Patterns of mating call preferences in túngara frogs, Physalaemus pustulosus

We examine acoustic mating preferences of a focal population at four different scales of divergence: within the population, between populations in the same genetic group, between populations in different genetic groups and between different species. At all scales there is substantial genetic divergence, variation in mating signals and preferences are influenced by signal variation. There is, however, no support for the hypothesis that mating preferences accumulate predictably with genetic distance. Females preferred the local conspecific call to the foreign conspecific call in about one-third of the experiments, and preferred the local call to all of the heterospecific calls tested. But there was no significant relationship between the variation in the strength of preference and genetic distance either among conspecific populations, or among heterospecific species. Thus, in this study macroevolutionary patterns are not apparent at the microevolutionary scale.     

Phylogenetic influence on mating call preferences in female túngara frogs, Physalaemus pustulosus

We evaluated how various phylogenetic models for estimating ancestral characters can influence studies of behavioural evolution. Previously we used a single model of evolution to estimate the values of call characters at ancestral nodes for the Physalaemus pustulosus species group and some close relatives (Ryan & Rand 1995, Science, 269, 390-392). We then synthesized these ancestral calls and measured the females' responses to such calls in phonotaxis experiments. We repeated the above procedure to determine the sensitivity of these results and conclusions to various models used to estimate the ancestral call characters. We asked whether: (1) different models gave different call estimates for the same nodes; (2) different call estimates at the same node were perceived as different by females; and (3) differences in female responses influenced previous conclusions. We used seven different models that varied in at least one of the following parameters: tree topology (bifurcating versus pectinate in-group trees), algorithms (local squared-change versus squared-change parsimony), tempo (gradual or punctuated evolution), and outgroups (two or three outgroup taxa used). Although different models often gave different call estimates for the same node, these different estimates often were not perceived as different by the females. These data reinforce our previous conclusions that: (1) the range of female preferences exceeds the known variation of the conspecific call; (2) females do not discriminate between the conspecific call and the call of their most recent ancestor; and (3) female responses may be context dependent, given that females differ in their responses to the same signal variation in discrimination and recognition experiments. Copyright 1999 The Association for the Study of Animal Behaviour.     

Hormonal state influences aspects of female mate choice in the Túngara Frog (Physalaemus pustulosus)

Females alter their mate choices as they transition through different reproductive stages; however, the proximal mechanisms for such behavioral fluctuation are unclear. In many taxa, as females transition through different reproductive stages, there is an associated change in hormone levels; therefore, we examined whether fluctuation in hormone levels serves as a proximal mechanism for within-individual variation in mate choice in female túngara frogs (Physalaemus pustulosus). We manipulated hormone levels of females by administering 0, 10, 100, 500 or 1,000 IU of human chorionic gonadotropin (HCG), which is a ligand for luteinizing hormone (LH) receptors and will therefore cause increased gonadal hormone production. Phonotaxis assays were conducted to measure three aspects of mate choice behavior before and after HCG administration; receptivity (response to a conspecific mate signal), permissiveness (response to a signal that is less attractive than conspecific signals) and discrimination (ability to discern signal differences). The probability of response to a conspecific and an artificial hybrid signal significantly increased at the highest HCG doses. The difference in mean response time between pre- and post-HCG tests was significantly different for both the receptivity and permissiveness tests among the five doses. Increased permissiveness, however, was not due to decreased discrimination because females could discriminate between calls even at the highest HCG doses. These hormonal manipulations caused the same behavioral pattern we reported in females as they transitioned through different reproductive stages (Lynch, K.S., Rand, A.S., Ryan, M.J., Wilczynski, W., 2005. Plasticity in female mate choice associated with changing reproductive states. Anim. Behav. 69, 689-699), suggesting that changes in hormone levels can influence the female's mate choice behavior.     

Fine-scale genetic pattern and evidence for sex-biased dispersal in the túngara frog, Physalaemus pustulosus

Túngara frogs (Physalaemus pustulosus) are a model system for sexual selection and communication. Population dynamics and gene flow are of major interest in this species because they influence speciation processes and microevolution, and could consequently provide a deeper understanding of the evolutionary processes involved in mate recognition. Although earlier studies have documented genetic variation across the species' range, attempts to investigate dispersal on a local level have been limited to mark-recapture studies. These behavioural studies indicated high mobility at a scale of several hundred metres. In this study we used seven highly polymorphic microsatellite loci to investigate fine-scaled genetic variation in the túngara frog. We analysed the influence of geographical distance on observed genetic patterns, examined the influence of a river on gene flow, and tested for sex-biased dispersal. Data for 668 individuals from 17 populations ranging in distance from 0.26 to 11.8 km revealed significant levels of genetic differentiation among populations. Genetic differentiation was significantly correlated with geographic distance. A river acted as an efficient barrier to gene flow. Several tests of sex-biased dispersal were conducted. Most of them showed no difference between the sexes, but variance of Assignment Indices exhibited a statistically significant male bias in dispersal.     

A laryngeal fibrous mass impacts the acoustics and attractiveness of a multicomponent call in túngara frogs (Physalaemus pustulosus)

The evolution of elaborate signals can emerge from changes in anatomical signaling structures. In the male túngara frog, a simple advertisement call (‘whine’) can be ornamented facultatively with a suffix (‘chuck’) to produce a more attractive complex call, or ‘whine-chuck’. A fibrous mass (FM1) supported by the vocal cords plays a role in chuck production. Here, we examine the effects of FM1 ablation on a large set of spectral and temporal features of both the whine and chuck and we test the hypothesis that FM1 ablation reduces call attractiveness to females. Both call components were impacted by FM1 ablation, but especially the suffix. The proportion of energy in the suffix’s odd compared to even harmonics diminished markedly as did the relative amplitude, effectively eliminating the chuck percept. FM1 ablation also reduced the whine’s frequency and its rate of frequency modulation. Moreover, post-surgery chucks no longer enhanced the attractiveness of the simple call and whines also appeared to diminish in attractiveness. Together, our results demonstrate that FM1 plays a role in the production of both call components in a way that stimulates the female auditory system and that the growth of FM1 had potentially positive sexual selection implications for the pre-existing simple call.     

Modelling the production of complex calls in the túngara frog (Physalaemus pustulosus)

Relatively few studies have used experimental manipulations to investigate the mechanics of vocal production in frogs and toads, even though many frogs produce complex signals with multiple components and/or nonlinearities. Modelling approaches can add to empirical studies by illuminating how various components of the vocal system interact to produce communication signals. In this study, we use bond graphs, a lumped-element modelling technique, to explore how the combination of active modulation of vocal production and the passive dynamics of a unique laryngeal structure result in the complex calls produced by males in an anuran model system. The túngara frog (Physalaemus (= Engystomops) pustulosus) produces advertisement calls with a ‘whine’ and a facultative ‘chuck’. Whines are amplitude and frequency modulated. The chuck is characterized by its spectral complexity and appears to contain a period doubling bifurcation resulting in subharmonics. In our model, we focus on how a fibrous mass attached to the vocal cords results in subharmonics in the chuck. Our models suggest that active (neural) modulation of the fibrous mass is not necessary for the transition between the spectral characteristics of the whine and chuck. Rather, it is possible that the vibratory mode of the fibrous mass and thus the vocal cords changes passively as a result of changes in airflow through the system.     

Biogeography of the túngara frog, Physalaemus pustulosus: a molecular perspective

Physalaemus pustulosus, a small leptodactylid frog with South American affinities, ranges across northern South America through Middle America to southern Mexico. To investigate its geographic variation and evolutionary origins, we analysed the presumptive gene products of 14 allozyme loci and sequenced a portion of the mitochondrial COI gene from individuals sampled throughout the distribution. Generally, allozyme dissimilarities and sequence divergences are correlated with each other and with geographic proximity. The greatest discontinuity in genetic variation was found between populations in Middle America vs. South America + Panama. Based on two Bayesian MCMC (Markov chain Monte Carlo) divergence time estimates involving two independent temporal constraints, the timing of the separation of northern and southern túngara frog lineages is significantly older than the time since completion of the current Panama land bridge. P. pustulosus first invaded Middle America from South America about 6-10 million years ago giving rise to the northern lineage. The southern lineage then invaded Panama independently after land bridge completion. Despite millions of years of independent evolution, the multilocus allozyme data revealed that western Panama populations represent a contact zone containing individuals with alleles from both groups present.     

The brain as a source of selection on the social niche: examples from the psychophysics of mate choice in túngara frogs

The main premise of this article is that various cognitive functions involved in signal analysis, memory, and decision making, all modulated by the animal's internal milieu, can generate selection for the forms of signals used in social interactions. Thus, just as an animal's view of its world, its Umwelt, determines how it interacts with its ecological niche, it can influence the evolution of its social niche. Thus, the brain is not only a landscape on which selection can act, but also it is a powerful source of selection on the animal's social niche.     

Estradiol induces sexual behavior in female túngara frogs

Steroid hormones play an important role in regulating vertebrate sexual behavior. In frogs and toads, injections of exogenous gonadotropins, which stimulate steroid hormone production, are often used to induce reproductive behavior, but steroid hormones alone are not always sufficient. To determine which hormonal conditions promote sexual behavior in female túngara frogs, we assessed the effect of hormone manipulation on the probability of phonotaxis behavior toward conspecific calls in post-reproductive females. We injected females with human chorionic gonadotropin (HCG), estradiol, estradiol plus progesterone, saline, or HCG plus fadrozole (an aromatase blocker) and tested their responses to mating calls. We found that injections of HCG, estradiol, and estradiol plus progesterone all increased phonotaxis behavior, whereas injections of saline or HCG plus fadrozole did not. Since injections of estradiol alone were effective at increasing phonotaxis behavior, we concluded that estradiol is sufficient for the expression of phonotaxis behavior. Next, to determine if estradiol-injected females display the same behavioral preferences as naturally breeding females, we compared mating call preferences of naturally breeding females to those of post-reproductive females injected with estradiol. We found that, when injected with estradiol, females show similar call preferences as naturally breeding females, although they were less likely to respond across multiple phonotaxis tests. Overall, our results suggest that estradiol is sufficient for the expression of sexual responses to mating calls in túngara frogs. To our knowledge, ours is the only study to find that estradiol alone is capable of promoting phonotaxis behavior in a frog.     

Phylogeny of the túngara frog genus Engystomops (= Physalaemus pustulosus species group; Anura: Leptodactylidae)

We present a phylogeny of the Neotropical genus Engystomops (= Physalaemus pustulosus species group) based on sequences of approximately 2.4 kb of mtDNA, (12S rRNA, valine-tRNA, and 16S rRNA) and propose a phylogenetic nomenclature. The phylogeny includes all described taxa and two unnamed species. All analyses indicate that Engystomops is monophyletic and contains two basal allopatric clades. Clade I (Edentulus) includes E. pustulosus and the Amazonian E. petersi + E. cf. freibergi. Clade II (Duovox) includes all species distributed in W Ecuador and NW Peru. Brevivox, a clade of small-sized species is strongly supported within Duovox. Populations of Engystomops pustulosus fall into two well-supported clades, each of which occupies two disjunct portions of the species range. Overall, our phylogeny is congruent with most previous hypotheses. This study is among the few published species-level phylogenies of Neotropical amphibians derived from molecular datasets. A review of the proportion of new species detected by similar studies suggests that the increasing use of molecular techniques will lead to the discovery of a vast number of species of Neotropical amphibians.     

Integration of sensory and motor processing underlying social behaviour in túngara frogs

Social decision making involves the perception and processing of social stimuli, the subsequent evaluation of that information in the context of the individual's internal and external milieus to produce a decision, and then culminates in behavioural output informed by that decision. We examined brain networks in an anuran communication system that relies on acoustic signals to guide simple, stereotyped motor output. We used egr-1 mRNA expression to measure neural activation in male túngara frogs, Physalaemus pustulosus, following exposure to conspecific and heterospecific calls that evoke competitive or aggressive behaviour. We found that acoustically driven activation in auditory brainstem nuclei is transformed into activation related to sensory-motor interactions in the diencephalon, followed by motor-related activation in the telencephalon. Furthermore, under baseline conditions, brain nuclei typically have correlated egr-1 mRNA levels within brain divisions. Hearing conspecific advertisement calls increases correlations between anatomically distant brain divisions; no such effect was observed in response to calls that elicit aggressive behaviour. Neural correlates of social decision making thus take multiple forms: (i) a progressive shift from sensory to motor encoding from lower to higher stages of neural processing and (ii) the emergence of correlated activation patterns among sensory and motor regions in response to behaviourally relevant social cues.     

The effects of time,space and spectrum on auditory grouping in túngara frogs

Male túngara frogs (Physalaemus pustulosus) produce complex calls consisting of two components, a ~350 ms FM sweep called the “whine” followed by up to seven ~40 ms harmonic bursts called “chucks”. In order to choose and locate a calling male, females attending to choruses must group call components into auditory streams to correctly assign calls to their sources. Previously we showed that spatial cues play a limited role in grouping: calls with normal spectra and temporal structure are grouped over wide angular separations (≤135°). In this study we again use phonotaxis to first test whether an alternative cue, the sequence of call components, plays a role in auditory grouping and second, whether grouping is mediated by peripheral or central mechanisms. We found that while grouping is not limited to the natural call sequence, it does vary with the relative onset times of the two calls. To test whether overlapping stimulation in the periphery is required for grouping, the whine and chuck were filtered to restrict their spectra to the sensitivity ranges of the amphibian and basilar papillae, respectively. For these dichotic-like stimuli, grouping still occurred (albeit only to 45° separation), suggesting that stream formation is mediated by central mechanisms.     

(Under)water love-linking mate choice and cognition in fish and frogs

Aims and Objectives This Special Column aims at complementing our knowledge and deepening our understanding of the complex processes involved in learning and neurobiological mechanisms in the context of sexual selection.     

Low temperature tolerance,cold hardening and acclimation in tadpoles of the neotropical túngara frog (Engystomops pustulosus)

Many frogs from temperate climates can tolerate low temperatures and increase their thermal tolerance through hardening and acclimation. Most tropical frogs, on the other hand, fail to acclimate to low temperatures. This lack of acclimation ability is potentially due to lack of selection pressure for acclimation because cold weather is less common in the tropics. We tested the generality of this pattern by characterizing the critical temperature minimum (CTMin), hardening, and acclimation responses of túngara frogs (Engystomops pustulosus). These frogs belong to a family with unknown thermal ecology. They are found in a tropical habitat with a highly constant temperature regime. The CTMin of the tadpoles was on average 12.5 °C. Pre-metamorphic tadpoles hardened by 1.18 °C, while metamorphic tadpoles hardened by 0.36 °C. When raised at 21 °C, tadpoles acclimated expanding their cold tolerance by 1.3 °C in relation to larvae raised at 28 °C. These results indicate that the túngara frog has a greatly reduced cold tolerance when compared to species from temperate climates, but it responds to cold temperatures with hardening and acclimation comparable to those of temperate-zone species. Cold tolerance increased with body length but cold hardening was more extensive in pre-metamorphic tadpoles than in metamorphic ones. This study shows that lack of acclimation ability is not general to the physiology of tropical anurans.     

Female calls in lek-mating birds: indirect mate choice, female competition for mates, or direct mate choice?

I tested predictions from ultimate hypotheses of why femalegreat snipe Gallinago media give loud calls when visiting leks,using observational data and playback experiments. One hypothesisis that calls might be used in female—female competitionfor popular males, either (1) in an aggressive context towarda specific female, or (2) toward females in general to defendthe male. It has also been suggested that female calls (3) may not have an adaptive function, the capability of vocalizingbeing explained as a correlated response to selection on malesinging. Further, calls might function as (4) a copulationsolicitation toward a specific male. Finally, calls might havea function in mate choice, either (5) in indirect mate choiceas a fertility advertisement to incite male fighting, or (6)in direct mate choice as a mate-sampling aid to provoke quality-revealing responses from males. Disproportionately many female calls wereuttered when no other females were present on a male's territoryand in territories of males without mating success, contradictinghypotheses 1 and 2. Female calls were not associated with copulation;calls generally occurred several days before copulations, contradictinghypotheses 4 and 5. Playback of female calls attracted malesand increased fighting among males, even if females were presentnearby, contradicting hypothesis 3. Males changed their ownsongs in response to playback, and the response was relatedto their mating success. Taken together, the results are onlyconsistent with one of the hypotheses considered—femalecalls may function as a mate-sampling aid used in direct matechoice.     

Geographic variation of genetic and behavioral traits in northern and southern tüngara frogs

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

Sexual selection in female perceptual space: how female túngara frogs perceive and respond to complex population variation in acoustic mating signals

Female preferences for male mating signals are often evaluated on single parameters in isolation or small suites of characters. Most signals, however, are composites of many individual parameters. In this study we quantified multivariate traits in the advertisement call of the túngara frog, Physalaemus pustulosus. We represented the calls in multidimensional scaling space and chose nine test calls to represent the range of population variation. We then tested females for phonotactic preference between calls in each pair of the nine test calls. We used statistics developed for paired comparisons in such "round robin" competitions to evaluate the null hypothesis of equal attractiveness, and to examine the degree to which females responded to calls as being different from or similar to one another in attractiveness. We then examined the attractiveness of each test call relative to all other test calls as a function of their location in multivariate acoustic space (the acoustic landscape) to visualize sexual selection on calls. Finally, we used methods from cognitive psychology to illustrate the females' perception of call attractiveness in multivariate space, and compared this perceptual landscape to the acoustic landscape of quantitative call variation. We show that correlations between individual call characters are not strong and thus there are few biomechanical constraints on their independent evolution. Most call variables differed among males, and there was high repeatability of call characters within males. Females often discriminated between pairs of calls from the population, and there were significant differences among calls in their attractiveness. Female preferences for calls were not stabilizing. The region of the acoustic landscape that was most attractive to females included the mean call but was not centered around it. The females' perceptual or preference landscape did not correlate with the call's acoustic landscape, and female perception of calls decreased rather than enhanced call differences.     

Female mate choice and male–male competition in Tonkean macaques: Who decides?

The theory of sexual selection predicts that females should be discriminatory in the choice of sexual partners. Females can express their choice in two ways. In direct mate choice, they show preferences for certain partners. In indirect mate choice, they select partners by displaying sexually attractive traits, thus eliciting contest competition between males. We focused on a primate species in which females advertise the timing of their ovulation and studied the balance between these two choice strategies. We tested predictions related to three hypotheses about direct and indirect female choice, namely the best‐male, graded‐signal and weak‐selectivity hypotheses. We investigated the sexual and agonistic interactions occurring during oestrous periods in five captive groups of Tonkean macaques (Macaca tonkeana). The results showed that dominant males used mate guarding to monopolise sexual access to parous females that were in the fertile stage of their reproductive cycle, while lower‐ranking males monitored only nulliparous females. The distribution of sexual presentations indicated that females accepted different types of partners, supporting the weak‐selectivity hypothesis regarding direct mate choice. The analysis of behavioural sequences revealed that mate‐guarding males used mild coercive behaviours to prevent females from mating with other males at conception time. The distribution of mounts showed that females mainly mated with dominant males, which leads us to argue that the best‐male hypothesis provides the most parsimonious explanation regarding indirect mate choice in Tonkean macaques. At the individual level, it may be concluded that male competitive strategies prevented females from exercising direct mate choice. At the evolutionary level, however, female sexual advertising and thus indirect choice promoted competition between males. The outcome is that indirect mate choice appears more important than direct mate choice in female Tonkean macaques.     

Neural Activity Patterns in Response to Interspecific and Intraspecific Variation in Mating Calls in the Túngara Frog

Background

During mate choice, individuals must classify potential mates according to species identity and relative attractiveness. In many species, females do so by evaluating variation in the signals produced by males. Male túngara frogs (Physalaemus pustulosus) can produce single note calls (whines) and multi-note calls (whine-chucks). While the whine alone is sufficient for species recognition, females greatly prefer the whine-chuck when given a choice.

Methodology/Principal Findings

To better understand how the brain responds to variation in male mating signals, we mapped neural activity patterns evoked by interspecific and intraspecific variation in mating calls in túngara frogs by measuring expression of egr-1. We predicted that egr-1 responses to conspecific calls would identify brain regions that are potentially important for species recognition and that at least some of those brain regions would vary in their egr-1 responses to mating calls that vary in attractiveness. We measured egr-1 in the auditory brainstem and its forebrain targets and found that conspecific whine-chucks elicited greater egr-1 expression than heterospecific whines in all but three regions. We found no evidence that preferred whine-chuck calls elicited greater egr-1 expression than conspecific whines in any of eleven brain regions examined, in contrast to predictions that mating preferences in túngara frogs emerge from greater responses in the auditory system.

Conclusions

Although selectivity for species-specific signals is apparent throughout the túngara frog brain, further studies are necessary to elucidate how neural activity patterns vary with the attractiveness of conspecific mating calls.