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.

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.

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

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

Keeping up with the neighbor: a novel mechanism of call synchrony in Neoconocephalus ensiger katydids.

During solo calling, pulse periods gradually changed by up to 15% over several minutes. Pairs of calling males synchronized their pulses. The pulse rate (10-14 Hz) was considerably faster than the rate of synchronized signal units in other insects (0.5-3 Hz). Within each pulse cycle, males made only small adjustments to their pulse period, leading to regular switches of leader and follower roles. Large-scale timing adjustments only occurred in response to large delays. Stimulation with single pulses had no predictable effect on the timing of the male's next pulse, resulting in a flat phase response curve. When entrained to a stimulus with a faster pulse period, males briefly interrupted calling; they resumed calling largely synchronized with the stimulus. Throughout the stimulus, males made gradual changes to their pulse period, similar to those during pair calling. After the stimulus ended, pulse periods increased over several minutes, but did not return to their pre-stimulus values. Thus social context and intrinsic state of the males influenced pulse period in Neoconocephalus ensiger. These results indicate that N. ensiger males synchronize calls by adjusting their intrinsic pulse period, instead of adjusting the timing of individual pulses, as described in other synchronizing insects.

Dynamic dendritic compartmentalization underlies stimulus-specific adaptation in an insect neuron.

In many neural systems, repeated stimulation leads to stimulus-specific adaptation (SSA), with responses to repeated signals being reduced while responses to novel stimuli remain unaffected. The underlying mechanisms of SSA remain mostly hypothetical. One hypothesis is that dendritic processes generate SSA. Evidence for such a mechanism was recently described in an insect auditory interneuron (TN-1 in Neoconocephalus triops). Afferents, tuned to different frequencies, connect with different parts of the TN-1 dendrite. The specific adaptation of these inputs relies on calcium and sodium accumulation within the dendrite, with calcium having a transient and sodium a tonic effect. Using imaging techniques, we tested here whether the accumulation of these ions remained limited to the stimulated parts of the dendrite. Stimulation with a fast pulse rate, which results in strong adaptation, elicited a transient dendritic calcium signal. In contrast, the sodium signal was tonic, remaining high during the fast pulse rate stimulus. These time courses followed the predictions from the previous pharmacological experiments. The peak positions of the calcium and sodium signals differed with the carrier frequency of the stimulus; at 15 kHz, peak locations were significantly more rostral than at 40 kHz. This matched the predictions made from neuroanatomical data. Our findings confirm that excitatory postsynaptic potentials rather than spiking cause the increase of dendritic calcium and sodium concentrations and that these increases remain limited to the stimulated parts of the dendrite. This supports the hypothesis of "dynamic dendritic compartmentalization" underlying SSA in this auditory interneuron.

Dendritic mechanisms contribute to stimulus-specific adaptation in an insect neuron.

Reduced neuronal activation to repetitive stimulation is a common feature of information processing in nervous systems. Such stimulus-specific adaptation (SSA) occurs in many systems, but the underlying neural mechanisms are not well understood. The Neoconocephalus (Orthoptera, Tettigoniidae) TN-1 auditory neuron exhibits an SSA-like process, characterized by reliably detecting deviant pulses after response cessation to common standard pulses. Therefore, TN-1 provides a model system to study the cellular mechanisms underlying SSA with an identified neuron. Here we test the hypothesis that dendritic mechanisms underlie TN-1 response cessation to fast-pulse rate repeated signals. Electrically stimulating TN-1 with either high-rate or continuous-current pulses resulted in a decreased ability in TN-1 to generate action potentials but failed to elicit cessation of spiking activity as observed with acoustic stimulation. BAPTA injection into TN-1 delayed the onset of response cessation to fast-pulse rate acoustic stimuli in TN-1 but did not eliminate it. These results indicate that calcium-mediated processes contribute to the fast cessation of spiking activity in TN-1 but are insufficient to cause spike cessation on its own. Replacing normal saline with low-Na(+) saline (replacing sodium chloride with either lithium chloride or choline chloride) eliminated response cessation, and TN-1 no longer responded selectively to the deviant pulses. Sodium-mediated potassium channels are the most likely candidates underlying sodium-mediated response suppression in TN-1, triggered by Na(+) influx in dendritic regions activated by acoustic stimuli. On the basis of these results, we present a model for a cellular mechanism for SSA in a single auditory neuron.

Auditory change detection by a single neuron in an insect.

The detection of novel signals in the auditory scene is an elementary task of any hearing system. In Neoconocephalus katydids, a primary auditory interneuron (TN-1) with broad spectral sensitivity, responded preferentially to rare deviant pulses (7 pulses/s repetition rate) embedded among common standard pulses (140 pulses/s repetition rate). Eliminating inhibitory input did not affect the detection of the deviant pulses. Detection thresholds for deviant pulses increased significantly with increasing amplitude of standard pulses. Responses to deviant pulses occurred when the carrier frequencies of deviant and standard were sufficiently different, both when the deviant had a higher or lower carrier frequency than the standard. Recordings from receptor neurons revealed that TN-1 responses to the deviant pulses did not depend on the population response strength of the receptors, but on the distribution of the receptor cell activity. TN-1 responses to the deviant pulse occurred only when the standard and deviant pulses were transmitted by different groups of receptor cells. TN-1 responses parallel stimulus specific adaptation (SSA) described in mammalian auditory system. The results support the hypothesis that the mechanisms underlying SSA and change-detection are located in the TN-1 dendrite, rather than the receptor cells.

Phonotaxis to male's calls embedded within a chorus by female gray treefrogs, Hyla versicolor.

During the reproductive season, male Hyla versicolor produce advertisement calls to attract females. Females exhibit phonotaxis and approach the individual callers, resulting in amplexus. For frogs that call from dense choruses, the extent to which and the range from which a male's advertisement call within a chorus can be heard by a receptive female leading to phonotaxis is unclear. We investigated females' responses to natural choruses in the field and found that they were attracted and showed directed orientation to breeding choruses at distances up to 100 m. To assess the role of acoustic cues in the directed orientation, we conducted acoustic playback experiments in the laboratory using conspecific call and noise as stimuli, as well as chorus sounds (that contained calls from a focal male) recorded at various distances, all played at naturalistic intensities. Using two response metrics (females' normalized response times and their phonotaxis trajectories) we found that, unlike the field experiments, females oriented and were attracted to chorus sounds from 1 to 32 m only, but not from >32 m, or to band-limited noise. Possible reasons for the observed difference in phonotaxis behavior in the two experimental conditions were discussed.

Selective phonotaxis in Neoconocephalus nebrascensis (Orthoptera: Tettigoniidae): call recognition at two temporal scales.

The calls of many Orthopteran species are comprised of a simple trill of pulses, the temporal pattern of which is important for call recognition. Male Neoconocephalus nebrascensis produce pulses with a temporal structure typical for the genus. However, they modify this pattern by grouping their pulses into verses, thereby creating a higher order temporal structure. The importance of the pulse pattern and verse structure for call recognition in N. nebrascensis was determined using a walking compensator. Females required the conspecific pulse pattern for call recognition, responding only when the intervals between pulses were short or absent. Females also required the verse structure for call recognition, and recognized the verse structure only when the amplitude modulation depth between verses and pauses exceeded 18 dB. We discuss that the verse recognition mechanism is a derived trait adapted for pre-mating isolation. We hypothesize that the unusually large amplitude modulation required for verse recognition forces males to synchronize their calls in order to preserve an attractive pattern. Call synchrony appears to be the outcome of cooperation, rather than competition, in this species.

Developmental plasticity of mating calls enables acoustic communication in diverse environments.

Male calls of the katydid Neoconocephalus triops exhibit substantial developmental plasticity in two parameters: (i) calls of winter males are continuous and lack the verse structure of summer calls and (ii) at equal temperatures, summer males produce calls with a substantially higher pulse rate than winter males. We raised female N. triops under conditions that reliably induced either summer or winter phenotype and tested their preferences for the call parameters that differ between summer and winter males. Neither generation was selective for the presence of verses, but females had strong preferences for pulse rates: only a narrow range of pulse rates was attractive. The attractive ranges did not differ between summer and winter females. Both male pulse rate and female preference for pulse rate changed with ambient temperature, but female preference changed more than the male calls. As a result, the summer call was attractive only at 25 degrees C, whereas the slower winter call was attractive only at 20 degrees C. Thus, developmental plasticity of male calls compensates for differences in temperature dependency between calls and preferences and enables the communication system to function in heterogeneous environments. The potential role of call plasticity during the invasion of new habitats is discussed.

Mechanisms and evolution of synchronous chorusing: emergent properties and adaptive functions in Neoconocephalus katydids (Orthoptera: Tettigoniidae).

Synchronous interactions arise in various animal species that rhythmically broadcast acoustic, vibratory, and visual signals. These interactions are characterized by a coincidence in both rate and phase of the rhythms of neighboring signalers. Theory predicts several ways in which synchronized rhythms may specifically benefit the interacting signalers. However, synchrony may also arise as an emergent property, a default phenomenon that is neither preferred by conspecific receivers evaluating the signals nor advantageous to the signalers themselves. Here, we examine several well-studied cases of acoustic synchrony in Neoconocephalus katydids (Orthoptera: Tettigoniidae), a New World genus wherein males broadcast loud advertisement songs. We report that call synchrony found in N. spiza and N. nebrascensis results from two rather different mechanisms of rhythm adjustment. Moreover, synchrony in the former species appears to represent an incidental byproduct of signal competition between evenly matched males, whereas in the latter species synchrony functions as a specific adaptation in which cooperating males ensure that critical call features can be perceived by females. We discuss the separate evolutionary trajectories that may have led to similar outcomes, synchronous chorusing by advertising males, in these closely related species.

A Post-pleistocene Calibrated Mutation Rate from Insect Museum Specimens.

Quantifying the age of recent species divergence events can be challenging in the absence of calibration points within many groups. The katydid species Neoconocephalus lyristes provides the opportunity to calibrate a post-Pleistocene, taxa specific mutation rate using a known biogeographic event, the Mohawk-Hudson Divide. DNA was extracted from pinned museum specimens of N. lyristes from both Midwest and Atlantic populations and the mitochondrial gene COI sequenced using primers designed from extant specimens. Coalescent analyses using both strict and relaxed molecular clock models were performed in BEAST v1.8.2. The assumption of a strict molecular clock could not be rejected in favor of the relaxed clock model as the distribution of the standard deviation of the clock rate strongly abutted zero. The strict molecular clock model resulted in an intraspecific calculated mutation rate of 14.4-17.3 %/myr, a rate substantially higher than the common rates of sequence evolution observed for insect mitochondrial DNA sequences. The rate, however, aligns closely with mutation rates estimated from other taxa with similarly recent lineage divergence times.

Population structure within the one-dimensional range of a coastal plain katydid.

Biogeography plays a significant role in species' dispersal, and in turn population structure, across the landscape. The North American katydid Neoconocephalus melanorhinus belongs to a genus with high mobility. Unlike other members of the genus, N. melanorhinus is a salt marsh specialist restricted to a narrow corridor along the Atlantic and Gulf coasts. In addition, their range crosses at least one known biogeographic barrier and possesses biogeographic characteristics of the stepping-stone as well as the hierarchical island model of dispersal. Using AFLP markers we searched for areas that conform to the predictions of isolation by distance and for areas of non-uniform increases in genetic variance, indicative of isolation by barrier. We found significant genetic differentiation between all twelve sampled sites. Isolation by distance was the predominant pattern of variation across their range. In addition, we saw possible evidence of two biogeographic barriers to gene flow, one at the Atlantic-Gulf divide and the other along the Gulf coast. We also observed a change in body size across the range. Body size, as measured by male hind femur length, correlated closely with latitude, a possible indication of differential selection across the species range.

Flight of the bumble bee: Buzzes predict pollination services.

Multiple interacting factors drive recent declines in wild and managed bees, threatening their pollination services. Widespread and intensive monitoring could lead to more effective management of wild and managed bees. However, tracking their dynamic populations is costly. We tested the effectiveness of an inexpensive, noninvasive and passive acoustic survey technique for monitoring bumble bee behavior and pollination services. First, we assessed the relationship between the first harmonic of the flight buzz (characteristic frequency) and pollinator functional traits that influence pollination success using flight cage experiments and a literature search. We analyzed passive acoustic survey data from three locations on Pennsylvania Mountain, Colorado to estimate bumble bee activity. We developed an algorithm based on Computational Auditory Scene Analysis that identified and quantified the number of buzzes recorded in each location. We then compared visual and acoustic estimates of bumble bee activity. Using pollinator exclusion experiments, we tested the power of buzz density to predict pollination services at the landscape scale for two bumble bee pollinated alpine forbs (Trifolium dasyphyllum and T. parryi). We found that the characteristic frequency was correlated with traits known to affect pollination efficacy, explaining 30-52% of variation in body size and tongue length. Buzz density was highly correlated with visual estimates of bumble bee density (r = 0.97), indicating that acoustic signals are predictive of bumble bee activity. Buzz density predicted seed set in two alpine forbs when bumble bees were permitted access to the flowers, but not when they were excluded from visiting. Our results indicate that acoustic signatures of flight can be deciphered to monitor bee activity and pollination services to bumble bee pollinated plants. We propose that applications of this technique could assist scientists and farmers in rapidly detecting and responding to bee population declines.

Character State Reconstruction of Call Diversity in the Neoconocephalus Katydids Reveals High Levels of Convergence.

The katydid genus Neoconocephalus is characterized by high diversity of the acoustic communication system. Both male signals and female preferences have been thoroughly studied in the past. This study used Bayesian character state reconstruction to elucidate the evolutionary history of diverse call traits, based on an existing, well supported phylogenetic hypothesis. The most common male call pattern consisted of continuous calls comprising one fast pulse rate; this pattern is the likely ancestral state in this genus. Three lines of call divergence existed among the species of the genus. First, four species had significantly slower pulse rates. Second, five species had alternating pulse periods, resulting in a double pulse rhythm. Third, several species had discontinuous calls, when pulses were grouped into rhythmically repeated verses. Bayesian character state reconstruction revealed that the double-pulse pattern likely evolved convergently five times; the slow pulse rate also evolved four times independently. Discontinuous calls have evolved twice and occur in two clades; each of which contains reversals to the ancestral continuous calls. Pairwise phylogenetically independent contrast analyses among the three call traits found no significant correlations among the character states of the different traits, supporting the independent evolution of the three call traits.

Non-parallel coevolution of sender and receiver in the acoustic communication system of treefrogs.

Advertisement calls of closely related species often differ in quantitative features such as the repetition rate of signal units. These differences are important in species recognition. Current models of signal-receiver coevolution predict two possible patterns in the evolution of the mechanism used by receivers to recognize the call: (i) classical sexual selection models (Fisher process, good genes/indirect benefits, direct benefits models) predict that close relatives use qualitatively similar signal recognition mechanisms tuned to different values of a call parameter; and (ii) receiver bias models (hidden preference, pre-existing bias models) predict that if different signal recognition mechanisms are used by sibling species, evidence of an ancestral mechanism will persist in the derived species, and evidence of a pre-existing bias will be detectable in the ancestral species. We describe qualitatively different call recognition mechanisms in sibling species of treefrogs. Whereas Hyla chrysoscelis uses pulse rate to recognize male calls, Hyla versicolor uses absolute measurements of pulse duration and interval duration. We found no evidence of either hidden preferences or pre-existing biases. The results are compared with similar data from katydids (Tettigonia sp.). In both taxa, the data are not adequately explained by current models of signal-receiver coevolution.

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.

Evolution of novel signal traits in the absence of female preferences in Neoconocephalus katydids (Orthoptera, Tettigoniidae).

UNLABELLED: BACKGROUND SIGNIFICANCE: Communication signals that function to bring together the sexes are important for maintaining reproductive isolation in many taxa. Changes in male calls are often attributed to sexual selection, in which female preferences initiate signal divergence. Natural selection can also influence signal traits if calls attract predators or parasitoids, or if calling is energetically costly. Neutral evolution is often neglected in the context of acoustic communication. METHODOLOGY/PRINCIPAL FINDINGS: We describe a signal trait that appears to have evolved in the absence of either sexual or natural selection. In the katydid genus Neoconocephalus, calls with a derived pattern in which pulses are grouped into pairs have evolved five times independently. We have previously shown that in three of these species, females require the double pulse pattern for call recognition, and hence the recognition system of the females is also in a derived state. Here we describe the remaining two species and find that although males produce the derived call pattern, females use the ancestral recognition mechanism in which no pulse pattern is required. Females respond equally well to the single and double pulse calls, indicating that the derived trait is selectively neutral in the context of mate recognition. CONCLUSIONS/SIGNIFICANCE: These results suggest that 1) neutral changes in signal traits could be important in the diversification of communication systems, and 2) males rather than females may be responsible for initiating signal divergence.

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

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

Molecular phylogenetics of the genus Neoconocephalus (orthoptera, tettigoniidae) and the evolution of temperate life histories.

BACKGROUND: The katydid genus Neoconocephalus (25+ species) has a prominent acoustic communication system and occurs in large parts of the Neotropics and Nearctic. This group has been subject of numerous behavioral, physiological, and evolutionary studies of its acoustic communication system. Two distinct life histories occur in this group: The tropical life history incorporates multiple generations/year and direct egg development without environmental triggers. Temperate life history is characterized by overwintering in the egg stage, cold trigger of egg development, and one generation/year. This study reconstructs the phylogenetic relationships within the genus to (1) determine the evolutionary history of the temperate life history, and (2) to support comparative studies of evolutionary and physiological problems in this genus. METHODOLOGY/PRINCIPAL FINDINGS: We used Amplified Fragment Length Polymorphisms (AFLP), and sequences of two nuclear loci and one mitochondrial locus to reconstruct phylogenetic relationships. The analysis included 17 ingroup and two outgroup species. AFLP and mitochondrial data provided resolution at the species level while the two nuclear loci revealed only deeper nodes. The data sets were combined in a super-matrix to estimate a total evidence tree. Seven of the temperate species form a monophyletic group; however, three more temperate species were placed as siblings of tropical species. CONCLUSIONS/SIGNIFICANCE: Our analyses support the reliability of the current taxonomic treatment of the Neoconocephalus fauna of Caribbean, Central, and North America. Ancestral state reconstruction of life history traits was not conclusive, however at least four transitions between life histories occurred among our sample of species. The proposed phylogeny will strengthen conclusions from comparative work in this group.