Biogeography and the evolution of acoustic communication in the polyploid North American grey treefrog complex.

After polyploid species are formed, interactions between diploid and polyploid lineages may generate additional diversity in novel cytotypes and phenotypes. In anurans, mate choice by acoustic communication is the primary method by which individuals identify their own species and assess suitable mates. As such, the evolution of acoustic signals is an important mechanism for contributing to reproductive isolation and diversification in this group. Here, we estimate the biogeographical history of the North American grey treefrog complex, consisting of the diploid Hyla chrysoscelis and the tetraploid Hyla versicolor, focusing specifically on the geographical origin of whole genome duplication and the expansion of lineages out of glacial refugia. We then test for lineage-specific differences in mating signals by applying comparative methods to a large acoustic data set collected over 52 years that includes >1500 individual frogs. Along with describing the overall biogeographical history and call diversity, we found evidence that the geographical origin of H. versicolor and the formation of the midwestern polyploid lineage are both associated with glacial limits, and that the southwestern polyploid lineage is associated with a shift in acoustic phenotype relative to the diploid lineage with which they share a mitochondrial lineage. In H. chrysoscelis, we see that acoustic signals are largely split by Eastern and Western lineages, but that northward expansion along either side of the Appalachian Mountains is associated with further acoustic diversification. Overall, results of this study provide substantial clarity on the evolution of grey treefrogs as it relates to their biogeography and acoustic communication.

Neuroethology of sound localization in anurans.

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

The Complex History of Genome Duplication and Hybridization in North American Gray Treefrogs.

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. Although 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 diploid Hyla chrysoscelis and the tetraploid H. 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, 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.

Anuran Vocal Communication: Effects of Genome Size, Cell Number and Cell Size.

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.

A multivariate analysis of genetic variation in the advertisement call of the gray treefrog, Hyla versicolor.

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.

Trade-offs and upper limits to signal performance during close-range vocal competition in gray tree frogs hyla versicolor.

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.

A precedence effect underlies preferences for calls with leading pulses in the grey treefrog, Hyla versicolor.

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.

Experimental analysis of multivariate female choice in gray treefrogs (Hyla versicolor): evidence for directional and stabilizing selection.

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.

Hormonal modulation of phonotaxis and advertisement-call preferences in the gray treefrog (Hyla versicolor).

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.

Phonotactic selectivity in two cryptic species of gray treefrogs: effects of differences in pulse rate, carrier frequency and playback level.

The 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. chrysoscelis when 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.

Advertisement-call modification, male competition and female preference in the bird-voiced treefrog Hyla avivoca.

Senders and receivers influence dynamic characteristics of the signals used for mate attraction over different time scales. On a moment-to-moment basis, interactions among senders competing for a mate influence dynamic characteristics, whereas the preferences of receivers of the opposite gender exert an influence over evolutionary time. We observed and recorded the calling patterns of the bird-voiced treefrog Hyla avivoca, to assess how the dynamic characters of calls vary during interactions among groups of males in a chorus. This question was also addressed using playback experiments with males. Playback experiments with females showed how changes in dynamic call properties are likely to affect male mating success. Frogs calling in pairs, groups, or in response to playbacks produced longer calls than did isolated males. During call overlap, males often increased the duration of the silent interval (gaps) between the pulses of their calls so that the pulses of the calls of two neighbors interdigitated. This change resulted in increased variability of pulse rate, a traditionally static acoustic property; however, males also produced high proportions of non-overlapped calls in which variability in pulse rate was low and had species-typical values. Females preferred long calls to short and average-duration calls, and non-overlapped calls to overlapped calls. Given a choice between pairs of overlapped calls, females preferred pairs in which the proportion of overlap was low and pairs in which the pulses of such calls interdigitated completely. The observed patterns of vocal competition thus reflect the preferences of conspecific females, which have influenced the evolution of the calling behavior of H. avivoca.

Preferences based on spectral differences in acoustic signals in four species of treefrogs (Anura: Hylidae).

Frogs 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.

Temporal order and the evolution of complex acoustic signals.

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.

Polyploids with different origins and ancestors form a single sexual polyploid species.

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.

Mid-frequency suppression in the green treefrog (Hyla cinerea): mechanisms and implications for the evolution of acoustic communication.

Advertisement calls of green treefrogs (Hyla cinerea) have two spectral peaks centered at about 1 kHz and 3 kHz. Addition of a component of intermediate frequency (1.8 kHz) to a synthetic call reduced its attractiveness to females relative to an alternative lacking this component. This mid-frequency suppression occurred over a 20-dB range of playback levels. Addition of other intermediate frequencies had weak effects on preferences at some playback levels, in some localities, and at lower-than-normal temperatures. These effects correlate well with the response properties of a population of low-frequency-tuned auditory neurons innervating the amphibian papilla. Males of a closely related species (H. gratiosa) produce calls with emphasized frequencies within the range of suppression in H. cinerea; however, suppression also occurred in localities well outside the area of geographical overlap with this species. Thus, previous speculation that mid-frequency suppression evolved to enhance species discrimination is probably incorrect. This phenomenon is more likely to reflect a general sensory bias in anurans and other vertebrates, tone-on-tone inhibition. Such negative biases, and other inhibitory mechanisms, almost certainly play an important role in the evolution of communication systems but have received far less attention than positive biases that enhance signal attractiveness.

Advertisement-call preferences in diploid-tetraploid treefrogs (Hyla chrysoscelis and Hyla versicolor): implications for mate choice and the evolution of communication systems.

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.

6-hydroxydopamine lesions in anuran amphibians: a new model system for Parkinson's disease?

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.

Roles of the auditory midbrain and thalamus in selective phonotaxis in female gray treefrogs (Hyla versicolor).

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.

Reproductive character displacement in the acoustic communication system of green tree frogs (Hyla cinerea).

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.