Male spot-legged treefrogs (Polypedates megacephalus) respond differently to diverse vocal combinations varied in units and orders.

Some vertebrate taxa, particularly birds and mammals, employ complex vocalisations composed of multiple vocal units for communication. Many anurans, especially treefrogs, appear to possess similar vocal capabilities; however, the complex combinatorial nature of amphibian vocal systems has been studied less extensively. In this study, we recorded 2002 calls from 75 male spot-legged treefrogs (Polypedates megacephalus) in natural choruses and conducted playback experiments to examine their responses to different call types. We found that males can produce rich compound calls by combining three vocal units-simple A, B, and C calls, consisting of A, B, and C notes, respectively. In natural choruses, they specifically generated different combinations (e.g., AB and BA, CAB, and CBA) of these calls using the same vocal units. In playback experiments, males responded differently to simple A, B, and C calls. Furthermore, male receivers exhibited distinct calling behaviors when presented with combination calls vs separate, ordered calls composed of identical components (e.g., CA vs C + A, AB vs BA, CAB vs CBA). These results suggest that spot-legged treefrogs may communicate using diverse vocal combinations that vary in both units and orders. This study enhances our understanding of vocal complexity in non-human animals.