Understanding why the frequencies of some species’
vocalizations are far above or below those predicted by body size
is key for explaining the remarkable diversity of mammalian vocal
behavior. Horses, among the largest terrestrial mammals, provide a
clear example of such deviation: their whinnies contain a very
high fundamental frequency (>1,000 Hz) in addition to a second,
lower one (∼200 Hz). While the lower fundamental frequency of
whinnies is readily attributed to vocal fold (laryngeal)
vibrations, the biomechanical processes underlying the production
of the higher one remain unknown. Using a combination of in
vivo and ex vivo data, including excised larynx
experiments with helium, computed tomography (CT) scans,
endoscopic examinations, and acoustic analysis of horses with
recurrent laryngeal neuropathy, we provide evidence that the high
fundamental frequency in horse whinnies is generated by an
aerodynamic whistle mechanism within the larynx, rather than vocal
fold tissue vibration. These separate laryngeal sources explain
the simultaneous production of low and high fundamental
frequencies in vocalizations (i.e., biphonation). Horse
biphonation likely evolved to convey multiple independent messages
concurrently, highlighting the role of anatomical and aerodynamic
adaptations in enhancing vocal complexity across species.