Abstract
The evolution of birdsong has been studied predominantly in diurnal passerines, leaving nocturnal species underexplored. Owls provide an exceptional model: their cryptic plumage contrasts with conspicuous vocalizations that function in territorial defense, in low-visibility environments. We investigated evolutionary drivers of acoustic diversity in Megascops , the largest New World owl genus, using phylogenetic comparative methods on the most comprehensive vocal dataset assembled for the group. We compiled 5,652 recordings from all Megascops species and Gymnasio nudipes , measured 40 acoustic variables from territorial songs, and analyzed their evolutionary patterns using an updated molecular phylogeny. Three traits showed significant phylogenetic signal (phrase duration, number of notes, and relative time at 5% of phrase energy), indicating evolutionary conservatism with clade-specific innovations overlaying a shared ancestral motif. Contrary to predictions from the Acoustic Adaptation Hypothesis, forest-dwelling species produced higher-pitched songs than those in open habitats. We propose that this deviation reflects eavesdropping avoidance: as small-bodied owls vulnerable to larger sympatric species, Megascops may favor higher frequencies that attenuate rapidly, reducing detectability. Additionally, forest species showed intra-phrase acceleration, which combined with higher frequencies could enhance distance assessment (ranging), allowing receivers to gauge intruder proximity, a critical adaptation for territorial, armed birds. Elevation showed complex associations, with higher altitudes correlating with increased frequencies, likely mediated by habitat structure. Sexual vocal dimorphism was associated with subtle spectral patterns rather than overall frequency differences, suggesting that sex-specific vocal divergence appears on fine-scale modulation capacity. Unexpectedly, body mass showed no correlation with any acoustic trait after phylogenetic correction. Our findings reveal that owl vocal evolution reflects a complex interplay between phylogenetic constraint and ecological pressures distinct from diurnal songbirds, highlighting the need to expand bioacoustics research beyond traditional model systems to understand communication under nocturnal, predation-driven conditions.