Abstract
How sound is generated in the hummingbird syrinx is largely unknown despite their complex vocal behavior. To fill this gap, syrinx anatomy of four North American hummingbird species were investigated by histological dissection and contrast-enhanced microCT imaging, as well as measurement of vocalizations in a heliox atmosphere. The placement of the hummingbird syrinx is uniquely located in the neck rather than inside the thorax as in other birds, while the internal structure is bipartite with songbird-like anatomical features, including multiple pairs of intrinsic muscles, a robust tympanum and several accessory cartilages. Lateral labia and medial tympaniform membranes consist of an extracellular matrix containing hyaluronic acid, collagen fibers, but few elastic fibers. Their upper vocal tract, including the trachea, is shorter than predicted for their body size. There are between-species differences in syrinx measurements, despite similar overall morphology. In heliox, fundamental frequency is unchanged while upper-harmonic spectral content decrease in amplitude, indicating that syringeal sounds are produced by airflow-induced labia and membrane vibration. Our findings predict that hummingbirds have fine control of labia and membrane position in the syrinx; adaptations that set them apart from closely related swifts, yet shows convergence in their vocal organs with those of oscines.