Two conserved vocal central pattern generators broadly tuned for fast and slow rates generate species-specific vocalizations in Xenopus clawed frogs

Across phyla, species-specific vocalizations are used by males to attract females. Functional analyses of the neural circuitry underlying behavior have been challenging, particularly in vertebrates. However, using an ex vivo brain preparation that produces fictive vocalizations, we previously identified anatomically distinct fast and slow central pattern generators (CPGs) that drive the fast and slow clicks of male courtship calls in male African clawed frogs, Xenopus laevis. To gain insight into the evolution of neural circuits underlying species-specific courtship calls, we extended this approach to four additional species, X. amieti, X. cliivi, X. petersii, and X. tropicalis, by developing ex vivo brain preparation from which fictive vocalizations are elicited in response to a chemical or electrical stimulus. We found that even though the courtship calls of different Xenopus species vary in their click rates and duration, the CPGs used to generate clicks are conserved across species. The fast CPGs found in male X. laevis, which critically rely on reciprocal connections between the parabrachial nucleus and the nucleus ambiguus, are conserved among species that produce fast clicks. Similarly, the slow CPGs found in the caudal brainstem of male X. laevis are shared among species that produce slow clicks. In addition, our results suggest that testosterone plays a role in organizing fast CPGs in fast-click species, but it does not appear to have the same effect in slow-click species. Moreover, we demonstrate that, unlike other vestigial neural circuits that remain latent, fast CPGs are not inherited by all species. Instead, they are possessed only by the species that produce fast clicks. The results suggest that species-specific calls of the genus Xenopus have evolved by utilizing conserved slow and/or fast CPGs inherited by each species. Fast and slow CPGs are broadly tuned to generate fast or slow clicks, and the organization of the former appears to be regulated by testosterone in a species-specific manner.