Behaviour-structure interplay drives acoustic signal divergence: emergence of multiple mechanisms in closely related...

Acoustic communication plays a central role in reproductive isolation, yet the mechanisms driving signal divergence among closely related species remain poorly understood. In male crickets, calling songs emerge from the interaction between the morphology of the stridulatory apparatus and the behaviour controlling forewing movements. Species of the genus Agnothecous are morphologically similar in their sound-producing structures, yet emit high-frequency calls spanning approximately 10-20 kHz, making this group a suitable model to investigate this interaction. We analysed 15 species using acoustic recordings, morphological measurements of the stridulatory apparatus, behavioural estimates of forewing kinematics, and comparative phylogenetic methods. Two distinct mechanisms of high-frequency song production were identified. Most species rely on harmonic amplification, in which stridulation generates a low fundamental frequency while resonant forewings selectively amplify one of its harmonics. In contrast, A. robustus and A. tapinopus independently evolved a high-speed stridulation mechanism, producing dominant frequencies directly through accelerated forewing closure. Although both mechanisms generate similar acoustic outputs, they differ in their biomechanical basis. Phylogenetic reconstructions indicate that harmonic amplification is ancestral in Agnothecous, with high-speed stridulation evolving convergently in larger-bodied species. Bayesian correlation analyses across 11 continuous traits show that body size, stridulatory file, harp dimensions, and wing-closing speed form a tightly correlated trait complex that jointly shapes dominant frequency and syllable structure. Together, these results suggest energetic trade-offs and possible irreversibility of high-frequency communication. More broadly, they illustrate how morphological variation and behavioural plasticity interact to drive acoustic diversification in closely related species.