As noticed by Darwin , every derived feature in an organism must have evolved from a pre-existing feature in its ancestors. Therefore, the current form and function of organism attributes are determined, to a great extent, by phyletic heritage of past events . This equally applies to evolutionary key innovations, which are not designed every time anew, but use available materials, that are themselves a product of millions of years of evolution. This has been firmly established by many studies showing that (i) novel morphological structures may often appear by deploying ancient genetic regulatory circuits [e.g. ] and (ii) existing genes or morphological structures can be recruited to perform completely new functions [e.g. [4, 5]] or to explore new approaches to carry out the same task . Evolution is an integrated and unitary process , and effective reuse of previous molecular or morphological structures through natural selection is subjected to historical constraints . Deciphering how a particular functional mechanism arises in an organism requires framing the question within an evolutionary context through a multidisciplinary approach  involving functional, morphological, and phylogenetic analyses that allow determining which morphological structures were involved, their evolutionary origin (i.e. homology), and the succession of steps that led to a successful end.
Sound production is a key feature in the behavior of different animals such as crickets, frogs, birds or bats . Calls normally serve as advertising signals to delimit territories or attract mates, and in more sophisticated cases can become a proxy of the mood of the individual . Thus, it is not surprising that such a critical function has been the subject of intensive selection through evolutionary history, and a wide variety of sound production mechanisms have evolved in different animals. In frogs, diversification of sound production mechanisms is intimately linked to and/or constrained by the evolution of vocal structures, which is necessarily connected to the evolution of the respiratory system. Despite the considerable diversity of calls and larynx morphologies among extant frogs, the majority of the species call by moving air from the lungs through the glottis . In most frog species, the laryngeal apparatus, which is suspended between the posteromedial processes of the hyoid (= thyrohyals), is a cartilaginous capsule composed of two arytenoid cartilages (each bearing one vocal cord), the cricoid cartilage and associated musculature .
A remarkable exception to the above-described general sound production and larynx morphological patterns occurs in the family Pipidae. The extant members of this family include the South American genus Pipa (Surinam toads) and the four African genera Hymenochirus, Silurana, Xenopus, and Pseudhymenochirus (African clawed frogs). The family Pipidae together with its sister group, the monotypic family Rhinophrynidae (Mexican burrowing toads, genus Rhinophrynus), form the superfamily Pipoidea . The origin of pipids dates back at least to the Mesozoic [e.g. [13–16]] with known fossils from the Cretaceous . Pipids represent a nice example of highly adapted form and function that evolved from an inherited frog bauplan, which is per se highly specialized within amphibians (and tetrapods), and restricted to limited variation . Pipids are the only fully aquatic group of frogs, and their derived morphology and biology are largely a product of adaptations to this lifestyle . One of these remarkable adaptations is the pipid sound production , with the structure and function of their larynx being radically different from those of other frogs [20, 21]. Pipids lack vocal cords, and their larynx is a greatly enlarged and (at least partially) ossified box made up by the cricoid cartilage and the tyrohyals, which do not form part of the larynx in non-pipid frogs. This box encloses the arytenoid cartilages which are modified into two bony rods . The sound production mechanism was described in detail for Xenopus borealis [20, 22], and it appears to be based on implosion of air into a vacuum formed by rapidly moving disk-like enlargements of the arytenoids. The sound is then amplified by the enlarged voice box that serves as an internal vocal sac [20, 22]. Sounds thus are produced without moving an air column, and therefore without externally visible movements of the flanks or throat. Similar motionless calling was also observed in Hymenochirus boettgeri , Pipa pipa , Pipa carvalhoi , Xenopus laevis , and most other pipids [[26, 27], pers. obs.]. However, Pseudhymenochirus was stated to produce sounds by a more conventional sound production mechanism based on moving air , although this behavior has so far not been documented in detail.
Despite their many derived features, in several respects pipids have been more extensively studied than any other group of frogs because Xenopus laevis and Silurana tropicalis have been used as model organisms in physiology, development, and cell and molecular biology [e.g., ]. Knowledge on the closest relatives of model organisms is crucial to interpret and understand the evolutionary origin of studied characters and functions, but remarkably the phylogenetic relationships of pipids have not been comprehensively assessed so far. The rather aberrant morphology of pipids was initially considered to be relatively ancestral among frogs, and many of pipid morphological characters were initially assumed to retain plesiomorphic states. However, now pipids are viewed as highly derived frogs  with many autapomorphies primarily related to their fully aquatic lifestyle . Almost all possible alternative phylogenetic relationships among pipid genera have been recovered based on either morphological [19, 29, 30] or molecular [13, 31, 32] data sets, and the position of Pipoidea with respect to all other frog lineages remains also equally contentious [13–16, 31, 33]. See Additional file 1 for a detailed discussion of previously proposed hypotheses.
Here, we analyze DNA sequences of complete mitochondrial genomes and of nine nuclear genes to produce a robust phylogeny of extant pipoids. We used this phylogenetic framework to gain insights on the evolution of the sound production mechanism in pipids. In this context, we show through behavioral observations that the calling mechanism of Pseudhymenochirus clearly involves the movement of an air column, as it occurs in non-pipid ancestors. Given the unambiguous derived position of Pseudhymenochirus within pipid phylogeny, a reversal to air-driven sound production in this genus is hypothesized. In addition, we provide strong morphological evidence from comparing diverse alizarin-stained frog larynges, which show that larynx structure in Pseudhymenochirus has clear pipid affinities. These observations taken together, allow us to suggest that the use of air in the sound production in Pseudhymenochirus is an evolutionary novelty that evolved by deploying the typical larynx structures of pipids.