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Polyploid speciation has played an important role in evolutionary history across the tree of life, yet there remain large gaps in our understanding of how polyploid species form and persist. While systematic studies have been conducted in numerous polyploid complexes, recent advances in sequencing technology have demonstrated that conclusions from data-limited studies may be spurious and misleading. The North American gray treefrog complex, consisting of the diploidHyla chrysoscelisand the tetraploidHyla versicolor, has long been used as a model system in a variety of biological fields, yet all taxonomic studies to date were conducted with only a few loci from nuclear and mitochondrial genomes. Here, we utilized anchored hybrid enrichment and high-throughput sequencing to capture hundreds of loci along with whole mitochondrial genomes to investigate the evolutionary history of this complex. We used several phylogenetic and population genetic methods, including coalescent simulations and testing of polyploid speciation models with Approximate Bayesian Computation (ABC), to determine that H. versicolor was most likely formed via autopolyploidization from a now extinct lineage of H. chrysoscelis. We also uncovered evidence of significant hybridization between diploids and tetraploids where they co-occur, and show that historical hybridization between these groups led to the re-formation of distinct polyploid lineages following the initial whole genome duplication event. Our study indicates that a wide variety of methods and explicit model testing of polyploid histories can greatly facilitate efforts to uncover the evolutionary history of polyploid complexes.

Significant variation in genome size occurs among anuran amphibians and can affect cell size and number. In the gray treefrog complex in North America, increases in cell size in autotriploids of the diploid (Hyla chrysoscelis) altered the temporal structure of mate-attracting vocalizations and auditory selectivity for these properties. Here, we show that the tetraploid species (Hyla versicolor) also has significantly fewer brain neurons than H. chrysoscelis. With regard to cell size in tissues involved in vocal communication, spinal motor neurons were larger in tetraploids than in diploids and comparable to differences in erythrocyte size; smaller increases were found in one of the three auditory centers in the torus semicircularis. Future studies should address questions about how environmental conditions during development affect cell numbers and size and the causal relationships between these cellular changes and the vocal communication system.

Sustained perfect synchrony of signal production by animals is unrealistic, but even near-synchronous signalling is extremely rare. Near-synchronous signalling has been documented in some orthopteran insects and fireflies, and one kind of frog. This study provides observations and analyses of sustained bouts of impressive near-synchronous calling by a terrestrial breeding frog from Australia, the hip-pocket frog (Assa darlingtoni). Males called in scattered clusters of several individuals from the rainforest floor of northern New South Wales. In eight of nine pairs of semi-isolated males, there was sustained near-synchronous calling in bouts consisting of 16–20 calls and lasting 5–10 min. There was extensive overlap of the pulsed calls, and calls of a lagging male began overlapping that of a leading male after 2–5 pulses of a leader’s call note. In five pairs, one male’s calls were usually in the leading position; in three pairs, leadership frequently switched between males. In the latter interact...

Genetic variation in sexual displays is crucial for an evolutionary response to sexual selection, but can be eroded by strong selection. Identifying the magnitude and sources of additive genetic variance underlying sexually selected traits is thus an important issue in evolutionary biology. We conducted a quantitative genetics experiment with gray treefrogs (Hyla versicolor) to investigate genetic variances and covariances among features of the male advertisement call. Two energetically expensive traits showed significant genetic variation: call duration, expressed as number of pulses per call, and call rate, represented by its inverse, call period. These two properties also showed significant genetic covariance, consistent with an energetic constraint to call production. Combining the genetic variance-covariance matrix with previous estimates of directional sexual selection imposed by female preferences predicts a limited increase in call duration but no change in call rate despite significant selection on both traits. In addition to constraints imposed by the genetic covariance structure, an evolutionary response to sexual selection may also be limited by high energetic costs of long-duration calls and by preferences that act most strongly against very short-duration calls. Meanwhile, the persistence of these preferences could be explained by costs of mating with males with especially unattractive calls.

Interspecific aggression has important consequences for ecological processes and the evolution of behavioral strategies. We examined interspecific aggressive interactions in the 2 gray tree frog species, Hyla chrysoscelis and Hyla versicolor. These species call side by side in the same ponds, and acoustic interference occurs because of the similar spectral characteristics of their vocalizations. The aggressive calls of these 2 species, although very similar, were statistically distinguishable: calls of H. chrysoscelis were more strongly amplitude modulated than those of H. versicolor. We used playbacks and staged interactions to characterize the behaviors of males exposed to heterospecific competitors or their signals. Males readily responded with aggression to heterospecific individuals and playbacks of their calls. These behaviors were qualitatively similar in both intraspecific and interspecific interactions, but there were some significant differences. First, males of H. chrysoscelis were more aggressive toward playbacks of conspecific advertisement calls than toward those of H. versicolor. There were no significant differences in this respect in H. versicolor. Second, interspecific interactions were usually more escalated than intraspecific interactions and more likely to end with the loser moving away from its opponent. Although neither species had an advantage in staged interactions, behavioral responses were asymmetrical because H. versicolor was more likely than H. chrysoscelis to initiate physical contact. Previous studies showed that H. versicolor suffers from a greater reduction in attractiveness than H. chrysoscelis in the presence of heterospecific call overlap. Thus, the asymmetries in aggressive behavior between the 2 species may be related to differential costs of heterospecific competition.

As animal contests escalate, variation in the performance of aggressive signaling behaviors can give important insights into contest dynamics. In anuran amphibians, males of numerous species utilize distinctive aggressive vocalizations during disputes over calling spaces. Little is known, however, about the causes and consequences of variation in aggressive-call characteristics. We analyzed recordings of calls made during staged aggressive interactions between male gray tree frogs, Hyla versicolor, to determine how variation in a key aggressive-call characteristic, dominant frequency, was affected by increasing contest escalation. We found that dominant frequencies of aggressive calls were lower than those of advertisement calls used to attract females. Furthermore, we found that males lowered their aggressive-call frequencies with increases in escalation. Winners tended to have lower-frequency aggressive calls than losers. We conclude that aggressive calls in H. versicolor are similar to the graded aggressive calls that have been described in several other species. This gradation may allow males to balance the energetic costs of producing lower-frequency calls with the benefits of efficiently repelling rival males. Other processes related to motivation and the physiological effects of participating in contests may also be responsible for the observed variation in aggressive-call frequency with contest escalation. Our results demonstrate that detailed experimental studies of aggressive calling behavior in anurans, which to this point have rarely been performed, are feasible and generate important insights relating to general problems in animal contest behavior and animal communication.

Call timing is an important component of the behavioral repertoire of many chorusing species that compete acoustically for mates. The costs and benefits of particular call-timing patterns may vary with factors in the social environment, yet few studies have examined the possibility of socially mediated plasticity in call timing. We studied the effects of competition on advertisement-call timing in the gray tree frog, Hyla versicolor, by manipulating intermale distances in staged interactions. We measured phase angles and the proportion of calls that overlapped as interacting pairs of males were moved closer to one another, simulating an increase in acoustic competition. We observed a shift in phase angle resulting in substantially higher levels of overlap as males were moved so that they were calling at extremely close range. At close range, males often engage in intense agonistic contests over the calling space and we found that call timing may play a role in these interactions. Although there were not major differences in the call-timing behavior of winners and losers of contests, we suggest that call overlap may be a general signal of aggressive motivation. Changes in call timing with changes in intermale distance may, therefore, be related to changes in the intended audience as competition escalates. Key wor ds: aggressive signal, anuran, call timing, competition, overlap. [Behav Ecol]

The canyon treefrog, Hyla arenicolor, is a wide-ranging hylid found from southwestern US into southern Mexico. Recent studies have shown this species to have a complex evolutionary history, with several phylogeographically distinct lineages, a probable cryptic species, and multiple episodes of mitochondrial introgression with the sister group, the H. eximia complex. We aimed to use genome wide AFLP markers to better resolve relationships within this group. As in other studies, our inferred phylogeny not only provides evidence for repeated mitochondrial introgression between H. arenicolor lineages and H. eximia/H. wrightorum, but it also affords more resolution within the main H. arenicolor clade than was previously achieved with sequence data. However, as with a previous study, the placement of a lineage of H. arenicolor whose distribution is centered in the Balsas Basin of Mexico remains poorly resolved, perhaps due to past hybridization with the H. eximia complex. Furthermore, the AFLP data set shows no differentiation among lineages from the Grand Canyon and Colorado Plateau despite their large mitochondrial sequence divergence. Finally, our results infer a well-supported sister relationship between this combined Colorado Plateau/Grand Canyon lineage and the Sonoran Desert lineage, a relationship that strongly contradicts conclusions drawn from the mtDNA evidence. Our study provides a basis for further behavioral and ecological speciation studies of this system and highlights the importance of multi-taxon (species) sampling in phylogenetic and phylogeographic studies.

Performance limitations on signal production constrain signal evolution. Variation in signaling performance may be related to signaler quality and therefore is likely to be a salient aspect of communication systems. When multiple signal components are involved in communication, there may be trade-offs between components, and performance can be measured as the degree to which signalers approach the upper limits of the trade-off function. We examined vocal performance in the gray tree frog Hyla versicolor, in which females prefer values of call duration and rate exceeding the usual range of variation within and among males. We recorded interactions between pairs of males calling on mobile platforms that allowed us to manipulate intermale distance and place males in highly competitive environments. We found that, although there was a clear upper boundary on the ability of males to maximize call duration and call rate simultaneously, call effort did not remain constant in this highly competitive situation. Our estimates of an upper limit to vocal performance were corroborated by analyses of calling behavior in the context of close-range mate attraction. We discuss potential constraints on signaling performance and the relevance of this measure of performance for both intrasexual and intersexual communication.

We surveyed the geographical variation in male advertisement calls of the wide-ranging canyon treefrog, Hyla arenicolor, and found large call differences among geographically distant lineages that had been characterized by a recent phylogeographical study. To test whether these call differences were biologically relevant and could allow reproductive isolation of different lineages should they come into secondary contact, we assessed female preference in a lineage occurring in southern Utah and north-western Arizona, USA. These females exhibited a strong preference for their own lineage's call type over the calls of two Mexican lineages, but not over the calls from the geographically nearest lineage. We also identified traits that female frogs probably use to discriminate between lineage-specific advertisement calls. Our behavioural results, together with recent molecular estimates of phylogenetic relationships among lineages, will guide future work addressing the evolutionary forces that have led to this biologically significant variation in male sexual signals. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ••, ••–••.

The temporal relationship between signals often has strong and repeatable influences on receiver behaviour. While several studies have shown that receivers prefer temporally leading signals, we show that the relative timing of signal elements within overlapping signals can also have repeatable influences on receiver responses. Female grey treefrogs, Hyla versicolor, preferred overlapping conspecific advertisement call alternatives in which pulses were in the leading position relative to pulses in an alternative. The preference was maintained even when the first pulse of the stimulus with leading pulses began after that of the call with following pulses. To rule out the possibility of masking interference of the pulse pattern, we used a split-pulse design in which the playback of two nonoverlapping pulse elements were synchronized from spatially separated speakers. Females were attracted to the source of the short (6 ms) leading pulse element, which did not attract females in isolation, even though its amplitude was 24 dB lower than the long (24 ms) following element, which did attract females in isolation. Taken together, our results fall within a range of phenomena that have been classified as precedence effects. However, to our knowledge, showing localization based on successive leading pulses rather than the very first-arriving pulse is a novel discovery for nonhuman animals.

Even simple biological signals vary in several measurable dimensions. Understanding their evolution requires, therefore, a multivariate understanding of selection, including how different properties interact to determine the effectiveness of the signal. We combined experimental manipulation with multivariate selection analysis to assess female mate choice on the simple trilled calls of male gray treefrogs. We independently and randomly varied five behaviorally relevant acoustic properties in 154 synthetic calls. We compared response times of each of 154 females to one of these calls with its response to a standard call that had mean values of the five properties. We found directional and quadratic selection on two properties indicative of the amount of signaling, pulse number, and call rate. Canonical rotation of the fitness surface showed that these properties, along with pulse rate, contributed heavily to a major axis of stabilizing selection, a result consistent with univariate studies showing diminishing effects of increasing pulse number well beyond the mean. Spectral properties contributed to a second major axis of stabilizing selection. The single major axis of disruptive selection suggested that a combination of two temporal and two spectral properties with values differing from the mean should be especially attractive.

To understand the variation in aggressiveness and factors important for contest outcome, we quantified and compared agonistic interactions of four field cricket species in eastern North America: Gryllus fultoni (Orthoptera; Gryllidae), G. vernalis, G. pennsylvanicus, and G. rubens. The most aggressive behavior that we observed, the grapple, was frequently displayed in agonistic trials of G. pennsylvanicus and G. rubens. By contrast, we never observed this behavior in trials involving G. fultoni and G. vernalis. Consequently, species was the only significant variable affecting the variation in aggressiveness, whereas size difference, age difference, and contest duration had no significant effect. In species with high levels of aggressiveness, G. pennsylvanicus and G. rubens, the factors that might be indicative of resource holding potential such as size or age difference seemed to be significant for contest outcome. In species with low levels of aggressiveness, G. fultoni and G. vernalis, however, there were indications that contest outcomes were determined by aggressiveness itself rather than the size and age differences between opponents. Markov chain analyses revealed that the difference in aggressiveness between species with high and low levels of aggressiveness lay in a sequence of escalating behaviors: antennal fencing, mandible flare, and grapple. The escalated state of aggressiveness characterized by this behavioral sequence in G. pennsylvanicus and G. rubens seems to be the ancestral state in the North American Gryllus phylogeny. We argue that the loss of a tendency to use burrows in G. fultoni and G. vernalis might be related to low levels of aggressiveness in these species.

Signal-timing adjustment is common in communally signaling species, and there is large variation in signal-timing formats found in nature. We conducted a survey of geographic variation in female signal-timing preferences and male signaling behavior of a tree frog to test predictions of two hypotheses about the sources of selection acting on signal-timing behavior. We found that female preferences are important in shaping male signal timing, affecting the absolute placement of signals relative to the temporal limits of female preferences. Variation in other signal characters, particularly signal period, also affects signal timing, albeit in a more complicated pattern: the influence of signal period on signal timing varied between populations. Overall, our findings indicate that the evolution of male signal-timing behavior is strongly influenced by female preferences and by an interaction with other aspects of male signaling behavior such as male signal period.

SUMMARYFrogs have two inner ear organs, each tuned to a different range of frequencies. Female treefrogs (Hylidae) of three species in which males produce calls with a bimodal spectrum (Hyla chrysoscelis, H. versicolor, H. arenicolor) preferred alternatives with a bimodal spectrum to alternatives with a single high-frequency peak. By contrast,females of H. avivoca, in which males produce calls with a single,high-frequency peak, preferred synthetic calls with a single high-frequency peak to calls with a bimodal spectrum. These results are consistent with the expectations of the matched-filter hypothesis and run counter to the predictions of the pre-existing bias hypothesis. At moderate to high playback levels (85–90 dB), females of H. avivoca and of two of three mtDNA-defined lineages of H. versicolor preferred unimodal signals with a high-frequency peak to those with a low-frequency peak. Females of H. chrysoscelis, H. arenicolor and the third lineage of H. versicolor did not show a preference, indicating that receiver mechanisms may be at least as evolutionarily labile as call structure. Spectral-peak preferences of gray treefrogs from Missouri, USA were intensity-dependent. Whereas females chose low-frequency calls at 65 dB spl, there was either no preference (H. chrysoscelis) or a preference for high-frequency calls (H. versicolor) at 85 and 90 dB spl. These non-linear effects indicate that there is an increasing influence of high-frequency energy on preferences as females approach calling males, and these results serve to emphasize that playback experiments conducted at a single level may have limited generality.

The evolution of complex signals may be favoured by hidden preferences or pre-existing sensory biases. Females of two species of grey treefrogs ( Hyla chrysoscelis and Hyla versicolor ) were tested with combinations of a conspecific advertisement call and acoustic appendages. Appendages consisted of aggressive calls and segments of advertisement calls from conspecific males and males of three other species and bursts of filtered noise. When a wide variety of these acoustic appendages followed the advertisement call, the resulting compound signal was often more attractive than the same advertisement call alone. When the same appendages led advertisement calls, however, the compound signal was never more attractive and sometimes less attractive. The order effect was especially strong in tests of H. versicolor in which advertisement-call duration was decreased. These results cannot be explained by a general pre-existing bias for extra stimulation per se . Rather, order and other effects may constrain the evolution and subsequent modification of complex and extravagant signals, examples of which have been reported for a wide range of taxa.

Two closely related wood-cricket species, Gryllus fultoni (Orthoptera: Gryllidae) and G. vernalis, produce similar calling songs, consisting of 3-pulse chirps. Analysis of field and laboratory recordings of calling songs showed that, after correction to a common temperature, there was a divergence in chirp and pulse rates between far allopatric populations of G. fultoni and populations sympatric with G. vernalis. To determine whether the divergence in calling songs potentially provides reproductive isolation between G. fultoni and G. vernalis throughout the temperature range over which these insects sing, we recorded calling songs of lab-reared G. fultoni and G. vernalis populations between 18 and 28°C. Mean chirp rate significantly differed between sympatric and far allopatric G. fultoni populations as well as between sympatric G. fultoni and G. vernalis populations. Although there was a significant difference in mean pulse rate between sympatric G. fultoni and G. vernalis populations, pulse rate did not differ between sympatric and far allopatric G. fultoni populations in the laboratory study. Considering the magnitudes of differences in calling song characters discriminated by females of G. fultoni and the mean differences and the variability in calling song characters between the two species, the joint difference in chirp and pulse rates may be adequate for species discrimination over most of the range at which these crickets breed.

We investigated the effects of overlap between the advertisement calls of two closely related and often-sympatric species of grey treefrogs on female phonotactic behaviour and signal selectivity. The tetraploid H. versicolor and the diploid H. chrysoscelis have advertisement calls consisting of trains of stereotyped pulses that are similar in their spectral composition. All females of H. versicolor from a population sympatric with H. chrysoscelis approached a speaker broadcasting synthetic advertisement calls modelled after conspecific calls when the calls of the two species alternated from speakers with a 90° angular separation with respect to the release point. Surprisingly, the same females almost exclusively approached the source of synthetic calls modelled after the heterospecific advertisement calls when these calls completely overlapped the conspecific stimuli. Female H. chrysoscelis from the same sympatric population approached the source of synthetic conspecific calls even when these calls were overlapped by the heterospecific stimuli, indicating that females in these closely related species may differ in their susceptibility to the negative consequences of call overlap. When female H. versicolor were presented with conspecific advertisement calls that were completely overlapped by those of H. chrysoscelis, many females failed to approach either signal source. Response times of females that did respond were also longer, and the direction of phonotaxis was shifted in the direction of the heterospecific signal source. These negative influences of acoustic interference were reduced or eliminated as the degree of overlap between the calls of the two species was reduced. The results demonstrate that the effects of signal overlap on female phonotactic behaviour may be complex.

Polyploidization is one of the few mechanisms that can produce instantaneous speciation. Multiple origins of tetraploid lineages from the same two diploid progenitors are common, but here we report the first known instance of a single tetraploid species that originated repeatedly from at least three diploid ancestors. Parallel evolution of advertisement calls in tetraploid lineages of gray tree frogs has allowed these lineages to interbreed, resulting in a single sexually interacting polyploid species despite the separate origins of polyploids from different diploids. Speciation by polyploidization in these frogs has been the source of considerable debate, but the various published hypotheses have assumed that polyploids arose through either autopolyploidy or allopolyploidy of extant diploid species. We utilized molecular markers and advertisement calls to infer the origins of tetraploid gray tree frogs. Previous hypotheses did not sufficiently account for the observed data. Instead, we found that tetraploids originated multiple times from extant diploid gray tree frogs and two other, apparently extinct, lineages of tree frogs. Tetraploid lineages then merged through interbreeding to result in a single species. Thus, polyploid species may have complex origins, especially in systems in which isolating mechanisms (such as advertisement calls) are affected directly through hybridization and polyploidy.

Two species of closely related wood cricket, Gryllus fultoni (Orthoptera: Gryllidae) and Gryllus vernalis, occur together in some parts of the eastern United States and have a similar calling song structure, consisting of three-pulse chirps. A previous study revealed that chirp rate in G. fultoni was highest (greatest difference vis-a-vis chirp rate in G. vernalis) in sympatric populations, intermediate in near allopatric populations that were located close to the sympatric zone, and lowest in allopatric populations. A similar trend was observed in pulse rate, but the mean values of this trait showed much more convergence than chirp rate at the low end of the range of calling temperatures. In this study, we investigated the song discrimination of females from sympatric and allopatric populations of G. fultoni at about 23°C, which is near the middle of the normal range of calling temperatures. We used both single-stimulus and two-stimulus playback experiments to learn if geographical differences in song preferences paralleled those in calling songs. Stimuli presented were representative of calling songs in three classes of G. fultoni populations (sympatric, near allopatric, and far allopatric), a calling song of G. vernalis, and three calling songs with parameter values that were intermediate with respect to those of the songs of far allopatric G. fultoni and G. vernalis. In the single-stimulus playbacks, females of all G. fultoni populations responded poorly if at all to the heterospecific stimulus. Females of sympatric and near allopatric populations responded poorly to all intermediate stimuli, but females of far allopatric populations frequently responded to these sounds. In the two-stimulus playbacks, females of sympatric and near allopatric populations generally discriminated against intermediate and heterospecific stimuli. However, females of far allopatric populations often did not discriminate against intermediate stimuli, whose characteristics resembled the calling songs of G. vernalis. The divergent pattern of female phonotactic discrimination between sympatric and far allopatric populations was thus generally congruent with the pattern of divergence in chirp and pulse rates and would be expected to significantly reduce heterospecific mating in sympatry. These geographical patterns of female song discrimination and male calling songs conform to a commonly used definition of reproductive character displacement.

Females are expected to use spectral properties of acoustic signals for mate choice because dominant frequency often provides reliable information about male size. The advertisement calls of grey treefrogs ( Hyla chrysoscelis and H. versicolor ) have two main frequency bands: the amplitude of the dominant, high-frequency band averages 7–10 dB greater than that of the low-frequency band. The frequencies of these peaks were negatively correlated with male body size in both species. I used two-speaker playbacks of synthetic calls to generate preference functions for both species based on simultaneous variation in the frequencies of the two bands and, for H. versicolor , on variation in single-component calls; frequencies of most of these stimuli fell within the range of the dominant, high-frequency band. When two-component sounds were used, preference functions were bimodal and broad, with peaks close to ( H. versicolor ) or slightly higher than ( H. chrysoscelis ) mean frequencies in conspecific calls. Because preferences were not strong, I predict that females exert only weak stabilizing or directional selection on male call frequencies. When single-component calls were used in tests of H. versicolor , the preference-function peak was shifted to a lower-than-average frequency within the high-frequency range. I hypothesize that such a frequency may better stimulate both of the inner ear organs, whose tuning roughly matches frequencies in the two bands in conspecific calls, than a single component of average or much lower-than-average frequency. Females of both species preferred calls with both spectral peaks to calls with a single, high-frequency peak, even when the low-frequency peak was attenuated by 24–30 dB. This result serves to emphasize that typically subdominant frequency components can have seemingly disproportionate effects on signal attractiveness.

Signals used for mate choice and receiver preferences are often assumed to coevolve in a lock-step fashion. However, sender-receiver coevolution can also be nonparallel: even if species differences in signals are mainly quantitative, females of some closely related species have qualitatively different preferences and underlying mechanisms. Two-alternative playback experiments using synthetic calls that differed in fine-scale temporal properties identified the receiver criteria in females of the treefrog Hyla chrysoscelis for comparison with female criteria in a cryptic tetraploid species (H. versicolor); detailed preference functions were also generated for both species based on natural patterns of variation in temporal properties. The species were similar in three respects: (1) pulses of constant frequency were as attractive as the frequency-modulated pulses typical of conspecific calls; (2) changes in preferences with temperature paralleled temperature-dependent changes in male calls; and (3) preference functions were unimodal, with weakly defined peaks estimated at values slightly higher than the estimated means in conspecific calls. There were also species differences: (1) preference function slopes were steeper in H. chrysoscelis than in H. versicolor; (2) preferences were more intensity independent in H. chrysoscelis than in H. versicolor; (3) a synergistic effect of differences in pulse rate and shape on preference strength occurred in H. versicolor but not in H. chrysoscelis; and (4) a preference for the pulse shape typical of conspecific calls was expressed at the species-typical pulse duration in H. versicolor but not in H. chrysoscelis. However, females of H. chrysoscelis did express a preference based on pulse shape when tested with longer-than-average pulses, suggesting a hypothesis that could account for some examples of nonparallel coevolution. Namely, preferences can be hidden or revealed depending on the direction of quantitative change in a signal property relative to the threshold for resolving differences in that property. The results of the experiments reported here also predict patterns of mate choice within and between contemporary populations. First, intraspecific mate choice in both species is expected to be strongly influenced by variation in temperature among calling males. Second, simultaneous differences in pulse rate and pulse shape are required for effective species discrimination by females of H. versicolor but not by females of H. chrysoscelis. Third, there is greater potential for sexual selection within populations and for discrimination against calls produced by males in other geographically remote populations in H. chrysoscelis than in H. versicolor.

We investigated the effects of dopamine depletion on acoustically guided behavior of anurans by conducting phonotaxis experiments with female gray treefrogs (Hyla versicolor) before and 90 min after bilateral injections of 3, 6, or 12 microg 6-hydroxydopamine (6-OHDA) into the telencephalic ventricles. In experiments with one loudspeaker playing back a standard artificial mating call, we analyzed the effects of 6-OHDA on phonotactic response time. In choice tests we measured the degree of distraction from the standard call (20 pulses/s) by three different variants with altered pulse-rate (30/s, 40/s, 60/s). Five days after experiments, brains were immunostained for tyrosine hydroxylase. Labeled neurons were counted in the suprachiasmatic nucleus, posterior tuberculum, interpeduncular nucleus, and locus coeruleus, and correlation between neuronal numbers and behavioral scores was tested. Response times increased together with 6-OHDA concentrations, which was mainly due to longer immobile periods before the animals started movement. In choice tests the most irrelevant stimulus (60/s) distracted 6-OHDA injected females from the standard stimulus, while sham injected controls were undistracted. The number of catecholaminergic neurons decreased with increasing 6-OHDA concentration in the suprachiasmatic nucleus, posterior tuberculum, and interpeduncular nucleus. The normalized number of immunoreactive neurons in the posterior tuberculum was positively correlated with phonotaxis scores in the one-speaker test, demonstrating that motor deficits are a function of tubercular cell loss. We conclude that bilateral 6-OHDA lesions in anuran amphibians cause motor (difficulty to start movements) as well as cognitive symptoms (higher distraction by irrelevant stimuli) that have also been described for human Parkinson patients.

Diencephalic and midbrain auditory nuclei are involved in the processing of auditory communication signals in anurans [Comparative Hearing: Fish and Amphibians, Springer-Verlag, New York, 1999, p. 218], but their exact roles in acoustically guided behavior, such as female phonotaxis, are unclear. To address this question, behavioral experiments were combined with lesions of dorsal thalamic nuclei and the midbrain torus semicircularis. Females were tested in two-alternative-forced-choice phonotactic experiments before and after a defined brain area was lesioned. During phonotactic tests, females had to choose between a “standard” synthetic call and one of three different variants, each of which had a single acoustic property (pulse rate, pulse rise-time, sound spectrum) that differed from the standard synthetic call. Results showed that dorsomedial thalamus lesions produced little or no effect on phonotaxis. In contrast, superficial and deep thalamus lesions, as well as lesions of the torus semicircularis, significantly decreased the number of phonotactic responses and increased the response time. Superficial thalamus lesions also abolished or reversed preferences for the standard call in the rise-time and sound spectrum tests. This effect is likely to have been caused by an imbalance in the stimulation of the thalamus by the low- and high-frequency pathways because these preferences were not affected in animals with more extensive lesions that included the superficial thalamus. Our data suggest that the torus semicircularis, but not the dorsal thalamus is crucial for phonotaxis in gravid, reproductively active females. Although dorsal thalamic nuclei seem to play a role in spectral sensitivity, they may additionally have motivational or attentional functions that contribute to achieving a state of phonotactic readiness.

Interactions between species can affect the evolution of their sexual signals, receiver selectivity, or both. One commonly expected outcome is reproductive character displacement, whereby adverse consequences of mismating select for greater differentiation of communication systems in areas of sympatry than in areas of allopatry. We found evidence of reproductive character displacement in the acoustic communication system of green tree frogs (Hyla cinerea). The strength of female preferences for the spectral properties of calls that distinguish conspecific calls from those of a closely related congener, H. gratiosa, was greater in areas of sympatry with H. gratiosa than in areas of allopatry. We also found subtle differences in advertisement calls and in the heights of male calling perches when we restricted our comparisons to localities in which H. gratiosa was also breeding (syntopy) with localities where this species was absent. Hyla cinerea and H. gratiosa show only weak genetic incompatibility, but the calls representative of interspecific hybrids were unattractive to females of both parental species. Hybrids might also be at an ecological disadvantages because of different habitat preferences of the two taxa. Thus, selection against production of less fit or less attractive hybrid or backcross offspring are probably the main causes responsible for the differences documented in this paper.

Evolutionary change in signals may occur within and between populations. A pre-requisite of signal evolution is that heritable traits vary sufficiently between individuals for selection to operate....

Some territorial animals display low levels of aggression towards a familiar territorial neighbour in its usual territory, but exhibit high levels of aggression towards neighbours in novel locations and unfamiliar individuals. Here, we report results from a field playback study that investigated whether territorial males of the North American bullfrog (Rana catesbeiana) could discriminate between the acoustic signals of simulated neighbours and strangers in the absence of contextual cues associated with a specific location. Following repeated exposures to synthetic bullfrog calls from a particular location, subjects responded significantly less aggressively to a familiar call, compared with an unfamiliar one, when both calls were broadcast from familiar and novel locations, indicating that bullfrogs could recognize a neighbour's calls independently of the contextual cues provided by the direction of the neighbour's territory. Subjects responded equally aggressively to unfamiliar calls broadcast from either a familiar or a novel location, which indicates that they could perceive unfamiliar calls as those of a stranger, regardless of where the stranger was encountered. Together, these two results provide evidence that a frog possesses a capacity for individual voice recognition.

Studies of mate attraction have traditionally employed one of two experimental methods: choice tests in which female preferences from among two or more signals are tabulated, or single-stimulus tests in which the female response to one signal is quantified. Choice tests have long been preferred for examining mate attraction in anurans. Inspired by Wagner's (1998, Animal Behaviour,55, 1029–1042) discussion of the strengths and weaknesses of the two experimental approaches, we used a single-stimulus design to examine mate attraction in two sibling species of treefrogs (Hyla versicolor and H. chrysoscelis). We quantified female responses based upon the relative time required to approach signals varying in pulse rate, pulse rise time and pulse number. The data were used to generate response functions providing a quantitative measure of female attraction to the stimuli. Comparisons with data from choice experiments reveal broad similarities, as well as properties of female responses that had not been detected with choice tests. The results are discussed with regard to female selectivity for call parameters that are likely to mediate sexual selection and homospecific pairing.

Despite intense interest in mate choice, relatively little is known about how individuals sample prospective mates. Indeed, a key issue is whether females sample males or simply mate with the first male encountered. We investigated mate sampling by female barking treefrogs (Hyla gratiosa). Females choosing mates in natural choruses did not move between males but instead mated with the first male they approached closely. Most females mated with the male closest to them at the start of their mate-choice process, and females were more likely to mate with the closest male when the distance to other males was large. These observations are consistent with the hypothesis that females do not sample potential mates but instead mate with the first male they distinguish from the rest of the chorus. To test this initial detection hypothesis, we conducted a playback experiment in which we offered females a choice between two calls, one of which was detectable above the background chorus sound at the female’s release point, and one of which became detectable only as females moved toward the initially detectable call. Females did not prefer the initially detectable call, thus ruling out the initial detection hypothesis and implicating sampling of potential mates by females. Based on the behavior of females in natural choruses, we hypothesize that females approach the chorus, move to locations where they are able to detect the calls of several males simultaneously, and choose a mate from among these males at some distance from the males. Such simultaneous sampling may be common in lekking and chorusing species, which have been the subjects of many studies of sexual selection. Key words: Hyla gratiosa, mate choice, mate sampling, simultaneous sampling, treefrogs. [Behav Ecol 13:472–480 (2002)]

In his book Descent of Man and Selection in Relation to Sex, Darwin (1) made a compelling argument that evolution could not be explained solely on the basis of differential survival (natural selection). In particular, traits that enhance an individual’s reproductive success (sexual selection) often decrease its chances of survival. For example, the risks of injury and death can be high for males that fight for direct access to females or for territories that contain resources needed by females. Courtship ornaments and displays, which often determine territorial ownership or mating success, are not only energetically costly but frequently result in increased chances of predation. Some of the most striking and well-documented examples of the latter phenomenon involve reductions in sexual ornaments and modifications of behavior that reduce the conspicuousness of courting individuals in areas where predation is high compared with areas where predators are absent or uncommon (2, 3). These changes almost certainly increase survivorship at the expense of less effective territorial defense and lower mating success. In PNAS, Steinberg et al. (4) found that males of a semiarboreal lizard, the brown anole (Anolis sagrei), on islands in the Bahamas where a predator was introduced show a significant reduction in the amplitude of head bobs compared with lizards from predator-free islands. This behavioral change reduces the communication range of these visual signals, thus lowering the chances of being detected by a predator. By the same token, such signals are likely to be less effective in repelling more distant territorial rivals and attracting potential mates. Whereas a common response to predation is to reduce the duration or frequency of their displays (5), the main change in A. sagrei on islands with predators was head-bob amplitude, a display component used in territorial defense and mate attraction.

Some territorial animals discriminate among neighbours and strangers based on individual differences in acoustic signals. Male North American bullfrogs, Rana catesbeiana, display this form of discrimination based on individual variation in advertisement calls. In this study, we investigated the acoustic basis of neighbour–stranger discrimination to determine how individual identity might be encoded by particular properties of bullfrog advertisement calls. We analysed patterns of within-male and between-male variability in 1078 bullfrog advertisement calls recorded from 27 territorial males. All call properties that we examined varied significantly among males. However, fundamental frequency and dominant frequency showed the lowest within-male variation and the highest repeatability between two recording sessions, and both properties were highly correlated with the first canonical root from discriminant function analyses, which typically accounted for 70–80% of the variability between males. We suggest that neighbour–stranger discrimination in bullfrogs is partially mediated by between-male differences in the spectral or fine temporal properties of advertisement calls.

We studied female mate choice by Hyla versicolor in three venues to examine how acoustic and spatial complexity, background noise, and the calling behavior of males might influence preferences manifest in previous laboratory two-stimulus choice tests. Our laboratory-based two-stimulus choice tests with and without broadcasts of chorus noise demonstrated that females prefer long calls relative to short calls when calling efforts of alternatives are equivalent. Background noise impaired the ability of females to discriminate in favor of longer over shorter calls, but the magnitude of the effect was small. Captures of females at eight speakers broadcasting 6- to 27-pulse calls at the edge of a pond revealed strong discrimination against only the shortest call variant. In natural choruses, females may only rarely encounter males using such unattractive vocalizations. Female phonotaxis at an artificial pond with caged and electronically monitored calling males also indicated that consequences of female preferences for temporal aspects of calling observed in two-stimulus choice tests are much attenuated in choruses and explain only small portions (

Whole-genome duplication is believed to have played a significant role in the early evolution and diversification of vertebrate animals. The establishment of newly arisen polyploid lineages of sexually reproducing animals requires assortative mating between polyploids. Here, we show that genome duplication can directly alter a phenotypic trait mediating mate choice in the absence of genotypic change. Our results suggest that the direct effect of polyploidy on behaviour is a consequence of increased cell size.

Territorial animals often exhibit relatively lower levels of aggression toward familiar territorial neighbors than toward strangers. Habituation to a neighbor or its communication signals has been proposed to account for this reduced aggression between adjacent territorial neighbors. The authors asked whether discrimination between neighbors and strangers by territorial male bullfrogs (Rana catesbeiana) could result from habituation of the aggressive response to repeated presentations of the acoustic communication signals of a simulated new neighbor calling from an adjacent territory. In 3 field playback experiments, the authors found evidence for 5 response characteristics that operationally define habituation. Moreover, aggressive response decrements persisted between nights of chorusing and were specific to an individually distinct property of male advertisement calls. The authors suggest that reduced aggression between neighboring territorial male bullfrogs could result from long-term, stimulus-specific habituation to the advertisement calls of a new neighbor.

Individuals of many territorial species discriminate between familiar territorial neighbors and unfamiliar strangers based on individual differences in acoustic signals. Many anuran amphibians are territorial and use primarily acoustic communication during social interactions, but evidence for acoustically mediated individual discrimination is available only for one species. As a first step in research designed to investigate individual discrimination in a second species of territorial frog, we examined patterns of within-male and among-male variability in 198 advertisement calls of 20 male green frogs, Rana clamitans. The aim was to determine which acoustic properties could be used by males to recognize their territorial neighbors and to estimate limits of reliability afforded by these properties for identifying different neighbors. All of the call properties that we examined exhibited significant inter-individual variation. Discriminant function analyses assigned between 52% and 100% of calls to the correct individual, depending on sample size and the call properties included in the model. This suggests that there is sufficient among-male variability to statistically identify individuals by their advertisement calls. The call properties of fundamental frequency and dominant frequency contributed the most towards discrimination among individuals. Based on their natural history and behavior and the results reported here, we suggest that male green frogs likely discriminate between strangers and adjacently territorial neighbors based on individual variation in advertisement calls.

Preference functions, which quantify preference strength relative to variation in male traits or signals, are central to understanding mechanisms and consequences of female choice. Female tree frogs (Hyla versicolor) choose mates on the basis of advertisement calls and prefer long calls to short calls. Here we show, in two experimental designs, that preference strength increased significantly as the difference in call duration was increased only if the absolute durations of alternative stimuli were below average. Hence preference strength was a non-linear function of duration, and females did not base preferences solely on the percentage difference in duration. In experiments simulating costly choice (unequal playback levels), non-linear effects were more pronounced than in the conventional design (equal playback levels). Repeated estimates of preference strength using the unequal-playback design revealed significant among-female variation. These patterns of preference suggest that selection by female choice for males producing calls of average duration over males producing very short calls is stronger than selection for males producing very long calls over males producing calls of average duration. Female preferences, especially in tests simulating a potentially costly choice, could reflect differences in the net benefits to females of mating with males producing calls of different duration. Key words: preference function, preference strength, advertisement calls, call duration, repeatability, Hyla versicolor. [Behav Ecol 11:663–669 (2000)]

We assessed the potential for several acoustic properties of the advertisement calls of male gray tree frogs to affect relative mating success by relating patterns of variation in these properties to minimum differences required to elicit female choice. Dynamic properties (pulse number, PN; call rate, CR; and duty cycle, DC, the ratio of call duration to call period) varied much more within bouts of calling than a static property (dominant frequency, DF) but nevertheless exhibited significant between male variation in three of four breeding seasons. Many multiply recorded males consistently produced calls with values substantially above or below mean values of males recorded on the same nights. Nightly ranges of variation in PN and CR were often greater than the minimum differences required to elicit female preferences in the laboratory. In most experiments, females chose high-PN or fast-CR calls over low-PN or slow-CR alternatives, respectively, even if the preferred stimuli were farther away or 6-10 dB lower in sound pressure level (SPL), provided that differences in PN or CR were 100%. Consistent with these results, females did not always choose the closer of two calling males in the field. Nightly ranges of variation in DF rarely equaled the minimum difference required to elicit SPL independent preferences. Females preferred a stimulus of high-PN and slow-CR over an alternative of low-PN or fast-CR with the same acoustic on-time; in two experiments, females chose calls of high-PN over low-PN alternatives even though the playback of the high-PN call was interrupted and the low-PN call was broadcast continuously. Thus, female preferences were not merely based on the total time of acoustic stimulation. Responses of females tested twice in the same experiment suggest that phenotypic variation in preference was limited in our study populations.

In nearly all female choice experiments with anurans, the values of acoustic properties of experimental stimuli have been fixed. In nature, however, a female usually experiences an array of calls in which acoustic properties, especially dynamic ones, differ between males in variability as well as in mean values. High variability per se does not make a synthetic call more or less attractive to female grey treefrogs, Hyla versicolor, than a non-variable or low-variability call with about the same mean values of pulse number per call, call period, or both. Females did prefer a low-variability alternative with a slightly higher number of sound pulses per unit time than the variable stimulus, suggesting that females can resolve small differences in mean values between the calls of different males despite considerable within-male variability.

SYNOPSIS. Insects and anurans show various patterns of selective responsiveness to playbacks of natural and synthetic sounds. Preferences and tuning of the auditory system most often match one or more strongly emphasized frequency components in long-range, mate-attracting signals typical of conspecific individuals, but exceptions, in which frequencies lower or higher than the mean are preferred, occur in some species. In some insects the most preferred frequencies may correspond to regions that maximize localizability of signals rather than regions of maximum sensitivity. Patterns of preference for fine-temporal properties are typically stabilizing within a population; females prefer values near the mean and few males produce calls with values that deviate sufficiently to make them less attractive than calls with average values. Preferences for gross temporal properties {e.g., the rate and duration of signaling) are usually highly directional, with much higher than mean values preferred. In anurans, call rate is a better predictor of male mating success than dominant frequency. Nevertheless, a variety of factors, especially close-range assessments that are common in insects, may modify or negate the advantages of producing long-range signals of high relative attractiveness. The evolutionary consequences of selective responsiveness in anurans and insects are discussed

Abstract. Because male treefrogs indiscriminately try to mate with other frogs of comparable size that move close to them, a major cause of heterospecific matings is thought to be inadvertent contact between a female of one species and a vocalizing male of another species. It follows that the probability of such mismatings would be reduced if females recognized the calls of sympatric, heterospecific males and avoided moving near those males during phonotactic approaches to vocalizing conspecific males. Gravid female treefrogs of three species were tested in situations in which they could avoid a source of heterospecific calls as they responded to conspecific calls. The results of these experiments provide no evidence that females avoid sources of heterospecific calls. Because females of all three species sometimes show positive phonotactic responses in 'no-choice' situations to the same kinds of heterospecific calls used in this study, these results suggest that the mechanisms responsible for homospecific pairing are qualitatively no different than those mediating intraspecific mate choice. The implications of these results for current debates about processes underlying mate choice are discussed.

Abstract. Females of the grey treefrog, Hyla chrysoscelis, from three areas where a genetically incompatible, sibling species, H. versicolor, also occurred, were much more likely to prefer synthetic sounds with fine-temporal properties typical of conspecific males than were females from two areas of remote allopatry. Specifically, more than 90% of the females from sympatric populations weighted a species-specific cue (pulse-repetition rate = pulse rate) more strongly than they did a cue (call duration) that mediates intraspecific mate choice, whereas only about 50% of the females from remote allopatric populations did so. These results suggest that selection for the avoidance of mating mistakes has acted on females in sympatric populations. If this is a general phenomenon, then the apparent rarity of reproductive character displacement may be explained at least in part by the fact that nearly all previous studies have focused solely on geographical patterns of variation in male courtship signals.

Genetic variation in sexual displays is crucial for an evolutionary response to sexual selection, but can be eroded by strong selection. Identifying the magnitude and sources of additive genetic variance underlying sexually selected traits is thus an important issue in evolutionary biology. We conducted a quantitative genetics experiment with gray treefrogs (Hyla versicolor) to investigate genetic variances and covariances among features of the male advertisement call. Two energetically expensive traits showed significant genetic variation: call duration, expressed as number of pulses per call, and call rate, represented by its inverse, call period. These two properties also showed significant genetic covariance, consistent with an energetic constraint to call production. Combining the genetic variance-covariance matrix with previous estimates of directional sexual selection imposed by female preferences predicts a limited increase in call duration but no change in call rate despite significant selection on both traits. In addition to constraints imposed by the genetic covariance structure, an evolutionary response to sexual selection may also be limited by high energetic costs of long-duration calls and by preferences that act most strongly against very short-duration calls. Meanwhile, the persistence of these preferences could be explained by costs of mating with males with especially unattractive calls.

Different acoustic properties of communication signals potentially encode different kinds of biologically significant information. Studies of frogs and toads have identified behaviorally relevant properties that correlate with a male's species, body size, energetic investment in calling and perhaps physical and evolutionary fitness. Behavioral experiments reveal that simultaneous variation of different acoustic properties can interact in unpredictable ways to determine the overall effectiveness of a signal.

Patterns of variation in the acoustic properties of advertisement calls of several species of treefrogs (family Hylidae) are related to patterns of female preferences for these same properties. Properties were unequivocally classified as static or dynamic based on within-male variability during bouts of calling. Static properties are highly stereotyped within males and between males in natural populations. Dynamic properties often change by as much as 100% during single bouts of calling; dynamic properties are usually much more variable among males in natural populations than are static properties. An analysis of the calling performance of individual males over the course of a breeding season indicated that at least one dynamic property, pulse number (or call duration) in Hyla versicolor, was sufficiently repeatable to differentiate between males. Playback experiments that employed synthetic calls showed that: (1) females exhibit patterns of preferences that should result in selection that is stabilizing or weakly directional on static properties, and highly directional on dynamic properties; and (2) the overall attractiveness of a signal is affected by variation in both static and dynamic properties. Similar results were found in laboratory and field studies of other taxa, primarily anurans and insects. Patterns of variability in call properties of males and in female preferences are discussed in terms of proximate mechanisms and theoretical models of the evolution of female choice.

SUMMARYThe two main spectral components of the advertisement calls of two species of North American gray treefrogs (Hyla chrysoscelis and H. versicolor) overlap broadly in frequency, and the frequency of each component matches the sensitivity of one of the two different auditory inner ear organs. The calls of the two species differ in the shape and repetition rate (pulse rate) of sound pulses within trills. Standard synthetic calls with one of these spectral peaks and the pulse rate typical of conspecific calls were tested against synthetic alternatives that had the same spectral peak but a different pulse rate. The results were generalized over a wide range of playback levels. Selectivity based on differences in pulse rate depended on which spectral peak was used in some tests, and greater pulse-rate selectivity was usually observed when the low-frequency rather than the high-frequency peak was used. This effect was more pronounced and occurred over a wider range of playback levels in H. versicolor than in H. chrysosceliswhen the pulse rate of the alternative was higher than that of the standard call. In tests at high playback levels with an alternative of 15 pulses s–1, however, females of H. versicolor showed greater selectivity for the standard call when the high-frequency rather than the low-frequency spectral peak was used. This last result may reflect the different ways in which females of the two species assess trains of pulses,and the broad implications for understanding the underlying auditory mechanisms are discussed.

Hormonal levels fluctuate during the breeding season in many anurans, but the identity of the hormones that modulate breeding behavior and their effects remain unclear. We tested the influence of a combined treatment of progesterone and prostaglandin on phonotaxis, the key proceptive reproductive behavior of female anurans. First, we found that female gray treefrogs (Hyla versicolor) treated with progesterone and prostaglandin exhibited phonotaxis to synthetic male advertisement signals significantly more often than animals treated with ringers vehicle or uninjected controls. Responsive females had greater levels of plasma progesterone and estradiol compared to both control groups, suggesting that these steroids may be promoting phonotaxis. Second, we found that the selectivity of hormonally-induced phonotaxis in H.versicolor was similar to that observed in freshly captured breeding animals. Females made the same choices between acoustic signals after hormone treatments in tests of frequency, call rate and pulse rate, compared to their responses without treatment immediately after collection from the breeding chorus. The preference for a longer call was, however, significantly weaker after hormonal induction of phonotaxis. Hormonally primed females were also less likely to respond in any test and took longer to respond than did freshly collected females. Consequently, our study shows how progesterone-prostaglandin induced phonotaxis in female treefrogs influences both the quality and quantity of phonotaxis, relative to that exhibited by naturally breeding females.

A fundamental assumption of the Hamilton and Zuk (1982) model of sexual selection is that parasite-free males should produce mate attracting displays that are substantially more vigorous and effective than those of their parasitized counterparts. Further, the model predicts that parasite-free males should be preferred as mating partners by females and, as a consequence, should have higher reproductive success than their parasitized competitors. Gray treefrogs ( Hyla versicolor ) constitute an ideal species for testing both assumptions and predictions of the Hamilton-Zuk model in that: (i) mate attraction by males requires the production of energetically costly calls and (ii) males of this species have been shown to harbor a wide range of helminth parasites. The present paper summarizes results of analyses of calling and mating behavior by male gray treefrogs in relation to their parasite infection levels. In general, these analyses revealed no consistent relationship between the number and kinds of parasites harbored by males and either the duration or duty-cycle (a direct measure of energetic cost) of their calls. Furthermore, males found in amplexus (i.e., males actually selected as mating partners by females) showed a slightly higher prevalence of parasite infection (but a slightly lower intensity of infection) than did a comparable sample of unmated males. In sum, the present study failed to support the major within-species prediction of the Hamilton-Zuk model of sexual selection and also calls into question one of the fundamental assumptions of the model.