Here we present the first detailed description of the vocal tract of a basal hummingbird; a species with the potential to illuminate how vocal learning has evolved. We identified a tracheobronchial syrinx located in the dorsal part of the neck. The syrinx appears to be comprised of heavily modified osseous structures, intrinsic syringeal musculature with a particular ventro-dorsal orientation and a pair of vibratory membranes in each of the sides of the syrinx. Further, we find one ossicle embedded in each of the medial vibratory membranes. This peculiar syringeal morphology allows the black jacobin to produce a vibrato that challenges the known limits of this acoustic feature.
Hummingbirds (family Trochilidae) are grouped with swifts (family Apodidae) and treeswifts (family Hemiprocnidae) in the order Apodiformes . Within hummingbirds, a comparative study that included the syrinx of the clades Hermits, Mangoes and Brilliant suggested that adjacent bronchi, highly modified bronchial half-rings, intrinsic muscles and tympanic ossicle are unique to hummingbirds within Apodiformes . A recent study that investigated the syrinx of the clade Bees, relatively recent radiation within hummingbirds , found also specialized pairs of accessory cartilages, one of them embedded in the medial vibratory membranes, and intrinsic muscles . Similarly to hummingbirds, the syrinx of the swifts is tracheobronchial with an osseous tympanum . However, the synrix of the swifts is placed inside of the thoracic cavity, have two pairs of extrinsic muscles (tracheolateralis and sternotrachealis) and no intrinsic muscles [35, 41,42,43]. Given the relatively basal placement of Topazes within hummingbirds  and the consistency of these characters in the hummingbird species reported to date, we suggest that the extrathoracic syrinx, heavily modified bronchial half-rings, tympanic ossicles, more than one pair of intrinsic muscles, lack of sterno-tracheal muscle and two pairs of vibratory membranes are synapomorphies of the family.
Decoupling from physiological noise and syrinx stabilization without ST muscles
The black jacobin’s syrinx is located outside of the thoracic cavity, in contrast to most of the birds that have their syringes inside the thoracic cavity , except for the roseate spoonbill (Platalea ajaja) . Hummingbirds are highly specialized for hovering: unsurprisingly, its flight muscles make up 25 to 30% of its body weight, a ratio that is more than that of any other bird family . The hummingbird’s enlarged flight muscles are combined with an enlarged heart, comprising about 2.5% of its body mass, which beat rate is the highest among birds [44, 45]. The syrinx location outside of the thoracic cavity potentially alleviates spatial constraints caused by the enlarged flight muscles and avoids mechanical disturbances from the cardiac muscles. Thus, we speculate that an extrathoracic syrinx may have allowed hummingbirds to evolve control over its syringeal biomechanics despite their unique adaptations for the hovering flight.
The black jacobin’s syrinx lacks the sterno-tracheal muscle (ST), in contrast to almost all other birds. Further exceptions are the nearly non-vocal New World vultures and the vocalizing tinamous Darwin’s Nothura (Nothura darwinii) . The function of ST is unclear, in species in which intrinsic muscles are absent it is hypothesized to function as syringeal adductor [43, 47,48,49]. For example in the tracheal syrinx of pigeons, the shortening of the ST brings its cartilages closer together, thereby closing the syringeal lumen . The adduction of the labia is crucial for sound production in general as it facilitates the build-up of the phonation threshold pressure (PTP), which is necessary for sound onset . Surprisingly, in species with intrinsic syrinx muscles, adduction is achieved by intrinsic musculature rather than ST and ST function is attributed to syringeal stabilization [17, 51]. Similarly, and congruent with our observations, the closing mechanism in black jacobins is probably realized through intrinsic musculature. Darwin’s nothura, which also lacks ST, has the membrane of the interclavicular air sac more caudal than in the other tinamous species and it is proposed to also stabilize the syrinx . Black jacobins seem to obtain syrinx stabilization through tight wrapping of the syrinx in several layers of soft tissue. These layers create a rigid frame that might also compensate for the lack of rib cage protection. While protecting the syrinx from its immediate environment, this tight wrapping also keeps the syringeal elements inside flexible and potentially aids in maintaining the differential pressure necessary for the onset of sound production [18, 22]. The most external of these layers may be an evagination of the interclavicular membrane that cranially encloses the syrinx within the interclavicular air sac, which has also been reported in other hummingbird species .
The syrinx displacement may also have had implications for muscle orientation. The intrinsic muscles of the black jacobin’s syrinx are oriented dorso-ventrally, while the intrinsic muscle fibers of most bird taxa run cranio-caudally, for example, the ventral syringeal muscle of songbirds [13, 16, 36]. Because all of the black jacobin’s intrinsic muscles are ventrally attached to the tympanum, but each of them is dorsally attached to a different point, they run dorso-ventrally on different angles. The general dorso-ventral orientation with differences in angulation might allow the black jacobin to control the mobile syringeal elements despite the lack of lateral stabilization provided by the STs in other taxa.
The extrathoracic disposition of the syrinx and accompanied absence of STs in hummingbirds [35, 40, 52], might have been one of the driving pressures for the evolution of intrinsic muscles, a key prerequisite of vocal learning.
Although cartilaginous formations were found embedded in the vibratory membranes of songbirds , tympanic ossicles have not been reported in any species other than hummingbirds [35, 40, 52]. The origin of tympanic ossicles is uncertain. Due to their medial position and proximity to the tympanum, they might be either modified bronchial half-rings or have originated from a tracheal ring. In humans, the prevalence of a small sesamoid bone in the knee has increased worldwide in the past century, probably as a dissipative response to increased mechanical forces due to the enlargement of leg bones and muscles . Similarly, increased tension in the labia might have led to the formation of tympanic ossicles in the black jacobin’s syrinx.
In addition to direct muscular activity, stiffness of vocal tissues depends on the elastic properties of the tissue itself [24, 54]. In songbirds, cartilage embedded in the medial labia (ML) both aids in the dissipation of the tension, avoiding rupture under high stress and modifies the elastic properties of the syrinx . In particular, the cartilage that connects with the muscle potentially supports a more gradual bending mechanism, which in turn allows uncoupling the control of amplitude and frequency . Similarly, this might be the function of the cartilaginous extension in the dorsal part of the ML and its embedded ossicles, the tympanic ossicles, in the black jacobin. This extension is connected by a thin strip of connective tissue to a few muscle fibers of the larger syringeal muscle; given this arrangement, direct muscular control of the extension seems likely.
The tympanic ossicles may contribute to achieving the black jacobin’s high fundamental frequency: they cause high local density and prevent an entire part of the ML from vibrating at all, thus shortening its length and increasing the fundamental frequency. In other words, the tympanic ossicles could be used as a secondary mechanism to gradually increase ML stiffness and reduce ML length. It is therefore likely that the cartilaginous extension of the ML in the black jacobin both shifted the elastic properties of the ML towards the optimal for high fundamental frequency by increasing ML density towards the muscle attachment site that directly controls ML stiffness, and shortened the vibratory part of the ML.
Extreme vocal performance
Black jacobins produce particularly rapidly-modulated vibrato sounds . The black jacobins’ vibratos oscillate periodically up and down with a frequency bandwidth of up to 3 kHz at a rate of about 250 Hz. This fast vibrato rate can be compared to that of other extreme vocal performances, such as of starlings (Sturnus vulgaris), a songbird whose muscle activity in the syrinx produces changes in sound amplitude at a repetition rate of 218 Hz . The musculature of the songbird’s syrinx belongs to a special class of muscles, called superfast muscles [56, 57], and can produce work at cycling limits of approximately 90 Hz to 250 Hz . In vitro preparations revealed that the superfast songbird muscles in the syrinx have the potential to function at cycle frequencies as fast as 250 Hz . Although direct electromyographic recordings of the syringeal musculature would be needed to confirm that the black jacobin’s vibrato rate of 250 Hz is a direct result of muscular control, this extremely fast performance suggests that the black jacobin’s syringeal muscles produce work on the upper limit of the superfast muscle activity reported to date  and that black jacobins may have muscle properties comparable to those of songbirds.
Biomechanics of sound production and implications for vocal learning in hummingbirds
Parrots and songbirds, two vocal learners, have a tracheal and a tracheobronchial syrinx, respectively, both with intrinsic musculature [17, 20, 36, 59]. The black jacobin’s syrinx, like that of all the other hummingbird species reported so far [35, 40, 52], is tracheobronchial, with three pairs of intrinsic muscles that are as complex as those of songbirds . The black jacobin’s multiple intrinsic muscles attach in close proximity to movable elements of its syringeal osseous elements (modified bronchial half-rings) to which the vibratory membranes (medial labia or lateral labia) are attached via cartilaginous extensions. These muscles seem to operate consecutively. For example, both lateral and medial labia are attached to the bronchial half-ring B2, where two large muscles are attached. At its cranial surface is the cranial syringeal muscle (CrS), and at its lateral part, the central syringeal muscle (CeS). Given the location and orientation of each muscle, we speculate that various amounts of contraction of each muscle might contribute gradually to distinct functions, such as the abduction of the ML and the stretching of the labia. Since the position and tension of the labia are directly related to distinct acoustic parameters, multiple muscles contributing to the same function creates redundancy in possible motor commands controlling acoustic parameters such as fundamental frequency. When the brain has multiple, rather than a single motor command available to achieve a certain vocal output, a redundant control space may simplify trial-and-error attempts during imitation in the vocal production learning process .
Hummingbirds and songbirds converge in their syrinx morphology, while parrots produce learned vocalizations with seemingly less complex syringeal musculature . However, parrot’s lingual articulation introduces a hitherto overlooked level of complexity to their vocal production system . Syrinx muscle complexity alone does not correlate with vocal learning . Nevertheless, the presence of intrinsic musculature when combined with further specializations leading to acoustic complexity may facilitate the evolution of neurological structures associated with vocal learning. Thus, we speculate that the more degrees of freedom are provided to the motor redundancy by peripheral adaptations for vocal production, the more likely a species is to follow on to the next evolutionary step towards the evolution of vocal learning.