Phonotaxis Experiments Research Articles

Elevational speciation in action? Restricted gene flow associated with adaptive divergence across an altitudinal gradient.

Evolutionary theory predicts that divergent selection pressures across elevational gradients could cause adaptive divergence and reproductive isolation in the process of ecological speciation. Although there is substantial evidence for adaptive divergence across elevation, there is less evidence that this restricts gene flow. Previous work in the boreal chorus frog (Pseudacris maculata) has demonstrated adaptive divergence in morphological, life history and physiological traits across an elevational gradient from approximately 1500-3000m in the Colorado Front Range, USA. We tested whether this adaptive divergence is associated with restricted gene flow across elevation - as would be expected if incipient speciation were occurring - and, if so, whether behavioural isolation contributes to reproductive isolation. Our analysis of 12 microsatellite loci in 797 frogs from 53 populations revealed restricted gene flow across elevation, even after controlling for geographic distance and topography. Calls also varied significantly across elevation in dominant frequency, pulse number and pulse duration, which was partly, but not entirely, due to variation in body size and temperature across elevation. However, call variation did not result in strong behavioural isolation: in phonotaxis experiments, low-elevation females tended to prefer an average low-elevation call over a high-elevation call, and vice versa for high-elevation females, but this trend was not statistically significant. In summary, our results show that adaptive divergence across elevation restricts gene flow in P. maculata, but the mechanisms for this potential incipient speciation remain open.

Female and male phonotactic responses and the potential effect of sexual selection on the advertisement calls of a frog

The outcome of sexual selection acting on a given sexual trait depends on the interaction between the strength, direction and form of both inter- and intrasexual selection processes. Therefore, determining female choice and male–male competition is necessary to gain a better understanding of how sexual selection acts on sexual traits. Anuran males produce advertisement vocalizations to attract females and to maintain other males at distance, providing a convenient opportunity to test the combined effect of inter- and intrasexual selection. Our main objective was to test experimentally potential effects of female choice and male–male competition and their interaction when acting as selective agents on the advertisement vocalizations of the rosy ground frog, Eupsophus roseus . We performed phonotaxis experiments on females and males and determined their behavioural responses to artificial signals synthesized based on the distribution of natural calls. The main results suggest that females' preference would favour vocalizations having lower frequencies and a harmonic composition in which the main harmonics of vocalizations have similar amplitudes. These preferences suggest that females could exert negative directional sexual selection on the frequency and stabilizing selection on the amplitude ratio. The responses of males were variable, suggesting that this type of male–male competition may not result in intrasexual selection favouring specific values of advertisement vocalizations. The occurrence of no clear preferences in males may result in a decrease in the potential effect of females' preferences, which could further contribute to variability in spectral components of calls.

Selective phonotaxis of female crickets under natural outdoor conditions

Acoustic mate choice in insects has been extensively studied under laboratory conditions, using different behavioural paradigms. Ideally, however, mate choice designs should reflect natural conditions, including the physical properties of the transmission channel for the signal. Since little is known about the discrimination ability of females between male song variants under natural conditions, we performed phonotaxis experiments with female field crickets (Gryllus bimaculatus) outdoors, using two-choice decisions based on differences in carrier frequency, sound pressure level, and chirp rate. For all three song parameters, minimum differences necessary for a significant preference between two song models were considerably larger outdoors compared to laboratory conditions. A minimum amplitude difference of 5 dB was required for a significant choice in the field, compared to only 1–2 dB reported for lab-based experiments. Due to the tuned receiver system, differences in carrier frequency equal differences in perceived loudness, and the results on choice for differences in carrier frequency corroborate those in amplitude. Similarly, chirp rate differences of 50 chirps/min were required outdoors compared to only 20 chirps/min in the lab. For predictions about patterns of sexual selection, future studies need to consider the different outcomes of mate choice decisions in lab and field trials.

Male vocal competition is dynamic and strongly affected by social contexts in music frogs

Male-male vocal competition in anuran species is critical for mating success; however, it is also highly time-consuming, energetically demanding and likely to increase predation risks. Thus, we hypothesized that changes in the social context would cause male vocal competition to change in real time in order to minimize the costs and maximize the benefits of competition. To test this hypothesis, we assessed the effect of repeating playbacks of either white noise (WN) or male advertisement calls on male call production in the Emei music frog (Babina daunchina), a species in which males build mud-retuse burrows and call from within these nests. Previous studies have shown that calls produced from inside burrows are highly sexually attractive (HSA) to females while those produced outside nests are of low sexual attractiveness (LSA). Results showed that most subjects called responsively after the end of WN playbacks but before the onset of conspecific call stimuli although call numbers were similar, indicating that while males adjusted competitive patterns according to the biological significance of signals, their competitive motivation did not change. Furthermore, these data indicate that the frogs had evolved the ability of interval timing. Moreover, when the inter-stimulus interval (ISI) between playbacks was varied, the subjects preferentially competed with HSA calls when the ISI was short (<4s) but responded equally to HSA and LSA calls if the ISI was long (≥4s), suggesting that males allocate competitive efforts depending on both the perceived sexual attractiveness of rivals and the time available for calling. Notably, approximately two-thirds of male calls occurred in response to HSA calls, a preference rate comparable to that previously found for females in phonotaxis experiments and consistent with the idea that the mechanisms underlying both the male's competitive responses to rivals and the female's preferences toward potential mates coevolved under the same selective pressure.

Selective phonotaxis to high sound-pulse rate in the cricket Gryllus assimilis

Calling song of the cricket Gryllus assimilis is unusual among Gryllus spp. in the high sound-pulse rate, ca. 80Hz, within its chirps. We asked whether, as in other cricket species, females were able to analyze such a high pulse rate. In phonotaxis experiments, females failed to respond to stimuli with pulse rates substantially higher or lower than the species-typical value, demonstrating that they are indeed selective for this parameter. We also examined how pulse rate was represented by modulation in firing rate of the neuron AN1, the main carrier of information about cricket-song-like stimuli to the brain. For attractive stimuli, i.e. with high pulse rates, modulation of AN1 firing rate through time was surprisingly modest. This suggests that the brain circuits that analyze AN1 spike trains might be more sensitive to slight variations in AN1 firing rate than their counterparts in more slowly singing species.

The influence of geographic heterogeneity in predation pressure on sexual signal divergence in an Amazonian frog species complex

Sexual selection plays an important role in mating signal divergence, but geographic variation in ecological factors can also contribute to divergent signal evolution. We tested the hypothesis that geographic heterogeneity in predation causes divergent selection on advertisement call complexity within the Engystomops petersi (previously Physalaemus petersi) frog species complex. We conducted predator phonotaxis experiments at two sites where female choice is consistent with call trait divergence. Engystomops at one site produces complex calls, whereas the closely related species at the other site produces simple calls. Bats approached complex calls more than simple calls at both sites, suggesting selection against complex calls. Moreover, bat predation pressure was greater at the site with simple calls, suggesting stronger selection against complex calls and potentially precluding evolution of complex calls at this site. Our results show that geographic variation in predation may play an important role in the evolution and maintenance of mating signal divergence.

Female preferences for spectral call properties in the western genetic lineage of Cope's gray treefrog (Hyla chrysoscelis)

Female frogs discriminate among potential mates based on individual variation in male advertisement calls. While considerable data have accumulated allowing comparisons of female preference functions among species, we still lack fundamental knowledge about how and why the shapes of preference functions for particular call properties vary among populations within all but a few species. Here, we report results from a study aimed at describing female preference functions for spectral call properties in Cope's gray treefrog (Hyla chrysoscelis). Widespread throughout the eastern half of North America, Cope's gray treefrog is the diploid member of the cryptic diploid-tetraploid Hyla versicolor species complex, and its populations are divided into two distinct genetic lineages (eastern and western). In this study of a western lineage population, we recorded and analyzed the spectral properties of 1000 advertisement calls from 50 males and conducted two-choice phonotaxis experiments to estimate a population-level preference function. Females preferred calls with average frequencies over calls with frequencies that were 2 or 3 semitones (1.4 or 2.1 standard deviations, respectively) lower than the population mean. We observed no behavioral discrimination between calls with average and higher-than-average frequencies. Preferences discriminating against low-frequency calls were weak and were abolished by attenuating the preferred average call by 3 dB. We discuss these results in light of previous studies of eastern lineage populations, geographic variation in female preference functions, and the potential adaptive value of discriminating against calls with low frequencies.

TESTS FOR CALL RESTORATION IN THE GRAY TREEFROG HYLA VERSICOLOR

ABSTRACT Phonemic restoration, a form of temporal induction, occurs when the human brain compensates for masked or missing portions of speech by filling in obscured or nonexistent sounds. We tested for temporal induction and related abilities in females of the Gray Treefrog Hyla versicolor. The number of pulses in calls is used by females for assessment of males. Accordingly, an ability to “restore” or interpolate between masked or otherwise sonically degraded portions of calls could help females during mate choice in noisy choruses. In phonotaxis experiments, we employed unmodified calls and those that had a centrally placed gap, a region overlapped by a portion of another call or filtered noise, or replaced with filtered noise. When offered call alternatives with equivalent numbers of clear pulses, we found that females discriminated against calls with gaps two or more times greater than the natural 25 ms interpulse interval. When a gap was replaced with a zone of call overlap or noise (so, again the call durations of the alternatives were unequal), females discriminated either in favour (overlap) of the modified stimuli or failed to discriminate (noise). However, when the unmodified and modified stimuli were the same duration, females discriminated against the latter. Normal calls were also chosen when paired against calls with multiple noise sections. Pulses formed from noise bursts were attractive, but less so than normal pulses. In single speaker tests, standardized rates of movement did not differ between calls containing noise segments of different duration. Our results therefore do not indicate that females of the Gray Treefrog employ a form of temporal induction that is fully restorative. However, the data indicate that acoustically anomalous sections of calls can retain attractive potential provided acoustic energy and pulses are present.

Male vocalizations, female discrimination and molecular phylogeny: multiple perspectives on the taxonomic status of a critically endangered Caribbean frog

Until recently, morphology has been the predominant basis on which taxonomic decisions have been made. Now, many sources of data inform decisions in taxonomy, yet few studies are available that directly compare the conclusions made on the basis of different datasets. The difficulty of reaching clear taxonomic decisions is further complicated by the existence of allopatric populations, which may differ from other populations in notable ways yet not be distinct evolutionary units. We analyzed differences at the molecular level based on sequences of two mitochondrial genes, analyzed acoustic differences in male vocalizations (nine variables) and conducted a phonotaxis experiment with females to assess the taxonomic status of two putative Caribbean frog species (Mannophryne olmonae and Mannophryne trinitatis, Aromobatidae), which some authors have indicated as conspecific. A 16S gene tree (75 sequences of 15 putative species, 530 bp), a parametric bootstrap test, and the results of acoustic comparisons suggested that these entities were evolutionarily distinct. However, in the phonotaxis experiment, females of either species did not display significant preference among the male vocalizations presented. On the basis of the bioacoustic data and the 16S gene tree, we conclude that these taxa are distinct and suggest that lack of selection for pre-mating isolation in allopatry explains the lack of discrimination shown by females. Phonotaxis experiments in taxa with acoustic means of mate attraction should continue to be useful in assessing the evolutionary independence of putative sympatric entities, but our results suggest that they should be employed and interpreted cautiously when applied to allopatric populations. To most accurately assess the boundaries of evolutionary lineages, a pluralistic approach, utilizing as many sources of data as possible, is desirable.

Sound transmission and the recognition of temporally degraded sexual advertisement signals in Cope's gray treefrog (Hyla chrysoscelis).

Acoustic communication signals degrade as they propagate between signalers and receivers. While we generally understand the degrading effects of sound propagation on the structure of acoustic signals, we know considerably less about how receivers make behavioral decisions based on the perception of degraded signals in sonically and structurally complex habitats where communication occurs. In this study of acoustic mate recognition in Cope's gray treefrog, Hyla chrysoscelis (Cope 1880), we investigated how the temporal structure of male advertisement calls was compromised by propagation in a natural habitat and how females responded to stimuli mimicking various levels of temporal degradation. In a sound transmission experiment, we quantified changes in the pulsed structure of signals by broadcasting synthetic calls during active choruses from positions where we typically encountered signalers, and re-recording the signals from positions where we typically encountered potential receivers. Our main finding was that the silent gaps between pulses become increasingly 'filled in' by background noise and reverberations as a function of increasing propagation distance. We also conducted female phonotaxis experiments to determine the threshold modulation depth required to elicit recognition of the pulsatile structure of the call. Females were surprisingly tolerant of degraded temporal structure, and there was a tendency for greater permissiveness at lower playback levels. We discuss these results in terms of presumed mechanisms of call recognition in complex environments and the acoustic adaptation hypothesis.

Testing an auditory illusion in frogs: perceptual restoration or sensory bias?

The human auditory system perceptually restores short deleted segments of speech and other sounds (e.g. tones) when the resulting silent gaps are filled by a potential masking noise. When this phenomenon, known as 'auditory induction', occurs, listeners experience the illusion of hearing an ongoing sound continuing through the interrupting noise even though the perceived sound is not physically present. Such illusions suggest that a key function of the auditory system is to allow listeners to perceive complete auditory objects with incomplete acoustic information, as may often be the case in multisource acoustic environments. At present, however, we know little about the possible functions of auditory induction in the sound-mediated behaviours of animals. The present study used two-choice phonotaxis experiments to test the hypothesis that female grey treefrogs, Hyla chrysoscelis, experience the illusory perceptual restoration of discrete pulses in the male advertisement call when pulses are deleted and replaced by a potential masking noise. While added noise restored some attractiveness to calls with missing pulses, there was little evidence to suggest that the frogs actually experienced the illusion of perceiving the missing pulses. Instead, the added noise appeared to function as an acoustic appendage that made some calls more attractive than others as a result of sensory biases, the expression of which depended on the temporal order and acoustic structure of the added appendages.

Multiple signals and male spacing affect female preference at cocktail parties in treefrogs.

Effective acoustic communication in the face of intense conspecific background noise constitutes a constant sensory challenge in chorusing and colonial species. An evolutionary approach suggests that behavioural and environmental constraints in these species should have shaped signal design and signalling behaviour to enable communication in noisy conditions. This could be attained both through the use of multicomponent signals and through short-term adjustments in the spatial separation of calling males. We investigated these two hypotheses in a chorusing anuran, the hylid Hyla arborea, through a series of phonotaxis experiments conducted within a six-speaker arena in a high background noise situation, by presenting females with male calls containing either single or multiple attractive call components, and by modifying distances between speakers. We found that female ability to discriminate attractive calls increased when several attractive call components were available, providing novel evidence that the use of multicomponent signals enhances communication in complex acoustic conditions. Signal discrimination in females also improved with speaker separation, demonstrating that within natural choruses, spatial unmasking conditioned by male density and spatial separation probably improves female discrimination of competing males. Implications of these results for the accuracy of mate choice within choruses are discussed.

Behavioral measures of signal recognition thresholds in frogs in the presence and absence of chorus-shaped noise

Anuran amphibians are superb animal models for investigating the mechanisms underlying acoustic signal perception amid high levels of background noise generated by large social aggregations of vocalizing individuals. Yet there are not well-established methods for quantifying a number of key measures of auditory perception in frogs, in part, because frogs are notoriously difficult subjects for traditional psychoacoustic experiments based on classical or operant conditioning. A common experimental approach for studying frog hearing and acoustic communication involves behavioral phonotaxis experiments, in which patterns of movement directed toward sound sources indicate the subjects' perceptual experiences. In this study, three different phonotaxis experiments were conducted using the same target signals and noise maskers to compare different experimental methods and analytical tools for deriving estimates of signal recognition thresholds in the presence or absence of "chorus-shaped noise" (i.e., artificial noise with a spectrum similar to that of real breeding choruses). Estimates of recognition thresholds based on measures of angular orientation, response probabilities, and response latencies were quite similar in both two-choice and no-choice phonotaxis tests. These results establish important baselines for comparing different methods of estimating signal recognition thresholds in frogs tested in various masking noise conditions.

Processing of clustered sensory signals in gray treefrogs (Hyla versicolor): Behavioral responses in the laboratory and field.

Anuran mating choruses may consist of hundreds of sympatric and conspecific males calling in addition to other biotic and abiotic noise, representing a complex auditory scene whose characteristics change with distance. Detecting and localizing individual males in the chorus poses a challenge to the female, especially at long ranges. We carried out a series of recordings and behavioral experiments that are aimed at determining the distance at which female treefrogs (Hyla versicolor) use the chorus as an acoustic cue for their orienting responses. We analyzed natural choruses recorded at distances of 1–100 m and used these to create stimuli for phonotaxis experiments evaluating how the chorus attracts and provides cues for localization by females. In addition, we performed phonotaxis tests in the field near natural choruses to compare the frog’s behavior under laboratory and field conditions. We found that females utilize acoustic cues for orientation toward choruses at a distance up to 50 m, but additional cues may be used to ensure orientation in the field at distances &amp;gt;50 m. Additional experiments revealed that temporal coherence of the male call, as opposed to a general reduction in stimulus intensity, was the most important factor in female phonotactic behavior.

Treatment with arginine vasotocin alters mating calls and decreases call attractiveness in male túngara frogs

The peptide hormone arginine vasotocin (AVT) and its mammalian homolog arginine vasopressin modulate a variety of social behaviors in vertebrates. In anurans, AVT influences the production of advertisement calls, the acoustic signals that males use to attract females and repel rival males. In this study, we investigate the effects of AVT on call characteristics in the túngara frog ( Physalaemus pustulosus). Túngara frogs produce a “whine” that is important for species recognition; they may also produce a second, attractive call component, the “chuck”. We used a field playback experiment to determine changes in male calling behavior following treatment with AVT. A previous study showed that AVT alters call rate and the production of chucks; in the current analysis, we focus on changes in the whine. Males produce shorter whines with higher initial frequencies following treatment with AVT. Call changes do not vary with a social stimulus. We also used female phonotaxis experiments to investigate the effects of call changes on female mate choice. Females disfavor the calls produced by males treated with exogenous AVT. We suggest that AVT influences motivation to call and the motor control of call production, but that over-stimulation of the vocal system limited the production of attractive calls in this experimental context.

A complex mechanism of call recognition in the katydid Neoconocephalus affinis (Orthoptera: Tettigoniidae)

Acoustic pattern recognition is important for bringing together males and females in many insect species. We used phonotaxis experiments on a walking compensator to study call recognition in the katydid Neoconocephalus affinis, a species with a double-pulsed call and an atypically slow pulse rate for the genus. Call recognition in this species is unusual because females require the presence of two alternating pulse amplitudes in the signal. A Fourier analysis of the stimulus-envelopes revealed that females respond only when both the first and second harmonics of the AM spectrum are of similar amplitude. The second harmonic is generated by the amplitude difference between the two pulses making up a pulse-pair. Females respond to double pulses that have been merged into a single pulse only if this amplitude modulation is preserved. Further experiments suggest that females use a resonance mechanism to recognize the pulse rate of the call, supporting a neural model of rate recognition in which periodic oscillations in membrane potential are used to filter the pulse rate of the signal. Our results illustrate how a reduction in pulse rate extends the opportunities for females to evaluate fine-scale temporal properties of calls, and provide further evidence for the importance of oscillatory membrane properties in temporal processing. The results are discussed with regard to evolutionary changes in call recognition mechanisms within the genus.

Parallel female preferences for call duration in a diploid ancestor of an allotetraploid treefrog

The gray treefrog species complex (Hyla chrysoscelis and H. versicolor) comprises a single allotetraploid species (H. versicolor) that arose multiple times from hybrid matings between an extant diploid species (H. chrysoscelis) and at least two other extinct diploid treefrogs. While previous studies have investigated female preferences for call duration in the tetraploid, we know little about these preferences in its putative diploid anscestors. Here, I report results from two-choice phonotaxis experiments investigating call duration preferences in H. chrysoscelis. Females preferred an average-length call over shorter-than-average calls (0.5-2.0 standard deviations [SD] below average), and they preferred longer-than-average calls over average or shorter-than-average calls if the difference in pulse number was at least 2.0 SD. When the amplitude of the longer alternative was attenuated by 6 dB, females still preferred an average-length call over a shorter-than-average call, but there was no preference for longer-than-average calls over an average call. In the presence of chorus noise, female preferences for both average and longer-than-average calls over shorter alternatives were weakened or reversed. Together, the results from this study reveal patterns of female preferences for call duration that are strikingly similar among two members of a species complex with a novel evolutionary history. In both species, female preferences are directional, nonlinear, and limited by environmental noise. Furthermore, these results also highlight the need for caution in studies of sexual selection when extrapolating from patterns of female preference obtained under ideal laboratory conditions to conclusions about how those preferences are expressed in the real world.

Mate choice and courtship signal differentiation promotes speciation in an Amazonian frog

Female mate choice influences the evolution of male courtship signals and may promote speciation when those sexually selected traits also have a function in species discrimination. Here, we assess interpopulation female mate choice by conducting phonotaxis experiments on a population of the Amazonian frog Engystomops petersi in Puyo, Ecuador. Our results show very strong behavioral isolation relative to 1 of 2 foreign populations. Puyo females strongly discriminate against La Selva in favor of Puyo or

Female preferences for multiple attributes in the acoustic signals of the Italian treefrog, Hyla intermedia

The advertisement call of frogs and toads is an example of multiple message signal because different acoustic properties encode different kinds of biologically significant information. In the Italian treefrog, Hyla intermedia, pulse rate and frequency have been found to be under stabilizing female preferences and to encode information important for mate recognition, whereas the number of calls per call group have been found to be under directional preferences and, thus, to be important for mate quality assessment. In this study, we investigate preferences for calls that differ simultaneously in frequency, pulse rate, and number of calls per call group, and we ask how these properties interact with each other in influencing female mating decisions. Results of two-choice phonotaxis experiments provide no evidence to support the hypothesis that females process multi-attribute signals in a hierarchical way. In contrast, the pattern of preferences is consistent with the ‘preference function’ hypothesis, that is, with the hypothesis that females rank signals along an ordinal scale of values and choose accordingly. Pulse rate and frequency influence mating preferences more than does the number of calls per call group. The interaction between pulse rate and frequency is not additive but multiplicative: small differences in either pulse rate or frequency that, alone, have no effects on female choice, interact synergistically so that their combination has strong influence on female preferences. A preference repeatability test shows strong among-female differences in preference for multi-attribute signals. We suggest that this result reveals not only a variation in attribute values among females, but also a variation in the way females weight and combine attribute values into a single preference score.

GEOGRAPHIC VARIATION OF GENETIC AND BEHAVIORAL TRAITS IN NORTHERN AND SOUTHERN TÚNGARA FROGS

We use a combination of microsatellite marker analysis and mate-choice behavior experiments to assess patterns of reproductive isolation of the túngara frog Physalaemus pustulosus along a 550-km transect of 25 populations in Costa Rica and Panama. Earlier studies using allozymes and mitochondrial DNA defined two genetic groups of túngara frogs, one ranging from Mexico to northern Costa Rica (northern group), the second ranging from Panama to northern South America (southern group). Our more fine-scale survey also shows that the northern and southern túngara frogs are genetically different and geographically separated by a gap in the distribution in central Pacific Costa Rica. Genetic differences among populations are highly correlated with geographic distances. Temporal call parameters differed among populations as well as between genetic groups. Differences in calls were explained better by geographic distance than by genetic distance. Phonotaxis experiments showed that females preferred calls of males from their own populations over calls of males from other populations in about two-thirds to three-fourths of the contrasts tested. In mating experiments, females and males from the same group and females from the north with males from the south produced nests and tadpoles. In contrast, females from the south did not produce nests or tadpoles with males from the north. Thus, northern and southern túngara frogs have diverged both genetically and bioacoustically. There is evidence for some prezygotic isolation due to differences in mate recognition and fertilization success, but such isolation is hardly complete. Our results support the general observation that significant differences in sexual signals are often not correlated with strong genetic differentiation.