The return to water in ancestral Xenopus was accompanied by a novel mechanism for producing and shaping vocal signals.

Listeners locate potential mates using species-specific vocal signals. As tetrapods transitioned from water to land, lungs replaced gills, allowing expiration to drive sound production. Some frogs then returned to water. Here we explore how air-driven sound production changed upon re-entry to preserve essential acoustic information on species identity in the secondarily aquatic frog genus Xenopus. We filmed movements of cartilage and muscles during evoked sound production in isolated larynges. Results refute the current theory for Xenopus vocalization, cavitation, and favor instead sound production by mechanical excitation of laryngeal resonance modes following rapid separation of laryngeal arytenoid discs. Resulting frequency resonance modes (dyads) are intrinsic to the larynx rather than due to neuromuscular control. Dyads are a distinctive acoustic signature. While their component frequencies overlap across species, their ratio is shared within each Xenopus clade providing information on species identity that could facilitate both conspecific localization and ancient species divergence. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Genetics, Morphology, Advertisement Calls, and Historical Records Distinguish Six New Polyploid Species of African Clawed Frog (Xenopus, Pipidae) from West and Central Africa.

African clawed frogs, genus Xenopus, are extraordinary among vertebrates in the diversity of their polyploid species and the high number of independent polyploidization events that occurred during their diversification. Here we update current understanding of the evolutionary history of this group and describe six new species from west and central sub-Saharan Africa, including four tetraploids and two dodecaploids. We provide information on molecular variation, morphology, karyotypes, vocalizations, and estimated geographic ranges, which support the distinctiveness of these new species. We resurrect Xenopus calcaratus from synonymy of Xenopus tropicalis and refer populations from Bioko Island and coastal Cameroon (near Mt. Cameroon) to this species. To facilitate comparisons to the new species, we also provide comments on the type specimens, morphology, and distributions of X. epitropicalis, X. tropicalis, and X. fraseri. This includes significantly restricted application of the names X. fraseri and X. epitropicalis, the first of which we argue is known definitively only from type specimens and possibly one other specimen. Inferring the evolutionary histories of these new species allows refinement of species groups within Xenopus and leads to our recognition of two subgenera (Xenopus and Silurana) and three species groups within the subgenus Xenopus (amieti, laevis, and muelleri species groups).

Evolution of advertisement calls in African clawed frogs.

For most frogs, advertisement calls are essential for reproductive success, conveying information on species identity, male quality, sexual state and location. While the evolutionary divergence of call characters has been examined in a number of species, the relative impacts of genetic drift or natural and sexual selection remain unclear. Insights into the evolutionary trajectory of vocal signals can be gained by examining how advertisement calls vary in a phylogenetic context. Evolution by genetic drift would be supported if more closely related species express more similar songs. Conversely, a poor correlation between evolutionary history and song expression would suggest evolution shaped by natural or sexual selection. Here, we measure seven song characters in 20 described and two undescribed species of African clawed frogs (genera Xenopus and Silurana) and four populations of X. laevis. We identify three call types - click, burst and trill - that can be distinguished by click number, call rate and intensity modulation. A fourth type is biphasic, consisting of two of the above. Call types vary in complexity from the simplest, a click, to the most complex, a biphasic call. Maximum parsimony analysis of variation in call type suggests that the ancestral type was of intermediate complexity. Each call type evolved independently more than once and call type is typically not shared by closely related species. These results indicate that call type is homoplasious and has low phylogenetic signal. We conclude that the evolution of call type is not due to genetic drift, but is under selective pressure.

Vocal competition in male Xenopus laevis frogs.

Male Xenopus laevis frogs produce underwater advertisement calls that attract gravid females and suppress calling by male competitors. Here we explore whether groups of males establish vocal ranks and whether auditory cues alone suffice for vocal suppression. Tests of male-male pairs within assigned groups reveal linear vocal dominance relations, in which each male has a defined rank. Both the duration over which males interact, as well as the number of competitive opportunities, affect linearity. Linear dominance across the group is stable for about 2 weeks; rank is dynamic. Males engage in physical interactions (clasping) while paired but clasping and vocal rank are not correlated. Playbacks of advertisement calls suppress calling and calls from high- and low-ranking males are equally effective. Thus, auditory cues alone suffice to suppress vocal behavior. Playback intensities equivalent to a nearby male advertising effectively suppress calling while low-intensity playbacks are either ineffective or stimulate vocal behavior. X. laevis advertisement calls are biphasic, composed of alternating fast and slow click trills. Approximately half the males tested are more vocally suppressed by all slow than by all fast trills; thus, these males can distinguish between the two phases. The fully aquatic family Pipidae diverged from terrestrial ancestors approximately 170 mya. Vocal suppression in the X. laevis mating system may represent the translation of an ancient anuran social strategy to underwater life.

Vocal communication between male Xenopus laevis.

This study focuses on the role of male-male vocal communication in the reproductive repertoire of the South African clawed frog, Xenopus laevis. Six male and two female call types were recorded from native ponds in the environs of Cape Town, South Africa. These include all call types previously recorded in the laboratory as well as one previously unidentified male call: chirping. The amount of calling and the number of call types increased as the breeding season progressed. Laboratory recordings indicated that all six male call types were directed to males; three of these were directed to both sexes and three were directed exclusively to males. Both female call types were directed exclusively to males. The predominant call type, in both field and laboratory recordings, was the male advertisement call. Sexual state affected male vocal behaviour. Male pairs in which at least one male was sexually active (gonadotropin injected) produced all call types, whereas pairs of uninjected males rarely called. Some call types were strongly associated with a specific behaviour and others were not. Clasped males always growled and clasping males typically produced amplectant calls or chirps; males not engaged in clasping most frequently advertised. The amount of advertising produced by one male was profoundly affected by the presence of another male. Pairing two sexually active males resulted in suppression of advertisement calling in one; suppression was released when males were isolated after pairing. Vocal dominance was achieved even in the absence of physical contact (clasping). We suggest that X. laevis males gain a reproductive advantage by competing for advertisement privileges and by vocally suppressing neighbouring males.

Estrogen receptor expression in laryngeal muscle in relation to estrogen-dependent increases in synapse strength.

In Xenopus laevis, the laryngeal neuromuscular synapse is the final effector for sexually differentiated song production. Females have stronger laryngeal synapses than males, and synapse strength is estrogen dependent. Estrogen-induced increases in synaptic strength require at least 3 weeks of exposure, suggesting that the hormone acts via a classical genomic mechanism involving the estrogen receptor (ER). The locus of the sex difference in synapse strength, determined using quantal analysis, is presynaptic, leading to the prediction that estrogen acts directly on vocal motor neurons. However, laryngeal motor neurons do not accumulate estrogen. Estrogen might instead affect motor neuron transmitter release via a retrograde signal from its target muscle. To test this hypothesis, we determined whether laryngeal muscle expresses ER. With RT-PCR using primers that recognize highly conserved domains of the ERalpha, mRNA products of the predicted size were amplified from laryngeal muscle as well as from other classical target tissues (forebrain and oviduct). Northern blots using a portion of the PCR product as primer revealed the same-sized band in oviduct and laryngeal muscle. Immunocytochemistry and Western blots confirmed the presence of ER protein in laryngeal muscle fibers and revealed several proteins in laryngeal muscle, brain and liver; among these was an approximately 66-kD protein - presumed to be full-length ER - that was the only one found in oviduct. Estrogen treatment of juveniles resulted in an upregulation of the 66-kD ER protein concomitant with an increase in quantal content. Taken together, these experiments strongly suggest that the ER is expressed by laryngeal muscle; this receptor could mediate estrogen-dependent changes in synaptic strength via retrograde signaling.

Estrogen receptors in Xenopus: duplicate genes, splice variants, and tissue-specific expression.

The estrogenic steroid hormones, acting primarily through the nuclear estrogen receptors ERalpha and ERbeta, regulate sexual differentiation in a wide variety of vertebrates. In the frog Xenopus laevis, estrogen regulates the strength of vocal neuromuscular synapses and contributes to the physiological basis of sexually differentiated songs. To understand the mechanisms by which estrogen produces these effects, we have characterized the ERs of X. laevis and their expression in laryngeal muscle and other tissues. We found a remarkable molecular diversity in the estrogen receptor population within individuals. First, we have identified two distinct ERalpha genes, xlERalpha1 and xlERalpha2, which represent, to our knowledge, the first discovery of retained duplicates of the ERalpha gene in any species. These two genes are highly conserved at the amino acid level but have distinct nucleotide sequences; moreover, ERalpha2 has no N-terminal domain. Cloning of ERalpha and ERbeta in the related species Xenopus tropicalis and phylogenetic analysis indicate that the two xlERalpha loci were generated by a duplication specific to the X. laevis lineage-most likely the genome duplication that led to a doubling of the X. laevis chromosome number about 30 million years ago. The primary ER expressed in X. laevis laryngeal muscle is the novel gene xlERalpha2; ERalpha1 is primarily expressed in liver, forebrain, and oviduct. Alternatively spliced transcripts of both xlERalpha1 and xlERalpha2 are also expressed in a tissue-specific manner. We propose that complementary spatial expression of these two genes and their alternatively spliced transcripts contributes to their conservation over such a long period of time, consistent with the subfunctionalization model for evolution after gene duplication.