studies showed not only that the frog ear is inherently directional by virtue of acoustical coupling or crosstalk between the two eardrums, but also confirmed that there are extratympanic pathways that affect directionality in the low-frequency region of the frog's hearing range. Feng's recordings in the midbrain also showed that directional information is enhanced by cross-midline inhibition. An important contribution toward the end of his career involved his participation in neuroethological research with a team of scientists working with frogs that produce ultrasonic calls.

Predicting and Measuring Decision Rules for Social Recognition in a Neotropical Frog.

AbstractMany animals use signals to recognize familiar individuals but risk making mistakes because the signal properties of different individuals often overlap. Furthermore, outcomes of correct and incorrect decisions yield different fitness payoffs, and animals incur these payoffs at different frequencies depending on interaction rates. To understand how signal variation, payoffs, and interaction rates shape recognition decision rules, we studied male golden rocket frogs, which recognize the calls of territory neighbors and are less aggressive to neighbors than to strangers. We first quantified patterns of individual variation in call properties and predicted optimal discrimination thresholds using signal variation. We then measured thresholds for discriminating between neighbors and strangers using a habituation-discrimination field playback experiment. Territorial males discriminated between calls differing by 9%-12% in temporal properties, slightly higher than the predicted thresholds (5%-10%). Finally, we used a signal detection theory model to explore payoff and interaction rate parameters and found that the empirical threshold matched those predicted under ecologically realistic assumptions of infrequent encounters with strangers and relatively costly missed detections of strangers. We demonstrate that receivers group continuous variation in vocalizations into discrete social categories and that signal detection theory can be applied to understand evolved decision rules.

Moderately elevated glucocorticoids increase mate choosiness but do not affect sexual proceptivity or preferences in female gray treefrogs.

Glucocorticoids (GCs) are rarely studied in the context of female mate choice, despite the expression of receptors for these products in sexual, sensory and decision-making brain areas. Here we investigated the effects of GC concentrations on three aspects of female sexual behavior in breeding Cope's gray treefrogs (Hyla chrysoscelis): proceptivity-a measure of sexual motivation, intraspecific mate preferences, and mate choosiness. To our knowledge this is the first experimental study on the endocrine basis of mate choosiness. We predicted that mate choosiness-forfeiting an initial mate preference to pursue a suddenly more attractive mate-would be particularly impacted by elevated GCs with moderate GC levels associated with greater choosiness. We found support for this predicted inverted-U relationship. Females in the control group (no injection) showed no change in choosiness across timepoints. In contrast, females in the vehicle, Low (20 ng g-1) and High (180 ng g-1) corticosterone groups exhibited a nominal decline in choosiness after injection, suggesting that the experience of injection has little or perhaps slightly suppressive effects on female choosiness. Females in the moderate dose group (60 ng g-1), however, exhibited a significant increase (>100%) in choosiness. Further, we found no effect of elevated GCs on sexual proceptivity or the species-typical preference for longer calls. These findings may reflect a buffering of primary sensory areas in the brain against elevated GCs. The recruitment of other cognitive processes during active decision-making, however, may facilitate GC modulation of mate choosiness, thereby promoting tactical plasticity at this critical life history juncture.

Neural basis of acoustic species recognition in a cryptic species complex.

Sexual traits that promote species recognition are important drivers of reproductive isolation, especially among closely related species. Identifying neural processes that shape species differences in recognition is crucial for understanding the causal mechanisms of reproductive isolation. Temporal patterns are salient features of sexual signals that are widely used in species recognition by several taxa, including anurans. Recent advances in our understanding of temporal processing by the anuran auditory system provide an opportunity to investigate the neural basis of species-specific recognition. The anuran inferior colliculus consists of neurons that are selective for temporal features of calls. Of potential relevance are auditory neurons known as interval-counting neurons (ICNs) that are often selective for the pulse rate of conspecific advertisement calls. Here, we tested the hypothesis that ICNs mediate acoustic species recognition by exploiting the known differences in temporal selectivity in two cryptic species of gray treefrog (Hyla chrysoscelis and Hyla versicolor). We examined the extent to which the threshold number of pulses required to elicit behavioral responses from females and neural responses from ICNs was similar within each species but potentially different between the two species. In support of our hypothesis, we found that a species difference in behavioral pulse number thresholds closely matched the species difference in neural pulse number thresholds. However, this relationship held only for ICNs that exhibited band-pass tuning for conspecific pulse rates. Together, these findings suggest that differences in temporal processing of a subset of ICNs provide a mechanistic explanation for reproductive isolation between two cryptic treefrog species.

Lung-to-ear sound transmission does not improve directional hearing in green treefrogs (Hyla cinerea).

Amphibians are unique among extant vertebrates in having middle ear cavities that are internally coupled to each other and to the lungs. In frogs, the lung-to-ear sound transmission pathway can influence the tympanum's inherent directionality, but what role such effects might play in directional hearing remains unclear. In this study of the American green treefrog (Hyla cinerea), we tested the hypothesis that the lung-to-ear sound transmission pathway functions to improve directional hearing, particularly in the context of intraspecific sexual communication. Using laser vibrometry, we measured the tympanum's vibration amplitude in females in response to a frequency modulated sweep presented from 12 sound incidence angles in azimuth. Tympanum directionality was determined across three states of lung inflation (inflated, deflated, reinflated) both for a single tympanum in the form of the vibration amplitude difference (VAD) and for binaural comparisons in the form of the interaural vibration amplitude difference (IVAD). The state of lung inflation had negligible effects (typically less than 0.5 dB) on both VADs and IVADs at frequencies emphasized in the advertisement calls produced by conspecific males (834 and 2730 Hz). Directionality at the peak resonance frequency of the lungs (1558 Hz) was improved by ∼3 dB for a single tympanum when the lungs were inflated versus deflated, but IVADs were not impacted by the state of lung inflation. Based on these results, we reject the hypothesis that the lung-to-ear sound transmission pathway functions to improve directional hearing in frogs.

Treefrogs exploit temporal coherence to form perceptual objects of communication signals.

For many animals, navigating their environment requires an ability to organize continuous streams of sensory input into discrete 'perceptual objects' that correspond to physical entities in visual and auditory scenes. The human visual and auditory systems follow several Gestalt laws of perceptual organization to bind constituent features into coherent perceptual objects. A largely unexplored question is whether nonhuman animals follow similar Gestalt laws in perceiving behaviourally relevant stimuli, such as communication signals. We used females of Cope's grey treefrog (Hyla chrysoscelis) to test the hypothesis that temporal coherence-a powerful Gestalt principle in human auditory scene analysis-promotes perceptual binding in forming auditory objects of species-typical vocalizations. According to the principle of temporal coherence, sound elements that start and stop at the same time or that modulate coherently over time are likely to become bound together into the same auditory object. We found that the natural temporal coherence between two spectral components of advertisement calls promotes their perceptual binding into auditory objects of advertisement calls. Our findings confirm the broad ecological validity of temporal coherence as a Gestalt law of auditory perceptual organization guiding the formation of biologically relevant perceptual objects in animal behaviour.

The paradox of hearing at the lek: auditory sensitivity increases after breeding in female gray treefrogs (Hyla chrysoscelis).

Both behavioral receptivity and neural sensitivity to acoustic mate attraction signals vary across the reproductive cycle, particularly in seasonally breeding animals. Across a variety of taxa receptivity to signals increases, as does peripheral auditory sensitivity, as females transition from a non-breeding to breeding condition. We recently documented decreases in receptivity to acoustic mate attraction signals and circulating hormone levels, but an increase in peripheral auditory sensitivity to call-like stimuli following oviposition in Cope's gray treefrogs (Hyla chrysoscelis). However, it is not known if changes in auditory sensitivity are confined to the frequency range of calls, or if they result from more generalized changes in the auditory periphery. Here, we used auditory brainstem responses (ABRs) to evaluate peripheral frequency sensitivity in female Cope's gray treefrogs before and after oviposition. We found lower ABR thresholds, greater ABR amplitudes, and shorter ABR latencies following oviposition. Changes were most pronounced and consistent at lower frequencies associated with the amphibian papilla, but were also detectable at higher frequencies corresponding to the tuning of the basilar papilla. Furthermore, only ABR latencies were correlated with circulating steroid hormones (testosterone). Changes in peripheral processing may result from changes in metabolic function or sensorineural adaptation to chorus noise.

The difference a day makes: Breeding remodels hearing, hormones and behavior in female Cope's gray treefrogs (Hyla chrysoscelis).

In seasonal breeders, there are behavioral, endocrine, and neural adaptations that promote the sexual receptivity of females and tune their sensory systems to detect and discriminate among advertising males and to successfully copulate. What happens immediately after this key life history event is unclear, but this transitional moment offers a window into the mechanisms that remodel sexual phenotypes. In this study of wild female Cope's gray treefrogs (Hyla chrysoscelis), we tested the hypothesis that oviposition results in a suite of coordinated changes in the sexual phenotype. Specifically, we predicted that sexual receptivity and discrimination behaviors would decline along with circulating concentrations of steroid hormones (corticosterone, estradiol, testosterone) and auditory sensitivity to the acoustic frequencies emphasized in male advertisement calls. We conducted these trait measurements before and after oviposition (ca. 24-h period). There was a 100% decrease in behavioral responsiveness after oviposition, and the concentrations of all three steroids plummeted during this brief window of time, especially testosterone. Moreover, higher concentrations of corticosterone-an important component of the endocrine stress response-were associated with longer response latencies, suggesting that adrenal hormones should be considered in future studies on the hormonal basis of mate choice. Counter to our prediction, auditory sensitivity increased following oviposition, and the amplitude of the auditory brainstem response was influenced by concentrations of estradiol. In pre-oviposition females auditory sensitivity diminished with increasing estradiol concentrations, while sensitivity increased with increasing estradiol concentrations in post-oviposition females, suggesting non-linear estrogenic modulation of peripheral auditory neural recruitment. Overall, our results indicate that there is considerable remodeling of behavioral output following oviposition that co-occurs with changes in both endocrine and sensory physiology.

Masking release in temporally fluctuating noise depends on comodulation and overall level in Cope's gray treefrog.

Many animals communicate acoustically in large social aggregations. Among the best studied are frogs, in which males form large breeding choruses where they produce loud vocalizations to attract mates. Although chorus noise poses significant challenges to communication, it also possesses features, such as comodulation in amplitude fluctuations, that listeners may be evolutionarily adapted to exploit in order to achieve release from masking. This study investigated the extent to which the benefits of comodulation masking release (CMR) depend on overall noise level in Cope's gray treefrog (Hyla chrysoscelis). Masked signal recognition thresholds were measured in response to vocalizations in the presence of chorus-shaped noise presented at two levels. The noises were either unmodulated or modulated with an envelope that was correlated (comodulated) or uncorrelated (deviant) across the frequency spectrum. Signal-to-noise ratios (SNRs) were lower at the higher noise level, and this effect was driven by relatively lower SNRs in modulated conditions, especially the comodulated condition. These results, which confirm that frogs benefit from CMR in a level-dependent manner, are discussed in relation to previous studies of CMR in humans and animals and in light of implications of the unique amphibian inner ear for considerations of within-channel versus across-channel mechanisms.

Nonlinear processing of a multicomponent communication signal by combination-sensitive neurons in the anuran inferior colliculus.

Diverse animals communicate using multicomponent signals. How a receiver's central nervous system integrates multiple signal components remains largely unknown. We investigated how female green treefrogs (Hyla cinerea) integrate the multiple spectral components present in male advertisement calls. Typical calls have a bimodal spectrum consisting of formant-like low-frequency (~0.9 kHz) and high-frequency (~2.7 kHz) components that are transduced by different sensory organs in the inner ear. In behavioral experiments, only bimodal calls reliably elicited phonotaxis in no-choice tests, and they were selectively chosen over unimodal calls in two-alternative choice tests. Single neurons in the inferior colliculus of awake, passively listening subjects were classified as combination-insensitive units (27.9%) or combination-sensitive units (72.1%) based on patterns of relative responses to the same bimodal and unimodal calls. Combination-insensitive units responded similarly to the bimodal call and one or both unimodal calls. In contrast, combination-sensitive units exhibited both linear responses (i.e., linear summation) and, more commonly, nonlinear responses (e.g., facilitation, compressive summation, or suppression) to the spectral combination in the bimodal call. These results are consistent with the hypothesis that nonlinearities play potentially critical roles in spectral integration and in the neural processing of multicomponent communication signals.

Sound source localization and segregation with internally coupled ears: the treefrog model.

Acoustic signaling plays key roles in mediating many of the reproductive and social behaviors of anurans (frogs and toads). Moreover, acoustic signaling often occurs at night, in structurally complex habitats, such as densely vegetated ponds, and in dense breeding choruses characterized by high levels of background noise and acoustic clutter. Fundamental to anuran behavior is the ability of the auditory system to determine accurately the location from where sounds originate in space (sound source localization) and to assign specific sounds in the complex acoustic milieu of a chorus to their correct sources (sound source segregation). Here, we review anatomical, biophysical, neurophysiological, and behavioral studies aimed at identifying how the internally coupled ears of frogs contribute to sound source localization and segregation. Our review focuses on treefrogs in the genus Hyla, as they are the most thoroughly studied frogs in terms of sound source localization and segregation. They also represent promising model systems for future work aimed at understanding better how internally coupled ears contribute to sound source localization and segregation. We conclude our review by enumerating directions for future research on these animals that will require the collaborative efforts of biologists, physicists, and roboticists.

Evolutionary adaptations for the temporal processing of natural sounds by the anuran peripheral auditory system.

Sensory systems function most efficiently when processing natural stimuli, such as vocalizations, and it is thought that this reflects evolutionary adaptation. Among the best-described examples of evolutionary adaptation in the auditory system are the frequent matches between spectral tuning in both the peripheral and central auditory systems of anurans (frogs and toads) and the frequency spectra of conspecific calls. Tuning to the temporal properties of conspecific calls is less well established, and in anurans has so far been documented only in the central auditory system. Using auditory-evoked potentials, we asked whether there are species-specific or sex-specific adaptations of the auditory systems of gray treefrogs (Hyla chrysoscelis) and green treefrogs (H. cinerea) to the temporal modulations present in conspecific calls. Modulation rate transfer functions (MRTFs) constructed from auditory steady-state responses revealed that each species was more sensitive than the other to the modulation rates typical of conspecific advertisement calls. In addition, auditory brainstem responses (ABRs) to paired clicks indicated relatively better temporal resolution in green treefrogs, which could represent an adaptation to the faster modulation rates present in the calls of this species. MRTFs and recovery of ABRs to paired clicks were generally similar between the sexes, and we found no evidence that males were more sensitive than females to the temporal modulation patterns characteristic of the aggressive calls used in male-male competition. Together, our results suggest that efficient processing of the temporal properties of behaviorally relevant sounds begins at potentially very early stages of the anuran auditory system that include the periphery.

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.

Auditory brainstem responses in Cope's gray treefrog (Hyla chrysoscelis): effects of frequency, level, sex and size.

Our knowledge of the hearing abilities of frogs and toads is largely defined by work with a few well-studied species. One way to further advance comparative work on anuran hearing would be greater use of minimally invasive electrophysiological measures, such as the auditory brainstem response (ABR). This study used the ABR evoked by tones and clicks to investigate hearing in Cope's gray treefrog (Hyla chrysoscelis). The objectives were to characterize the effects of sound frequency, sound pressure level, and subject sex and body size on ABRs. The ABR in gray treefrogs bore striking resemblance to ABRs measured in other animals. As stimulus level increased, ABR amplitude increased and latency decreased, and for responses to tones, these effects depended on stimulus frequency. Frequency-dependent differences in ABRs were correlated with expected differences in the tuning of two sensory end organs in the anuran inner ear (the amphibian and basilar papillae). The ABR audiogram indicated two frequency regions of increased sensitivity corresponding to the expected tuning of the two papillae. Overall, there was no effect of subject size and only small effects related to subject sex. Together, these results indicate the ABR is an effective method to study audition in anurans.

Assessing stimulus and subject influences on auditory evoked potentials and their relation to peripheral physiology in green treefrogs (Hyla cinerea).

Anurans (frogs and toads) are important models for comparative studies of communication, auditory physiology, and neuroethology, but to date, most of our knowledge comes from in-depth studies of a relatively small number of model species. Using the well-studied green treefrog (Hyla cinerea), this study sought to develop and evaluate the use of auditory evoked potentials (AEPs) as a minimally invasive tool for investigating auditory sensitivity in a larger diversity of anuran species. The goals of the study were to assess the effects of frequency, signal level, sex, and body size on auditory brainstem response (ABR) amplitudes and latencies, characterize gross ABR morphology, and generate an audiogram that could be compared to several previously published audiograms for green treefrogs. Increasing signal level resulted in larger ABR amplitudes and shorter latencies, and these effects were frequency dependent. There was little evidence for an effect of sex or size on ABRs. Analyses consistently distinguished between responses to stimuli in the frequency ranges of the three previously-described populations of afferents that innervate the two auditory end organs in anurans. The overall shape of the audiogram shared prominent features with previously published audiograms. This study highlights the utility of AEPs as a valuable tool for the study of anuran auditory sensitivity.