The return to water in ancestral Xenopus was accompanied by a novel mechanisms for producing and shaping vocal signals
Species-specific vocal signals allow listeners to locate potential mates. During the tetrapod transition from water to land, lungs replaced gills, allowing expiration to drive sound production. Several groups, e.g. cetaceans and some frogs, then returned to water. Here we explore how air-driven sound production changed upon re-entry and how essential acoustic information on species identity was preserved in the secondarily aquatic frog Xenopus. We filmed movements of cartilage and muscles during evoked sound production in isolated larynges. Our results refute the current theory for Xenopus vocalization, cavitation, and instead favor sound production by mechanical excitation of laryngeal resonance modes following rapid separation of laryngeal arytenoid discs. The resulting frequency resonance modes (dyads) are intrinsic to the larynx rather than due to neuromuscular control. We show that dyads are a distinctive acoustic signature across species. While dyad component frequencies overlap across species, their ratio is shared within each Xenopus clade and thus provide information on species identity, potentially facilitating both conspecific localization and ancient species divergence.