Virtual and augmented reality: New tools for visualizing, analyzing, and communicating complex morphology.

Virtual and augmented reality (VR/AR) are new technologies with the power to revolutionize the study of morphology. Modern imaging approaches such as computed tomography, laser scanning, and photogrammetry have opened up a new digital world, enabling researchers to share and analyze morphological data electronically and in great detail. Because this digital data exists on a computer screen, however, it can remain difficult to understand and unintuitive to interact with. VR/AR technologies bridge the analog-to-digital divide by presenting 3D data to users in a very similar way to how they would interact with actual anatomy, while also providing a more immersive experience and greater possibilities for exploration. This manuscript describes VR/AR hardware, software, and techniques, and is designed to give practicing morphologists and educators a primer on using these technologies in their research, pedagogy, and communication to a wide variety of audiences. We also include a series of case studies from the presentations and workshop given at the 2019 International Congress of Vertebrate Morphology, and suggest best practices for the use of VR/AR in comparative morphology.

Linking structure and function in the vertebrate respiratory system: A tribute to August Krogh.

High rates of pulmonary gas exchange require three things: 1) that gases at the contact surface of the lung's capillaries are replenished rapidly from the environment; 2) that this surface is large and thin; 3) that the capillaries are effectively perfused with blood. In spite of this uniform requirement, lungs have evolved complex and highly diverse architectures, but we have a poor understanding of the drivers of this diversity. Here, I briefly discuss some of the diversity in gross anatomical features directing airflow in avian and non-avian reptiles. I also review new insights into the cellular anatomy of the blood-gas barrier, which in mammals is composed of specialized endothelial as well as epithelial cells.

Anatomy, ontogeny, and evolution of the archosaurian respiratory system: A case study on Alligator mississippiensis and Struthio camelus.

The avian lung is highly specialized and is both functionally and morphologically distinct from that of their closest extant relatives, the crocodilians. It is highly partitioned, with a unidirectionally ventilated and immobilized gas-exchanging lung, and functionally decoupled, compliant, poorly vascularized ventilatory air-sacs. To understand the evolutionary history of the archosaurian respiratory system, it is essential to determine which anatomical characteristics are shared between birds and crocodilians and the role these shared traits play in their respective respiratory biology. To begin to address this larger question, we examined the anatomy of the lung and bronchial tree of 10 American alligators (Alligator mississippiensis) and 11 ostriches (Struthio camelus) across an ontogenetic series using traditional and micro-computed tomography (µCT), three-dimensional (3D) digital models, and morphometry. Intraspecific variation and left to right asymmetry were present in certain aspects of the bronchial tree of both taxa but was particularly evident in the cardiac (medial) region of the lungs of alligators and the caudal aspect of the bronchial tree in both species. The cross-sectional area of the primary bronchus at the level of the major secondary airways and cross-sectional area of ostia scaled either isometrically or negatively allometrically in alligators and isometrically or positively allometrically in ostriches with respect to body mass. Of 15 lung metrics, five were significantly different between the alligator and ostrich, suggesting that these aspects of the lung are more interspecifically plastic in archosaurs. One metric, the distances between the carina and each of the major secondary airways, had minimal intraspecific or ontogenetic variation in both alligators and ostriches, and thus may be a conserved trait in both taxa. In contrast to previous descriptions, the 3D digital models and CT scan data demonstrate that the pulmonary diverticula pneumatize the axial skeleton of the ostrich directly from the gas-exchanging pulmonary tissues instead of the air sacs. Global and specific comparisons between the bronchial topography of the alligator and ostrich reveal multiple possible homologies, suggesting that certain structural aspects of the bronchial tree are likely conserved across Archosauria, and may have been present in the ancestral archosaurian lung.

Parental Care, Destabilizing Selection, and the Evolution of Tetrapod Endothermy.

Parental care has evolved convergently an extraordinary number of times among tetrapods that reproduce terrestrially, suggesting strong positive selection for this behavior in the terrestrial environment. This review speculates that destabilizing selection on parental care, and especially embryo incubation, drove the convergent evolution of many tetrapod traits, including endothermy.

Computational Fluid Dynamics Reveals a Unique Net Unidirectional Pattern of Pulmonary Airflow in the Savannah Monitor Lizard (Varanus exanthematicus).

This report models pulmonary airflow in the savannah monitor (Varanus exanthematicus) using computational fluid dynamics simulations, which are based on computed tomography data. Simulations were validated by visualizing the flow of aerosolized lipids in excised lungs with good but not perfect agreement. The lung of this lizard has numerous successive bronchi branching off a long intrapulmonary bronchus, which are interconnected by intercameral perforations. Unidirectional flow has been documented in the lateral secondary bronchi of the savannah monitor, but patterns of airflow in the rest of the lung remain unknown, hindering our understanding of the evolution of pulmonary patterns of airflow in tetrapods. These results indicate that the lung contains a unique net unidirectional flow, where the overall flow scheme is similar during expiration and late inspiration, but dissimilar during early inspiration. Air is transported net caudally through the intrapulmonary bronchus and net craniad through secondary bronchi, much like the pattern of flow in birds. The simulations show that many chambers feature flow in multiple directions during parts of the respiratory cycle, but some regions also show robust unidirectional airflow. Air moves craniad through secondary bronchi and between adjacent secondary bronchi through intercameral perforations. The first secondary bronchus, the hilar-cranial bronchus, contains tidal flow that may improve ventilation of the central and dorsal lung parenchyma. These results expand our understanding of flow patterns in varanid lungs and suggest lungs with net unidirectional flow as an evolutionary pathway between tidal flow and complete unidirectional flow in multicameral lungs. Anat Rec, 2020. © 2019 American Association for Anatomy Anat Rec, 303:1768-1791, 2020. © 2019 American Association for Anatomy.

Lithophagy Prolongs Voluntary Dives in American alligators (Alligator mississippiensis).

Many vertebrates ingest stones, but the function of this behavior is not fully understood. We tested the hypothesis that lithophagy increases the duration of voluntary dives in juvenile American alligators (Alligator mississippiensis). After ingestion of granite stones equivalent to 2.5% of body weight, the average duration of dives increased by 88% and the maximum duration increased by 117%. These data are consistent with the hypothesis that gastroliths serve to increase specific gravity, and that the animals compensate by increasing lung volume, thereby diving with larger stores of pulmonary oxygen.

Bone Microvasculature Tracks Red Blood Cell Size Diminution in Triassic Mammal and Dinosaur Forerunners.

Vertebrate red blood cells (RBCs) display a range of sizes, spanning orders of magnitude in volume in different clades [1]. The importance of this size variation to diffusion during exercise is reinforced by functional links between RBC and capillary diameters [2, 3]. Small RBCs, such as those of mammals (which lack nuclei) and birds, contribute to shorter diffusion distances and permit relatively fast O2 uptake kinetics [4]. Although constraints on RBC size have been tied to the cell's need to attend capillary sizes for effective gas diffusion [3], as well as to genome size evolution [5, 6], major questions persist concerning patterns of RBC size evolution and its paleobiological significance. Here, we evaluate the relationship between RBC sizes and bone histometry and use microstructural evidence to trace their evolution in a phylogeny of extinct tetrapods. We find that several fossilizable aspects of bone microstructure, including the sizes of vascular and lacunar (cellular) spaces, provide useful indicators of RBC size in tetrapods. We also show that Triassic non-mammalian cynodonts had reduced and densely packed vascular canals identical to those of some mammals and likely accommodated smaller, more mammal-like RBCs. Reduced channel diameters accommodating smaller RBCs predated the origin of crown mammals by as much as 70 million years. This discovery offers a new proxy for the physiologic status of the mammal and avian stem groups and contextualizes the independent origins of their increased activity metabolism.

Unidirectional pulmonary airflow in vertebrates: a review of structure, function, and evolution.

Mechanisms explaining unidirectional pulmonary airflow in birds, a condition where lung gases flow in a consistent direction during both inspiration and expiration in some parts of the lung, were suggested as early as the first part of the twentieth century and unidirectional pulmonary airflow has been discovered recently in crocodilians and squamates. Our knowledge of the functional anatomy, fluid dynamics, and significance of this trait is reviewed. The preponderance of the data indicates that unidirectional airflow is maintained by means of convective inertia in inspiratory and expiratory aerodynamic valves in birds. The study of flow patterns in non-avian reptiles is just beginning, but inspiratory aerodynamic valving likely also plays an important role in controlling flow direction in these lungs. Although highly efficient counter and cross-current blood-gas exchange arrangements are possible in lungs with unidirectional airflow, very few experiments have investigated blood-gas exchange mechanisms in the bird lung and blood-gas arrangements in the lungs of non-avian reptiles are completely unknown. The presence of unidirectional airflow in non-volant ectotherms voids the traditional hypothesis that this trait evolved to supply the high aerobic demands of flight and endothermy, and there is a need for new scenarios in our understanding of lung evolution. The potential value of unidirectional pulmonary airflow for allowing economic lung gas mixing, facilitating lung gas washout, and providing for adequate gas exchange during hypoxic conditions is discussed.

Similarity of Crocodilian and Avian Lungs Indicates Unidirectional Flow Is Ancestral for Archosaurs.

Patterns of airflow and pulmonary anatomy were studied in the American alligator (Alligator mississippiensis), the black caiman (Melanosuchus niger), the spectacled caiman (Caiman crocodilus), the dwarf crocodile (Osteolaemus tetraspis), the saltwater crocodile (Crocodylus porosus), the Nile crocodile (Crocodylus niloticus), and Morelet's crocodile (Crocodylus moreletii). In addition, anatomy was studied in the Orinoco crocodile (Crocodylus intermedius). Airflow was measured using heated thermistor flow meters and visualized by endoscopy during insufflation of aerosolized propolene glycol and glycerol. Computed tomography and gross dissection were used to visualize the anatomy. In all species studied a bird-like pattern of unidirectional flow was present, in which air flowed caudad in the cervical ventral bronchus and its branches during both lung inflation and deflation and craniad in dorsobronchi and their branches. Tubular pathways connected the secondary bronchi to each other and allowed air to flow from the dorsobronchi into the ventrobronchi. No evidence for anatomical valves was found, suggesting that aerodynamic valves cause the unidirectional flow. In vivo data from the American alligator showed that unidirectional flow is present during periods of breath-holding (apnea) and is powered by the beating heart, suggesting that this pattern of flow harnesses the heart as a pump for air. Unidirectional flow may also facilitate washout of stale gases from the lung, reducing the cost of breathing, respiratory evaporative water loss, heat loss through the heat of vaporization, and facilitating crypsis. The similarity in structure and function of the bird lung with pulmonary anatomy of this broad range of crocodilian species indicates that a similar morphology and pattern of unidirectional flow were present in the lungs of the common ancestor of crocodilians and birds. These data suggest a paradigm shift is needed in our understanding of the evolution of this character. Although conventional wisdom is that unidirectional flow is important for the high activity and basal metabolic rates for which birds are renowned, the widespread occurrence of this pattern of flow in crocodilians indicates otherwise. Furthermore, these results show that air sacs are not requisite for unidirectional flow, and therefore raise questions about the function of avian air sacs.

The Evolution of Unidirectional Pulmonary Airflow.

Conventional wisdom holds that the avian respiratory system is unique because air flows in the same direction through most of the gas-exchange tubules during both phases of ventilation. However, recent studies showing that unidirectional airflow also exists in crocodilians and lizards raise questions about the true phylogenetic distribution of unidirectional airflow, the selective drivers of the trait, the date of origin, and the functional consequences of this phenomenon. These discoveries suggest unidirectional flow was present in the common diapsid ancestor and are inconsistent with the traditional paradigm that unidirectional flow is an adaptation for supporting high rates of gas exchange. Instead, these discoveries suggest it may serve functions such as decreasing the work of breathing, decreasing evaporative respiratory water loss, reducing rates of heat loss, and facilitating crypsis. The divergence in the design of the respiratory system between unidirectionally ventilated lungs and tidally ventilated lungs, such as those found in mammals, is very old, with a minimum date for the divergence in the Permian Period. From this foundation, the avian and mammalian lineages evolved very different respiratory systems. I suggest the difference in design is due to the same selective pressure, expanded aerobic capacity, acting under different environmental conditions. High levels of atmospheric oxygen of the Permian Period relaxed selection for a thin blood-gas barrier and may have resulted in the homogeneous, broncho-alveolar design, whereas the reduced oxygen of the Mesozoic selected for a heterogeneous lung with an extremely thin blood-gas barrier. These differences in lung design may explain the puzzling pattern of ecomorphological diversification of Mesozoic mammals: all were small animals that did not occupy niches requiring a great aerobic capacity. The broncho-alveolar lung and the hypoxia of the Mesozoic may have restricted these mammals from exploiting niches of large body size, where cursorial locomotion can be advantageous, as well as other niches requiring great aerobic capacities, such as those using flapping flight. Furthermore, hypoxia may have exerted positive selection for a parasagittal posture, the diaphragm, and reduced erythrocyte size, innovations that enabled increased rates of ventilation and more rapid rates of diffusion in the lung.

New insight into the evolution of the vertebrate respiratory system and the discovery of unidirectional airflow in iguana lungs.

The generally accepted framework for the evolution of a key feature of the avian respiratory system, unidirectional airflow, is that it is an adaptation for efficiency of gas exchange and expanded aerobic capacities, and therefore it has historically been viewed as important to the ability of birds to fly and to maintain an endothermic metabolism. This pattern of flow has been presumed to arise from specific features of the respiratory system, such as an enclosed intrapulmonary bronchus and parabronchi. Here we show unidirectional airflow in the green iguana, a lizard with a strikingly different natural history from that of birds and lacking these anatomical features. This discovery indicates a paradigm shift is needed. The selective drivers of the trait, its date of origin, and the fundamental aerodynamic mechanisms by which unidirectional flow arises must be reassessed to be congruent with the natural history of this lineage. Unidirectional flow may serve functions other than expanded aerobic capacity; it may have been present in the ancestral diapsid; and it can occur in structurally simple lungs.

Unidirectional pulmonary airflow patterns in the savannah monitor lizard.

The unidirectional airflow patterns in the lungs of birds have long been considered a unique and specialized trait associated with the oxygen demands of flying, their endothermic metabolism and unusual pulmonary architecture. However, the discovery of similar flow patterns in the lungs of crocodilians indicates that this character is probably ancestral for all archosaurs--the group that includes extant birds and crocodilians as well as their extinct relatives, such as pterosaurs and dinosaurs. Unidirectional flow in birds results from aerodynamic valves, rather than from sphincters or other physical mechanisms, and similar aerodynamic valves seem to be present in crocodilians. The anatomical and developmental similarities in the primary and secondary bronchi of birds and crocodilians suggest that these structures and airflow patterns may be homologous. The origin of this pattern is at least as old as the split between crocodilians and birds, which occurred in the Triassic period. Alternatively, this pattern of flow may be even older; this hypothesis can be tested by investigating patterns of airflow in members of the outgroup to birds and crocodilians, the Lepidosauromorpha (tuatara, lizards and snakes). Here we demonstrate region-specific unidirectional airflow in the lungs of the savannah monitor lizard (Varanus exanthematicus). The presence of unidirectional flow in the lungs of V. exanthematicus thus gives rise to two possible evolutionary scenarios: either unidirectional airflow evolved independently in archosaurs and monitor lizards, or these flow patterns are homologous in archosaurs and V. exanthematicus, having evolved only once in ancestral diapsids (the clade encompassing snakes, lizards, crocodilians and birds). If unidirectional airflow is plesiomorphic for Diapsida, this respiratory character can be reconstructed for extinct diapsids, and evolved in a small ectothermic tetrapod during the Palaeozoic era at least a hundred million years before the origin of birds.

The pulmonary anatomy of Alligator mississippiensis and its similarity to the avian respiratory system.

Using gross dissections and computed tomography we studied the lungs of juvenile American alligators (Alligator mississippiensis). Our findings indicate that both the external and internal morphology of the lungs is strikingly similar to the embryonic avian respiratory system (lungs + air sacs). We identified bronchi that we propose are homologous to the avian ventrobronchi (entobronchi), laterobronchi, dorsobronchi (ectobronchi), as well as regions of the lung hypothesized to be homologous to the cervical, interclavicular, anterior thoracic, posterior thoracic, and abdominal air sacs. Furthermore, we suggest that many of the features that alligators and birds share are homologous and that some of these features are important to the aerodynamic valve mechanism and are likely plesiomorphic for Archosauria.

Subglottal pressure and fundamental frequency control in contact calls of juvenile Alligator mississippiensis.

Vocalization is rare among non-avian reptiles, with the exception of the crocodilians, the sister taxon of birds. Crocodilians have a complex vocal repertoire. Their vocal and respiratory system is not well understood but appears to consist of a combination of features that are also found in the extremely vocal avian and mammalian taxa. Anatomical studies suggest that the alligator larynx is able to abduct and adduct the vocal folds, but not to elongate or shorten them, and is therefore lacking a key regulator of frequency, yet alligators can modulate fundamental frequency remarkably well. We investigated the morphological and physiological features of sound production in alligators. Vocal fold length scales isometrically across a wide range of alligator body sizes. The relationship between fundamental frequency and subglottal pressure is significant in some individuals at some isolated points, such as call onset and position of maximum fundamental frequency. The relationship is not consistent over large segments of the call. Fundamental frequency can change faster than expected by pressure changes alone, suggesting an active motor pattern controls frequency and is intrinsic to the larynx. We utilized a two-mass vocal fold model to test whether abduction and adduction could generate this motor pattern. The fine-tuned interplay between subglottal pressure and glottal adduction can achieve frequency modulations much larger than those resulting from subglottal pressure variations alone and of similar magnitude, as observed in alligator calls. We conclude that the alligator larynx represents a sound source with only two control parameters (subglottal pressure and vocal fold adduction) in contrast to the mammalian larynx in which three parameters can be altered to modulate frequency (subglottal pressure, vocal fold adduction and length/tension).

Evolution of the dinosauriform respiratory apparatus: new evidence from the postcranial axial skeleton.

Examination of the thoracic rib and vertebral anatomy of extant archosaurs indicates a relationship between the postcranial axial skeleton and pulmonary anatomy. Lung ventilation in extant crocodilians is primarily achieved with a hepatic piston pump and costal rotation. The tubercula and capitula of the ribs lie on the horizontal plane, forming a smooth thoracic "ceiling" facilitating movement of the viscera. Although the parietal pleura is anchored to the dorsal thoracic wall, the dorsal visceral pleura exhibits a greater freedom of movement. The air sac system and lungs of birds are associated with bicapitate ribs with a ventrally positioned capitular articulation, generating a rigid and furrowed rib cage that minimizes dorsoventral changes in volume in the dorsal thorax. The thin walled bronchi are kept from collapsing by fusion of the lung to the thorax on all sides. Data from this study suggest a progression from a dorsally rigid, heterogeneously partitioned, multichambered lung in basal dinosauriform archosaurs towards the small entirely rigid avian-style lung that was likely present in saurischian dinosaurs, consistent with a constant volume cavum pulmonale, thin walled parabronchi, and distinct air sacs. There is no vertebral evidence for a crocodilian hepatic piston pump in any of the taxa reviewed. The evidence for both a rigid lung and unidirectional airflow in dinosauriformes raises the possibility that these animals had a highly efficient lung relative to other Mesozoic vertebrates, which may have contributed to their successful radiation during this time period.

On the evolution of arterial vascular patterns of tetrapods.

The factors that explain the diverse arrangement of the major arteries of tetrapods are not known. Here, I aim to illuminate some of the underpinnings of these patterns. I review the variation in the sauropsid left, right, and dorsal aortae regarding the origin of the gastrointestinal blood vessels and the relative diameters of left and right aortae where they join together to form the dorsal aorta. I focus on these features because the quality of blood that flows through these aortae can vary depending on the state of cardiac shunting and the size of the vessel can provide insight into the quantity of blood borne by the vessels. I then place the information in a phyletic, historical, and ecological context. The plesiomorphic pattern is for the gastrointestinal vessels to arise as segmental arteries from the dorsal aorta, which is formed from the confluence of left and right aortae with similar diameters. The pattern is well conserved with only two major variations. First, in several clades of reptiles (testudines, crocodilians, lizards of the genera Varanus and Hydrosaurus) a substantial portion of the gastrointestinal arteries arises from the left aorta, leaving the diameter of the left aorta smaller than the right at their confluence. I hypothesize that this vascular arrangement facilitates growth by allowing more alkaline blood to flow to the somatic (body wall) and appendicular circulations, which may promote bone deposition and inhibit resorption, whereas hypercapnic, acidic blood flows to the digestive viscera, which may provide CO(2) as a substrate for the synthesis of gastric acid, bicarbonate, fatty acids, glutamine, purine rings, as well as glucose from lactate. Second, in some snakes and lizards with snake-like body forms, such as Amphisbaenidae, the diameters of left and right aortae are asymmetrical at their confluence with the left aorta exceeding the right, but in members of the amphibian order Gymnophiona the right generally exceeds the left. This condition is associated with asymmetrical development of the lungs.

The provenance of alveolar and parabronchial lungs: insights from paleoecology and the discovery of cardiogenic, unidirectional airflow in the American alligator (Alligator mississippiensis).

Birds and mammals evolved greater aerobic abilities than their common ancestor had. This required expansion of the cardiopulmonary system's capacity for gas exchange, but while directional selection for this expanded capacity resulted in extremely similar avian and mammalian hearts, strikingly different lungs arose, and the reasons for this divergence in lung morphology are not understood. In birds, gas exchange occurs in the lungs as air moves through small tubes (parabronchi) in one direction; in mammals, air flows tidally into and out of the alveoli. Here, I present a scenario for the origin of both the alveolar and parabronchial lungs that explains when and how they could have arisen by a gradual sequence of steps. I argue that (1) the alveolar lung evolved in the late Paleozoic, when high levels of atmospheric oxygen relaxed selection for a thin blood-gas barrier within the lung; (2) unidirectional flow originated in the ectothermic ancestral archosaur, the forerunner of birds and crocodilians, to enable the heart to circulate pulmonary gases during apnea. This hypothesis would be supported by a demonstration of unidirectional flow in the lungs of crocodilians, the extant sister taxon of birds. Airflow in the lungs of juvenile alligators was measured during apnea using dual thermistor flowmeters, and cardiac activity was measured with electrocardiography. Coincident with each heartbeat, a pulse of air flowed in the pulmonary conduit under study with a bias in the direction of movement, yielding a net unidirectional flow. These data suggest the internal structures requisite for unidirectional flow were present in the common ancestors of birds and crocodilians and may have preadapted the lungs of archosaurs to function advantageously during the oxygen-poor period of the early Mesozoic.

Leptin integrates vertebrate evolution: from oxygen to the blood-gas barrier.

The following are the proceedings of a symposium held at the Second International Congress for Respiratory Science in Bad Honnef, Germany. The goals of the symposium were to delineate the blood-gas barrier phenotype across vertebrate species; to delineate the interrelationship between the evolution of the blood-gas barrier, locomotion and metabolism; to introduce the selection pressures for the evolution of the surfactant system as a key to understanding the physiology of the blood-gas barrier; to introduce the lung lipofibroblast and its product, leptin, which coordinately regulates pulmonary surfactant, type IV collagen in the basement membrane and host defense, as the cell-molecular site of selection pressure for the blood-gas barrier; to drill down to the gene regulatory network(s) involved in leptin signaling and the blood-gas barrier phenotype; to extend the relationship between leptin and the blood-gas barrier to diving mammals.

Unidirectional airflow in the lungs of alligators.

The lungs of birds move air in only one direction during both inspiration and expiration through most of the tubular gas-exchanging bronchi (parabronchi), whereas in the lungs of mammals and presumably other vertebrates, air moves tidally into and out of terminal gas-exchange structures, which are cul-de-sacs. Unidirectional flow purportedly depends on bellowslike ventilation by air sacs and may have evolved to meet the high aerobic demands of sustained flight. Here, we show that air flows unidirectionally through parabronchi in the lungs of the American alligator, an amphibious ectotherm without air sacs, which suggests that this pattern dates back to the basal archosaurs of the Triassic and may have been present in their nondinosaur descendants (phytosaurs, aetosaurs, rauisuchians, crocodylomorphs, and pterosaurs) as well as in dinosaurs.

The importance of the M. diaphragmaticus to the duration of dives in the American alligator (Alligator mississippiensis).

We tested the hypothesis that the crocodilian M. diaphragmaticus extends the duration of dives by disabling this muscle in a group of juvenile American alligators and comparing the duration of their dives to the duration of the dives of animals in which the muscle remained intact. We studied the groups while they were fasting, 1h after they had eaten a meal with a density that was either greater or less than water, and at 22 and 28 degrees C. We found that the duration of dives was longer for the control group compared to animals without a functional M. diaphragmaticus, both when fasting and after having consumed the denser meal. The warmer temperature significantly decreased the duration of the dives for both groups, as did eating in general. The preponderance of these data indicates that transection of the diaphragmaticus reduced time spent underwater, but the mechanism for this reduction is unknown. Lack of a functional diaphragmaticus could impair the animals' ability to inspire sufficient air to support the dive, but we think this explanation is unlikely because both groups were able to float at the surface and thus needed to reduce lung volume to dive. An alternative explanation is that the effect on duration is a consequence of an impairment of a locomotor rather than ventilatory function of the muscle.