Female Gray Treefrogs Research Articles

Personality affects female mate choice: frogs displaying more consistent bold behaviors are choosier

Abstract Mate choice is an important cause of natural and sexual selection and drives the evolution and elaboration of male ornaments. Yet mate choice decisions are often neither consistent nor uniform, and a range of factors have been identified to influence variation between and within individuals. A potential source of variation influencing preferences and/or choosiness is animal personality, that is, repeatable among-individual differences in behaviors. Not only may individuals differ in average personality phenotype but also vary in how consistently they display said personality. Distinguishing between personality and consistency is important because both aspects are potentially naturally selected traits that may yet influence how sexually selected ornaments are evaluated. Here, we use a predator evasion assay to test whether there is variation in boldness among female gray treefrogs (Hyla versicolor), and then examine whether personality traits (spectrum of shy to bold) are correlated with choosiness for longer duration calls. We document substantial and repeatable between-individual variation in boldness, suggesting the presence of animal personality. Results also reveal that the consistency with which females expressed boldness is independent from average personality phenotype and that it is correlated with choosiness: more consistently bold females were choosier.

Sensory biases in response to novel complex acoustic signals in male and female grey treefrogs, Hyla chrysoscelis.

The sensory bias hypothesis proposes that female preferences for male sexual signalling traits evolved in contexts other than mating. Individuals of both sexes may experience similar selection pressures in these contexts; thus males may have similar biases to females for variation in signal traits. We tested this prediction in the grey treefrog, Hyla chrysoscelis, in which males produce simple advertisement calls, but females are more attracted to certain novel complex stimuli. We recorded males' responses to playbacks of both simple advertisement calls and complex calls consisting of the advertisement call with an acoustic appendage (filtered noise, or heterospecific call pulses) either leading or following the call. We tested females' preferences for the same stimuli in phonotaxis tests. We found evidence for a sensory bias in both sexes: males gave more aggressive calls in response to complex stimuli and females sometimes preferred complex over simple calls. These biases were not universal and depended on both temporal order and appendage characteristics, but how these effects manifested differed between the sexes. Ultimately, our approach of studying biases of both sexes in response to novel mating signals will shed light on the origin of mating preferences, and the mechanisms by which sensory biases operate.

Female gray treefrogs maintain mate choice decisions under predation threat, but adjust movements to reduce conspicuousness during mate approach

Female gray treefrogs maintain mate choice decisions under predation threat, but adjust movements to reduce conspicuousness during mate approach

Interval-counting neurons allow gray treefrogs to make the right choice

At the right time of year across the eastern USA, from Texas to Maine, wetlands come alive with the sound of trilling eastern gray treefrogs (Hyla versicolor), each hoping to attract the attention of a nearby female. However, male eastern gray treefrogs are not the only vociferous suitors in the neighbourhood; they share their wetland breeding grounds with another close relative, Cope's gray treefrogs (Hyla chrysoscelis). ‘The two species are visually indistinguishable’, says Saumya Gupta from the University of Minnesota, USA. Yet, the consequences of a Cope's gray treefrog inadvertently selecting an eastern gray treefrog partner would be disastrous; their offspring rarely survive and, if they do, they are infertile. Fortunately, eastern and Cope's gray treefrogs barely make this rookie mistake, thanks to their trilling croaks. Even though the trill structures of their amorous calls are similar, they are sufficiently different – eastern gray treefrogs trill a little more slowly than their Cope's cousins – for females to avoid selecting the wrong mate. Yet, no one was sure exactly how these closely related species distinguish between each other's trills. However, Gupta and Mark Bee (University of Minnesota) had a hunch. They knew that a unique set of neurons in frog brains – called interval-counting neurons – are often tuned to the trill rate of their own species’ calls. Could these neurons hold the key to female eastern gray treefrogs rebuffing Cope's gray treefrog serenades and vice versa?Collecting female gray treefrogs in amorous embraces with their male partners from the Tamarack Nature Center in Minnesota, USA, Gupta first had to identify which species each frog belonged to. ‘I played mating calls of the eastern and Cope's gray treefrogs from two separate loudspeakers and identified the species based on which speaker it approached’, she says. Next, Gupta simulated a series of gray treefrog trills composed of individual pulses – 10 ms pulses, 20 ms apart for the Cope's gray treefrogs and 30 ms pulses separated by 60 ms for the eastern gray treefrogs. After recreating the distinctive pulse signatures of both species, she played a range of trills to the females, from a single pulse up to 16 repetitions, recording the number of pulses when the female's attention switched from indifference to attraction as she hopped toward the loudspeaker.Comparing the females’ reactions, it was clear that the Cope's gray treefrogs tended to become interested in trills composed of ∼8, 10 ms pulses when the calls were softer (65 dB SPL) while trills of ∼7.5 pulses became attractive when louder (85 dB SPL). In contrast, the eastern gray treefrog females only required ∼5, 30 ms pulses to get their attention, falling to 3.5 pulses when the volume was higher. But how would the interval-counting neurons in the treefrog brain respond to each species-specific serenade?This time, Rishi Alluri and Gary Rose from the University of Utah, USA, played simulated trills with increasing numbers of pulses to the frogs while painstakingly recording when interval-counting neurons in the frog brain began sending electrical nerve signals. Eventually, the duo identified a subset of neurons in the Cope's gray treefrog brain tuned to fire when they heard trills of at least 6–8, 10 ms pulses. Meanwhile, the eastern gray treefrogs’ interval-counting neurons responded strongly when the frogs heard trills containing at least 3–5, 30 ms pulses.‘Interval-counting neurons play key roles in decoding information about species identity in frogs’, says Gupta, placing the neurons at the hub of the mechanism protecting eastern and Cope's gray treefrogs from interbreeding, keeping them faithful to their own species’ serenades.

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.

Taxis bold as love: the influence of aggressive calls on acoustic attraction of female gray treefrogs, Hyla versicolor

We studied female phonotaxis in gray treefrogs to learn how male aggressive calling influences female choice of a mate from the perspective of the aggressive signaler and his male target. Although aggressive calls on their own attracted some females, when allowed to choose, all females preferred advertisement calls to aggressive calls. We wanted to ascertain also whether such calls could reduce the attractiveness of a nearby advertising male relative to an advertising male that was not close to a male giving aggressive calls. If so, it would be a good reason for a male to move away from a male giving aggressive calls. We found that females were indifferent to aggressive calls which were broadcast following and adjacent to a source of advertisement calls when the choice alternative was a more distant source of advertisement calls. However, this was not always the case when aggressive calls and advertisement calls overlapped. Angular separation from a source of overlapping aggressive calls did not improve the attractiveness of advertisement calls relative to advertisement calls adjacent to a source of such aggressive calls. In a final test, we simulated an aggressive exchange with a winner and a retreating loser. Females allowed to eavesdrop failed to discriminate. Thus, there may be no cost to moving away and a potential gain in terms of reduced acoustic interference. Loud calls or physical intrusions by conspecific males may cause male frogs to give aggressive calls. Although acoustic features of aggressive vocalizations may be well-described, we are largely ignorant of their utility and potential costs to producing and nearby males. We investigated whether the production of aggressive calls would compromise the attractiveness of male gray treefrogs to potential mates. Choruses of frogs may be especially suitable venues for eavesdropping, a phenomenon also ripe for investigation. Accordingly, we tested whether females exposed to simulated aggressive vocal exchanges would discriminate in favor of putative winners. We found that aggressive calling can reduce the signaler’s attractiveness but, lacking acoustic interference, not that of a neighbor. Females failed to discriminate between a simulated retreating loser and winner suggesting that such outcomes are irrelevant to females or that their ability to eavesdrop is limited.

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.

Effects of intracerebroventricular arginine vasotocin on a female amphibian proceptive behavior.

Mate choice decisions of animals show significant variability-both among and within individuals. Clearly, such variability can profoundly impact individual fitness, as well as subtly alter sexual selection processes, but we know little about the neural mechanisms underlying such variability. We examined the influence of the neuropeptide arginine vasotocin (AVT) on the strength of attraction of female gray treefrogs (Hyla versicolor) showing positive phonotaxis to the call of a conspecific male. Female treefrogs received intracerebroventricular injections with either saline, AVT (five doses), or the AVT receptor antagonist Manning compound (two doses). By 30min after injection, AVT significantly increased the speed with which females approached the speaker, at doses of 1, 10 and 50ng per frog. At the highest dose, the average speed was doubled. The AVT antagonist significantly inhibited phonotaxis at both doses (50 and 100ng). The effects of AVT on treefrog phonotaxis were shorter lived (disappearing within 60-90min), compared to Manning compound (effects persisted at least 90min). These findings support the hypothesis that endogenous AVT is critical to the display of female phonotaxis behavior. AVT may thus contribute to variability in female mate choices by modulating proceptive behaviors.

Differential effects of sound level and temporal structure of calls on phonotaxis by female gray treefrogs, Hyla versicolor.

We investigated how communication distance influenced the efficacy of communication by studying the effects of two attributes of male chorus sounds, namely, reduction in sound level and degradation of temporal sound structure, on attraction and accuracy of female phonotaxis in gray treefrogs, Hyla versicolor. For this, we conducted acoustic playback experiments, using synthetic calls and natural calls recorded at increasing distances from a focal male as stimuli. We found that the degradation of temporal structure had a greater effect on signal attractiveness than did the reduction in sound level, and that increasing sound level preferentially affected the attractiveness of proximally recorded calls, with less temporal degradation. Unlike signal attraction, accuracy of female localization increased systematically with the sound level. These results suggest that the degradation of temporal fine structure from both the chorus and signal-environmental effects imposes a limit for effective communication distances for female treefrogs in nature.

The Waiting and Mating Game: Condition Dependent Mate Sampling in Female Gray Treefrogs (Hyla versicolor)

Strong sexual selection by receivers can lead to the evolution of elaborate courtship behaviors in signalers. However the process by which receivers sample signalers and execute mate choice under complex signaling conditions—and thus the realized strength of sexual selection—is poorly understood. Moreover, receivers can vary in condition, which can further influence mate sampling strategies. Using wild female frogs we tested two hypotheses at the intersection of these important problems: that some of the individual variation in mate sampling is explained by (1) the reproductive urgency hypothesis, which predicts that receivers in a more urgent reproductive state will sample mates less and/or (2) the reproductive investment hypothesis, which predicts that receivers that have invested less in the current reproductive effort will sample mates less. Eastern gray treefrogs, Hyla versicolor, were collected in amplexus and repeatedly tested for phonotaxis behavior using a dynamic playback assay. To evaluate if hormonal mechanisms explained variation in the mate sampling, three steroid hormones, estradiol, progesterone, and corticosterone, were collected using a noninvasive water-borne hormone assay, validated for this species in the present study. Finally, we measured clutch size (investment) and the duration of time required for each female to oviposit after being reunited with their male mate (urgency). We found repeatability in many of the behaviors, including mate sampling. We found that females with higher concentrations estradiol and corticosterone made quicker choices, and that females with higher progesterone sampled mates more. We also found that female frogs in a more urgent reproductive state had lower concentrations of progesterone and estradiol, thereby providing the first evidence of a relationship between gonadal hormones and reproductive urgency. Collectively we found some support for the reproductive urgency but not the investment hypothesis. Thus, even though a female frog’s reproductive readiness is a highly transient life history stage, fine scale variation in her reproductive timeline could mitigate the strength of directional selection.

Mate choice strategies in a spatially-explicit model environment.

Decisions about the choice of a mate can greatly impact both individual fitness and selection processes. We developed a novel agent-based model to investigate two common mate choice rules that may be used by female gray treefrogs (Hyla versicolor). In this model environment, female agents using the minimum-threshold strategy found higher quality mates and traveled shorter distances on average, compared with female agents using the best-of-n strategy. Females using the minimum-threshold strategy, however, incur significant lost opportunity costs, depending on the male population quality average. The best-of-n strategy leads to significant female:female competition that limits their ability to find high quality mates. Thus, when the sex ratio is 0.8, best-of-5 and best-of-2 strategies yield mates of nearly identical quality. Although the distance traveled by females in the mating task varied depending on male spatial distribution in the environment, this did not interact with female choice for the best-of-n or minimum-threshold strategies. By incorporating empirical data from the frogs in this temporally- and spatially-explicit model, we thus show the emergence of novel interactions of common decision-making rules with realistic environmental variables.

Is it one call or two? Perception of inter-pulse and inter-call intervals in the grey treefrog, Hyla versicolor

Female grey treefrogs, Hyla versicolor, treat a call with an anomalously large gap between call pulses as relatively unattractive. However, whether such a ‘call’ is treated (or perhaps perceived) as a single call or two separate calls, an issue relevant to the problem of perceptual segregation of calls of different males, was unclear. We tested whether there could be gaps shorter than a typical inter-call interval that would be sufficient to elicit the latter percept by offering females a choice between a 5-pulse call and 13-pulse call separated by a 1575-ms gap and these pulse strings separated by shorter gaps. Preliminary results suggested that perception of inter-call gaps was categorical, and the distributions of such gaps between males in choruses were compatible with this finding. However, when we also offered females call alternatives designed to specifically test the hypothesis of categorical perception, predictions were only sometimes met.

Modality interactions alter the shape of acoustic mate preference functions in gray treefrogs.

Sexual selection takes place in complex environments where females evaluating male mating signals are confronted with stimuli from multiple sources and modalities. The pattern of expression of female preferences may be influenced by interactions between modalities, changing the shape of female preference functions, and thus ultimately altering the selective landscape acting on male signal evolution. We tested the hypothesis that the responses of female gray treefrogs, Hyla versicolor, to acoustic male advertisement calls are affected by interactions with visual stimuli. We measured preference functions for several call traits under two experimental conditions: unimodal (only acoustic signals presented), and multimodal (acoustic signals presented along with a video-animated calling male). We found that females were more responsive to multimodal stimulus presentations and, compared to unimodal playbacks, had weaker preferences for temporal call characteristics. We compared the preference functions obtained in these two treatments to the distribution of male call characteristics to make inferences on the strength and direction of selection expected to act on male calls. Modality interactions have the potential to influence the course of signal evolution and thus are an important consideration in sexual selection studies.

Species-specificity of temporal processing in the auditory midbrain of gray treefrogs: interval-counting neurons.

Interval-counting neurons (ICNs) respond after a threshold number of sound pulses have occurred with specific intervals; a single aberrant interval can reset the counting process. Female gray treefrogs, Hyla chrysoscelis and H. versicolor, discriminate against synthetic 'calls' possessing a single interpulse interval 2-3 three times the optimal value, suggesting that ICNs are important for call recognition. The calls of H. versicolor consist of pulses that are longer in duration, rise more slowly in amplitude and are repeated at a slower rate than those of H. chrysoscelis. Results of recordings from midbrain auditory neurons in these species include: (1) ICNs were found in both species and their temporal selectivity appeared to result from interplay between excitation and inhibition; (2) band-pass cells in H. versicolor were tuned to slower pulse rates than those in H. chrysoscelis; (3) ICNs that were selective for slow-rise pulse shape were found almost exclusively in H. versicolor, but fast-rise-selective neurons were found in both species, and (4) band-suppression ICNs in H. versicolor showed response minima at higher pulse rates than those in H. chrysoscelis. Selectivity of midbrain ICNs for pulse rise time and repetition rate thus correlate well with discriminatory abilities of these species that promote reproductive isolation.

Female gray treefrogs, Hyla versicolor, are responsive to visual stimuli but unselective of stimulus characteristics.

The visual ecology of nocturnal anurans is poorly understood, but there is growing evidence that vision plays a role in important behaviors such as mate choice. While several recent studies have demonstrated that females are responsive to visual cues when selecting mates, the forces responsible for these preferences are unknown. We investigated the responsiveness of female gray treefrogs, Hyla versicolor, to video playbacks of calling conspecific males in which we varied attributes of the vocal sac, a conspicuous visual characteristic of calling males and a target of female choice in other species. Females responded surprisingly strongly to the video playbacks, but did so indiscriminately with respect to variation in vocal sac characteristics. We followed up on these results with a series of additional tests that examined female responses to abstract stimuli. Females continued to respond to such stimuli, leading us to conclude that their behavior was related to a generalized phototactic response. Because of this, we were unable to make conclusions regarding female preferences for vocal sac characteristics. Nonetheless, our results are significant in two respects. First, we illustrate that despite much effort into improving video playback methodologies, challenges remain, and we offer our experimental design as a method to ensure that appropriate conclusions can be drawn from such studies. Second, we argue that the female phototactic response has potentially significant behavioral implications and in general the consequences of anuran visual preferences deserve further investigation.

Spatial release from masking improves sound pattern discrimination along a biologically relevant pulse-rate continuum in gray treefrogs

Many frogs form large choruses during their mating season in which males produce loud advertisement calls to attract females and repel rival males. High background noise levels in these social aggregations can impair vocal perception. In humans, spatial release from masking contributes to our ability to understand speech in noisy social groups. Here, we tested the hypothesis that spatial separation between target signals and ‘chorus-shaped noise’ improves the ability of female gray treefrogs (Hyla chrysoscelis) to perform a behavioral discrimination task based on perceiving differences in the pulsatile structure of advertisement calls. We used two-stimulus choice tests to measure phonotaxis (approach toward sound) in response to calls differing in pulse rate along a biologically relevant continuum between conspecific (50 pulses s−1) and heterospecific (20 pulses s−1) calls. Signals were presented in quiet, in colocated noise, and in spatially separated noise. In quiet conditions, females exhibited robust preferences for calls with relatively faster pulse rates more typical of conspecific calls. Behavioral discrimination between calls differing in pulse rate was impaired in the presence of colocated noise but similar between quiet and spatially separated noise conditions. Our results indicate that spatial release from energetic masking facilitates a biologically important temporal discrimination task in frogs. We discuss these results in light of previous work on spatial release from masking in frogs and other animals.

Restoration of call attractiveness by novel acoustic appendages in grey treefrogs

Ethologists have often reported preferences for novel signals, especially if they are more extravagant than normal signals. Such preferences presumably reflect sensory biases that may promote the evolution of both novel and complex signals. We tested behavioural responses of female grey treefrogs, Hyla versicolor, to novel complex calls in relation to the response properties of previously described temporally selective neurons in the auditory midbrain. As predicted by the selectivity of interval-counting neurons, females discriminated against synthetic advertisement calls containing a gap, a missed pulse or a pulse of abnormally short duration. The addition of a novel tonal appendage to such defective calls often resulted in partial restoration of the attractiveness of the signal. The restorative effect occurred only when an appendage with a higher amplitude followed (rather than led) the defective call. Our results show how the consideration of proximate mechanisms can provide insights about the evolution of complex signals; the behavioural results, in turn, suggest new ways of assessing the response properties of the auditory system.

Dip listening and the cocktail party problem in grey treefrogs: signal recognition in temporally fluctuating noise

Dip listening refers to our ability to catch brief "acoustic glimpses" of speech and other sounds when fluctuating background noise levels momentarily decrease. Exploiting dips in natural fluctuations of noise contributes to our ability to overcome the "cocktail party problem" of understanding speech in multi-talker social environments. We presently know little about how nonhuman animals solve analogous communication problems. Here, we asked whether female grey treefrogs (Hyla chrysoscelis) might benefit from dip listening in selecting a mate in the noisy social setting of a breeding chorus. Consistent with a dip listening hypothesis, subjects recognized conspecific calls at lower thresholds when the dips in a chorus-like noise masker were long enough to allow glimpses of nine or more consecutive pulses. No benefits of dip listening were observed when dips were shorter and included five or fewer pulses. Recognition thresholds were higher when the noise fluctuated at a rate similar to the pulse rate of the call. In a second experiment, advertisement calls comprising six to nine pulses were necessary to elicit responses under quiet conditions. Together, these results suggest that in frogs, the benefits of dip listening are constrained by neural mechanisms underlying temporal pattern recognition. These constraints have important implications for the evolution of male signalling strategies in noisy social environments.

Sperm depletion and a test of the phenotype-linked fertility hypothesis in Gray Treefrogs (Hyla versicolor)

The phenotype-linked fertility hypothesis (PLFH) predicts that ejaculate characteristics correlate with male phenotype, and that females may select for exaggerated phenotypes to guarantee high fertilization success. Female Gray Treefrogs ( Hyla versicolor LeConte, 1825) prefer males with long-duration calls and (or) calls produced at a fast rate. I tested the prediction that call duration or call rate were indicators of sperm number or viability. To assess whether female H. versicolor might benefit by identifying males with low sperm numbers, I examined the effect of mating history on sperm number and looked for evidence of sperm depletion. My study found no support for the PLFH in relation to call quality, as neither sperm number nor viability was related to call duration. I found that sperm stores are severely reduced after a single mating, and that sperm stores of males calling late in the season are often more similar to those of depleted males than unmated males. Thus, calling males may attempt to attract females without the sperm numbers necessary to fertilize the entire clutch. However, selection for females to identify males with high fertilization success may be low when all other factors that affect reproductive success (e.g., genetic benefits) are considered.

Finding Your Mate at a Cocktail Party: Frequency Separation Promotes Auditory Stream Segregation of Concurrent Voices in Multi-Species Frog Choruses

Vocal communication in crowded social environments is a difficult problem for both humans and nonhuman animals. Yet many important social behaviors require listeners to detect, recognize, and discriminate among signals in a complex acoustic milieu comprising the overlapping signals of multiple individuals, often of multiple species. Humans exploit a relatively small number of acoustic cues to segregate overlapping voices (as well as other mixtures of concurrent sounds, like polyphonic music). By comparison, we know little about how nonhuman animals are adapted to solve similar communication problems. One important cue enabling source segregation in human speech communication is that of frequency separation between concurrent voices: differences in frequency promote perceptual segregation of overlapping voices into separate “auditory streams” that can be followed through time. In this study, we show that frequency separation (ΔF) also enables frogs to segregate concurrent vocalizations, such as those routinely encountered in mixed-species breeding choruses. We presented female gray treefrogs (Hyla chrysoscelis) with a pulsed target signal (simulating an attractive conspecific call) in the presence of a continuous stream of distractor pulses (simulating an overlapping, unattractive heterospecific call). When the ΔF between target and distractor was small (e.g., ≤3 semitones), females exhibited low levels of responsiveness, indicating a failure to recognize the target as an attractive signal when the distractor had a similar frequency. Subjects became increasingly more responsive to the target, as indicated by shorter latencies for phonotaxis, as the ΔF between target and distractor increased (e.g., ΔF = 6–12 semitones). These results support the conclusion that gray treefrogs, like humans, can exploit frequency separation as a perceptual cue to segregate concurrent voices in noisy social environments. The ability of these frogs to segregate concurrent voices based on frequency separation may involve ancient hearing mechanisms for source segregation shared with humans and other vertebrates.