Vocalizations are ancient behaviours that require the complex coordination of breath and display. Understanding how laryngeal anatomy shapes vocalization provides insights into this diversity, its mechanisms and their evolution. Rodents are ideal for exploring this variation because of their diverse mechanisms and vocal structures. Here, we describe the laryngeal morphology and sound production mechanisms underlying the vocalizations of Alston's singing mouse (Scotinomys teguina) and compare these results to those of other vocalizing mammals. We reconstructed the three-dimensional laryngeal morphology with micro-computed tomography, recorded laryngeal sound production using high-speed video and investigated frequency control using surgical ablations. We found that singing mice use a whistle mechanism that uniquely relies on the inflation of an enlarged air sac called the ventral pouch. Song frequency can be controlled by pouch volume, airflow and cricothyroid muscle action. Singing mouse laryngeal morphology and vocal mechanism are distinct from those of other Neotomids; singing mice appear to use inflation-mediated whistles for both distant and close exchanges. Inflatable air sacs have evolved repeatedly for sound modulation and filtering. Our results indicate a novel role for these structures in being required to generate sound. Together, our results expand on an emerging story of how biomechanic and morphological variation contributes to vocal diversity.

Understanding vertebrate sound production offers powerful insights into constraint and convergence in evolution. Sound production falls into two broad categories: muscle-driven and airflow-driven. Muscle-driven mechanisms, widespread among fish, are tightly limited by muscle contraction rates, producing lower-frequency signals that often radiate poorly. In contrast, airflow-driven mechanisms, especially those using myoelastic aerodynamic (MEAD) principles, overcome these limits, enabling long-distance communication across a vast frequency range. MEAD-based laryngeal sound production dominates in tetrapods and convergently evolved in novel vocal organs in birds and toothed whales—all supporting complex, long-distance communication and new acoustic niches. In this review, we show how physical and physiological constraints shaped convergent innovations, positioning MEAD as a central evolutionary strategy underlying the remarkable diversity of vertebrate sounds.

Neural tissues are exceptionally sensitive to oxygen deprivation and rely on a dense network of blood vessels to support their extraordinarily high metabolic demands for oxygen, nutrients and clearance of waste products1-4. In birds, one of the metabolically most demanding neural tissue-the retina-lacks internal blood vessels5,6. This raises the question of how such a metabolically demanding neural tissue can function without blood perfusion. Here we show that, while the photoreceptor outer segments in the outer retina have access to oxygen, the inner bird retina operates under chronic anoxia, supported by anaerobic glycolysis in the retinal neurons. We provide evidence that the pecten oculi-a uniquely vascularized structure in the vitreous humour of birds, the function of which has been debated for centuries5-9-supplies the anoxic inner retina with glucose and removes lactic acid. We suggest that the pecten's metabolic support of the bird retina's anoxia tolerance enabled first the evolution of a thick cell-dense, avascular retina, which secondarily served as an exaptation enabling retinal function during high-altitude migrations.

Abstract Dexterous manipulation of objects relies on precise coordination between anatomical elements. In seed-eating birds, seeds are manipulated and dehusked using both the beak and tongue, but the functional roles and coordination of these structures remain unresolved. Here, we quantified the 3D movements of the upper beak, lower beak, tongue, and seed in a hard-biting and a weak-biting songbird species using X-ray Reconstruction of Moving Morphology (XROMM) and measured contractile properties of their primary jaw muscles. We show that the tongue serves as the main tool for seed rotation, transport, and stabilization. Multi-dimensional, high-frequency movements of the upper and lower beaks reveal that efficient seed processing depends on high mobility of the kinetic avian skull. Strong and weak biters differ in feeding kinematics and jaw muscle speeds, suggesting ecological specialization of cranial mechanics. The complexity, precision, and tight coordination of beak and tongue motions show that the avian cranium rivals the dexterity of the primate hand despite limited degrees of freedom.

Among air-breathing tetrapods, the most common sound production mechanism is flow-induced self-sustained tissue oscillation, aka voiced sound production, driven by inherently nonlinear physical processes. Some signature features like deterministic chaos have received particular attention in bioacoustics as nonlinear phenomena (NLP). However, one type of NLP that extends frequency ranges and enriches timbres has received much less focus in comparative bioacoustics: vocal registers. Controlled by muscle activity, vocal registers constitute distinct periodic vibratory states of vocal tissues. Transitions between vocal registers often lead to abrupt fundamental frequency jumps, which are, e.g., deliberately used in human alpine yodelling, for example. Theoretical work suggests that register transitions are caused by saddle-node-in-limit-cycle bifurcations. Here, we review the biophysical underpinnings of vocal registers and what signatures they leave in vocal fold kinematics and acoustics in the best studied species: humans. Apart from human speech and song, registers have been described only in a few animal taxa, but the occurrence of signature features suggests that vocal registers could be much more common across vertebrates than currently appreciated. We suggest that registers are a fundamental trait of voice production and that they are favoured in selection because they vastly extend and diversify the acoustic signalling space. This article is part of the theme issue 'Nonlinear phenomena in vertebrate vocalizations: mechanisms and communicative functions.'

Physics-based simulators for neuromechanical control of virtual animals have the potential to significantly enhance our understanding of intricate structure-function relationships in neuromuscular systems, their neural activity and motor control. However, a key challenge is the accurate prediction of the forces that muscle fibers produce based on their complex patterns of electrical activity ("spike trains") while preserving model simplicity for broader applicability. In this study, we present a chemomechanical, three-dimensional finite-element muscle model - JiSuJi (pronounced jì sù jī, meaning "ultrafast muscle" in Chinese) - that efficiently and accurately predicts muscle forces from naturalistic spike trains. The model's performance is validated against songbird vocal muscles, a particularly fast and therefore challenging muscle type. Our results demonstrate that JiSuJi accurately predicts both isometric and non-isometric muscle forces across a variety of naturalistic neural activity patterns. JiSuJi furthermore outperforms state-of-the-art muscle simulators for accuracy, while maintaining computational efficiency. Simulating muscle behavior offers a promising approach for investigating the underlying mechanisms of neuro-muscular interactions and precise motor control, especially in the fast-contracting muscles of animal model systems.

Birds evolved a novel vocal organ, the syrinx, that exhibits a high anatomical diversity. In the few species investigated, the syrinx can contain up to three pairs of functional syringeal vocal folds, acting as independent sound sources, and eight pairs of muscles. This rich variety in vocal structures and motor control results in a wide range of nonlinear phenomena (NLPs) and interactions that are distinct to avian vocal physiology, with many fascinating mechanisms yet to be discovered. Here, we review the occurrence of classical signatures of nonlinear dynamics, such as NLPs, including frequency jumps and transitions to chaos in birds. However, birds employ several additional unique tricks and transitions of inherent nonlinear dynamical nature that further enrich their vocal dynamics and are relevant for understanding the motor control of their vocalizations. Particularly, saddle-node in limit cycle (SNILC) bifurcations can switch sounds from tonal to harmonically rich and change the physiological control of fundamental frequency. In mammalian phonation, these bifurcations are mostly explored in the context of register transitions but could be equally relevant to altering vocal fold dynamical behaviour. Due to their diverse anatomy compared to mammals, birds provide unique opportunities to explore rich nonlinear dynamics in vocal production. This article is part of the theme issue ‘Nonlinear phenomena in vertebrate vocalizations: mechanisms and communicative functions’.

Birds and mammals converged upon the same physical mechanism of vocal fold vibration1,2 to produce the wide variety of communicative vocal signals critical for their reproduction, social interactions, survival, and speciation.3,4,5 Recent work reported high-frequency (7-11 kHz) vocalizations in zebra finches,6 termed "heat" or "incubation" calls, that are suggested to have close-range communicative relevance in the context of global warming.6,7 However, their acoustics are poorly described, and by what biophysical mechanism they are produced remains unknown. We recorded heat-associated vocalizations in adult zebra finches in vivo and showed that they are extremely soft, frequency-modulated vocalizations with source levels of 13.9 ± 3.3 dB sound pressure level (SPL) at 1 m and dominant frequencies of 6.8 ± 0.6 kHz. Through in vitro experiments, we establish that these vocalizations are aerodynamic whistles produced inside the avian larynx, not syrinx, during inspiration. Respiratory air flow during whistle production is higher than during regular song and consistent with thermal panting for evaporative cooling.7,8 Laryngeal geometry and dimensional flow analysis suggest that these whistles are laminar-flow whistles that occur when a flow boundary layer is in a transition phase from laminar to turbulent flows.9,10,11 Our data imply that in earlier experiments,6,7 playbacks were conducted at 30 dB above the physiologically relevant playback level (i.e., several magnitudes). Furthermore, heat whistles are at least 35 dB below the behavioral perception limit even of adults12 with more sensitive hearing than juveniles.13,14 Therefore, we show-contra previous works6,7-that heat whistles cannot function as adaptive signals or cues in parent-embryo communication. VIDEO ABSTRACT.

Avian vocalizations are produced by precisely coordinated motion of the respiratory, syringeal and upper vocal tract systems. Syringeal muscles are controlled with unprecedented resolution, down to independent control of individual muscle fibres. However, we currently lack an anatomical description of syrinx muscles at single fibre resolution. Here, we combined a micron-resolution synchrotron X-ray CT scan of the zebra finch syrinx with micro-dissections of independent specimens to resolve syrinx muscle morphology at individual muscle fibre level. We define two new, previously unknown muscles and update the fibre trajectories and attachment sites of three previously described muscles. Our new insights into the fine anatomy of syrinx muscles show that not one, but both avian vocal folds can be directly controlled by contracting syrinx muscles. Thus, our data reveal novel anatomical complexity with consequences for the biomechanics and motor control of sound production. This article is part of the theme issue 'The biology of the avian respiratory system'.

Identifying variation in vocal morphology and sound production mechanisms is essential to understanding vocal diversity. Rodents provide an ideal system for exploring this variation as they use multiple sound production mechanisms and have novel vocal structures whose morphology varies interspecifically. Here, we describe the laryngeal morphology and identify the sound production mechanism of Alston’s singing mouse ( Scotinomys teguina ), which produce stereotyped songs. We used micro-computed tomography to examine laryngeal morphology and manipulated excised larynges and surgically ablated a laryngeal muscle to determine sound production mechanism and frequency control. Laryngeal manipulations indicated that a whistle mechanism, likely an edge tone or shallow cavity, produces song. Singing mouse whistles are unique compared to other rodents because they rely on the inflation of an enlarged intralaryngeal air sac called the ventral pouch. Whistle frequency can be controlled by ventral pouch inflation, laryngeal airflow, and by cricothyroid muscle action. Cricothyroid ablation inhibited frequency modulation in vivo , suggesting that singing mice use this muscle during singing. Singing mouse laryngeal morphology and vocal mechanism are distinct from other Neotomids; instead of using vocal fold oscillations for loud, long-distance calls and whistles for close-range interactions, singing mice appear to use whistles for distant and close exchanges by inflating their intralaryngeal air sac. Air sacs have evolved repeatedly among vocalizing mammals and our results indicate a new role for these structures in generating sound. Together, our results expand on an emerging story of how biomechanic and morphological variation contributes to vocal diversity. ### Competing Interest Statement The authors have declared no competing interest. National Science Foundation, https://ror.org/021nxhr62, IOS-1457350, IOS-1556975 National Institute of Health, R01 M190270174 EEB Graduate Program, University of Texas at Austin IB Joint Graduate Program Fellowship at the University of Texas at Austin

Voice is a major means of communication for humans, non-human mammals and many other vertebrates like birds and anurans. The physical and physiological principles of voice production are described by two theories: the MyoElastic-AeroDynamic (MEAD) theory and the Source-Filter Theory (SFT). While MEAD employs a multiphysics approach to understand the motor control and dynamics of self-sustained vibration of vocal folds or analogous tissues, SFT predominantly uses acoustics to understand spectral changes of the source via linear propagation through the vocal tract. Because the two theories focus on different aspects of voice production, they are often applied distinctly in specific areas of science and engineering. Here, we argue that the MEAD and the SFT are linked integral aspects of a holistic theory of voice production, describing a dynamically coupled system. The aim of this manuscript is to provide a comprehensive review of both the MEAD and the source-filter theory with its nonlinear extension, the latter of which suggests a number of conceptual similarities to sound production in brass instruments. We discuss the application of both theories to voice production of humans as well as of animals. An appraisal of voice production in the light of non-linear dynamics supports the notion that it can be best described with a systems view, considering coupled systems rather than isolated contributions of individual sub-systems.

Acoustic communication relies critically on the receiver’s ability to hear. In precocial bird species hearing can already be functional during embryonic stages in the egg, while in altricial bird species hearing typically starts after hatching. Recent research suggests that zebra finch embryos, despite being altricial, have functional hearing already in the egg to engage in parent-embryo acoustic communication and in anthropogenic noise detection. However, their auditory sensitivity during early development remains unknown. Here, we measure auditory brainstem responses over early postnatal development and show that zebra finch hatchlings are deaf even to loud, broadband sounds with hearing responses emerging between 4-8 days after hatching. Auditory sensitivity develops progressively and reaches adult levels by day 20, contradicting the notion of early parent-to-embryo communication in zebra finches. Additionally, egg vibrations induced by sound remain far below detection thresholds of vibrotactile senses. The striking timing coincidence between maturation of the peripheral auditory system and the onset of song learning suggests that hearing functionality may gate the onset of vocal learning in zebra finches.

Birds and mammals converged upon the same physical mechanism of vocal fold vibration to produce their broad range of voiced sounds critical to communication. The frequency range of vocal fold vibration is limited per species by biophysical constraints to 3-4 octaves. However, recent work reported vocalizations in zebra finches with apparent fundamental frequencies of 7-11 kHz that far exceed the range of regular calls and song (0.5-1.5 kHz). These "heat" or "incubation" calls are suggested to have close-range communicative relevance in the global temperature rise context, but their acoustics are poorly described and by what biophysical mechanism they are produced remains unknown. We recorded heat calls in adult zebra finches in vivo and show they are extremely soft, frequency-modulated calls with source levels of 13.9 ± 3.3 dB SPL at one meter with dominant frequencies of 6.8 ± 0.6 kHz. Through a series of in vitro experiments, we establish that these calls are aerodynamic whistles produced inside the avian larynx, not syrinx, during inspiration. Respiratory air flow during whistle production is an order of magnitude higher than song and consistent with thermal panting for evaporative cooling. Laryngeal geometry and dimensional flow analysis suggest that these whistles are laminar flow whistles that occur when a flow boundary layer is in a transition phase from laminar to turbulent flows. Birds, like some rodents, are thus able to produce both voiced sounds and aerodynamical whistles in their vocal tract. ### Competing Interest Statement The authors have declared no competing interest.

ABSTRACT Vocalisations play a key role in the communication behaviour of many vertebrates. Vocal production requires extremely precise motor control, which is executed by superfast vocal muscles that can operate at cycle frequencies over 100 Hz and up to 250 Hz. The mechanical performance of these muscles has been quantified with isometric performance and the workloop technique, but owing to methodological limitations we lack a key muscle property characterising muscle performance, the force–velocity relationship. Here, we quantified the force–velocity relationship in zebra finch superfast syringeal muscles using the isovelocity technique and tested whether the maximal shortening velocity is different between males and females. We show that syringeal muscles exhibit high maximal shortening velocities of 25L0 s−1 at 30°C. Using Q10-based extrapolation, we estimate they can reach 37–42L0 s−1 on average at body temperature, exceeding other vocal and non-avian skeletal muscles. The increased speed does not adequately compensate for reduced force, which results in low power output. This further highlights the importance of high-frequency operation in these muscles. Furthermore, we show that isometric properties positively correlate with maximal shortening velocities. Although male and female muscles differ in isometric force development rates, maximal shortening velocity is not sex dependent. We also show that cyclical methods to measure force–length properties used in laryngeal studies give the same result as conventional stepwise methodologies, suggesting either approach is appropriate. We argue that vocal behaviour may be affected by the high thermal dependence of superfast vocal muscle performance.

Hearing is critical for vocal communication, and in birds its onset is linked to egg hatching. Hearing develops after hatching in early hatching (altricial) species, while already functional in the egg in later hatching (precocial) species. Recent work suggests that altricial zebra finches exhibit parent-to-embryo acoustic communication, requiring functional, high-frequency hearing already in the egg. Yet, the early postnatal development of their auditory sensitivity remains unknown. By measuring hearing and vibration-detection capacity during postnatal development in zebra finches, we show that zebra finch hatchlings are deaf even to loud, broadband sounds. Auditory nerve sensitivity develops gradually in a low-to-high frequency manner. Auditory nerve responses show adult-like properties only after 20 days from hatching. Our data provide evidence against the plausibility of early parent-to-offspring acoustic communication in zebra finches and suggest that hearing functionality may set the onset of vocal learning in zebra finches.### Competing Interest StatementThe authors have declared no competing interest.

Experimental and modelling data from the paper "Evolutionary novelties underlie sound production in baleen whales."

AbstractVoice production of humans and most mammals is governed by the MyoElastic-AeroDynamic (MEAD) principle, where an air stream is modulated by self-sustained vocal fold oscillation to generate audible air pressure fluctuations. An alternative mechanism is found in ultrasonic vocalizations of rodents, which are established by an aeroacoustic (AA) phenomenon without vibration of laryngeal tissue. Previously, some authors argued that high-pitched human vocalization is also produced by the AA principle. Here, we investigate the so-called “whistle register” voice production in nine professional female operatic sopranos singing a scale from C6 (≈ 1047 Hz) to G6 (≈ 1568 Hz). Super-high-speed videolaryngoscopy revealed vocal fold collision in all participants, with closed quotients from 30 to 73%. Computational modeling showed that the biomechanical requirements to produce such high-pitched voice would be an increased contraction of the cricothyroid muscle, vocal fold strain of about 50%, and high subglottal pressure. Our data suggest that high-pitched operatic soprano singing uses the MEAD mechanism. Consequently, the commonly used term “whistle register” does not reflect the physical principle of a whistle with regard to voice generation in high pitched classical singing.