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.     

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.     

Female mate choice and the potential for ornament evolution in túngara frogs Physalaemus pustulosus

The potential for ornament evolution in response to sexual selection rests on the interaction between the permissive-ness or selectivity of female preferences and the constraints on male development of signaling related traits. We investigate the former by determining how latent female preferences either exaggerate the magnitude of current traits (I.e. Elaborations) or favor novel traits (I.e. Innovations). In tungara frogs, females prefer complex mating calls (whine-chucks) to simple calls (whine only). The whine is critical for mate recognition while the chuck further enhances the attractiveness of the call. Here we use a combina-tion of synthetic and natural stimuli to examine latent female preferences. Our results show that a diversity of stimuli, including conspecific and heterospecific calls as well as predator-produced and human-made sounds, increase the attractiveness of a call when added to a whine. These stimuli do not make simple calls more attractive than a whine-chuck, however. In rare cases we found stimuli that added to the whine decrease the attractiveness of the call. Overall, females show strong preferences for both elaborations and innovations of the chuck. We argue that the emancipation of these acoustic adornments from mate recognition allows such female permissiveness, and that male constraints on signal evolution are probably more important in explaining why males evolved their specific adornment. Experimentally probing latent female preferences for stimuli out of the species' range is a useful means to gain insights about the potential of female choice to influence signal evolution and thus the astounding diversity in male sexually-selected traits.     

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.     

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.     

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.     

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.     

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.     

Foam nest components of the túngara frog: a cocktail of proteins conferring physical and biological resilience

The foam nests of the túngara frog (Engystomops pustulosus) form a biocompatible incubation medium for eggs and sperm while resisting considerable environmental and microbiological assault. We have shown that much of this behaviour can be attributed to a cocktail of six proteins, designated ranaspumins (Rsn-1 to Rsn-6), which predominate in the foam. These fall into two discernable classes based on sequence analysis and biophysical properties. Rsn-2, with an amphiphilic amino acid sequence unlike any hitherto reported, exhibits substantial detergent-like surfactant activity necessary for production of foam, yet is harmless to the membranes of eggs and spermatozoa. A further four (Rsn-3 to Rsn-6) are lectins, three of which are similar to fucolectins found in teleosts but not previously identified in a land vertebrate, though with a carbohydrate binding specificity different from previously described fucolectins. The sixth, Rsn-1, is structurally similar to proteinase inhibitors of the cystatin class, but does not itself appear to exhibit any such activity. The nest foam itself, however, does exhibit potent cystatin activity. Rsn-encoding genes are transcribed in many tissues of the adult frogs, but the full cocktail is present only in oviduct glands. Combinations of lectins and cystatins have known roles in plants and animals for defence against microbial colonization and insect attack. Túngara nest foam displays a novel synergy of selected elements of innate defence plus a specialized surfactant protein, comprising a previously unreported strategy for protection of unattended reproductive stages of animals.     

Building a home from foam—túngara frog foam nest architecture and three-phase construction process

Frogs that build foam nests floating on water face the problems of over-dispersion of the secretions used and eggs being dangerously exposed at the foam : air interface. Nest construction behaviour of túngara frogs, Engystomops pustulosus, has features that may circumvent these problems. Pairs build nests in periodic bursts of foam production and egg deposition, three discrete phases being discernible. The first is characterized by a bubble raft without egg deposition and an approximately linear increase in duration of mixing events with time. This phase may reduce initial over-dispersion of foam precursor materials until a critical concentration is achieved. The main building phase is marked by mixing events and start-to-start intervals being nearly constant in duration. During the final phase, mixing events do not change in duration but intervals between them increase in an exponential-like fashion. Pairs joining a colonial nesting abbreviate their initial phase, presumably by exploiting a pioneer pair''s bubble raft, thereby reducing energy and material expenditure, and time exposed to predators. Finally, eggs are deposited only in the centre of nests with a continuously produced, approximately 1 cm deep egg-free cortex that protectively encloses hatched larvae in stranded nests.     

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.     

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.     

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.     

Immunocytochemical evidence for α-melanocyte-stimulating hormone in the hypothalamus of the frog Rana esculenta

Summary The distribution of immunoreactive -melanocyte-stimulating hormone (-MSH) within the brain of the frog, Rana esculenta, has been studied on adjacent serial sections using an indirect immunofluorescence technique. Immunoreactive cell bodies are found in the anterior part of the preoptic nucleus and in some ventral subependymal cerebrospinal fluid-contacting elements, and in the nucleus infundibularis ventralis. Numerous -MSH-like immunoreactive fibers are present in the preoptic area, in the pars ventralis of the tuber cinereum, and in the outer layer of the median eminence. This staining pattern is completely eliminated after preabsorbing the antiserum with the corresponding antigen, but blocking tests with -MSH-related peptides do not lead to any change in the immunoreaction. From these results it may be inferred that an -MSH-like system is present in the hypothalamic neurosecretory area of R. esculenta, and is probably related to its hypophysiotropic functions.The results are compared to the distribution of -MSH within the hypothalamus of reptiles and mammals.This work was supported by a grant from the M.P.I. (60%)     

Does sex-biased dispersal account for the lack of geographic and host-associated differentiation in introduced populations of an aphid parasitoid?

Host recognition and use in female parasitoids strongly relies on host fidelity, a plastic behavior which can significantly restrict the host preferences of parasitoids, thus reducing the gene flow between parasitoid populations attacking different insect hosts. However, the effect of migrant males on the genetic differentiation of populations has been frequently ignored in parasitoids, despite its known impact on gene flow between populations. Hence, we studied the extent of gene flow mediated by female and male parasitoids by assessing sibship relationships among parasitoids within and between populations, and its impact on geographic and host‐associated differentiation in the aphid parasitoid Aphidius ervi. We report evidences of a high gene flow among parasitoid populations on different aphid hosts and geographic locations. The high gene flow among parasitoid populations was found to be largely male mediated, suggested by significant differences in the distribution of full‐sib and paternal half‐sib dyads of parasitoid populations.     

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.