An XROMM Study of Food Transport and Swallowing in Channel Catfish.

Most predatory ray-finned fishes swallow their food whole, which can pose a significant challenge, given that prey items can be half as large as the predators themselves. How do fish transport captured food from the mouth to the stomach? Prior work indicates that, in general, fish use the pharyngeal jaws to manipulate food into the esophagus, where peristalsis is thought to take over. We used X-Ray Reconstruction of Moving Morphology to track prey transport in channel catfish (Ictalurus punctatus). By reconstructing the 3D motions of both the food and the catfish, we were able to track how the catfish move food through the head and into the stomach. Food enters the oral cavity at high velocities as a continuation of suction and stops in the approximate location of the branchial basket before moving in a much slower, more complex path toward the esophagus. This slow phase coincides with little motion in the head and no substantial mouth opening or hyoid depression. Once the prey is in the esophagus, however, its transport is surprisingly tightly correlated with gulping motions (hyoid depression, girdle retraction, hypaxial shortening, and mouth opening) of the head. Although the transport mechanism itself remains unknown, to our knowledge, this is the first description of synchrony between cranial expansion and esophageal transport in a fish. Our results provide direct evidence of prey transport within the esophagus and suggest that peristalsis may not be the sole mechanism of esophageal transport in catfish.

Bifunctional Role of the Sternohyoideus Muscle During Suction Feeding in Striped Surfperch, Embiotoca lateralis.

In ray-finned fishes, the sternohyoideus (SH) is among the largest muscles in the head region and, based on its size, can potentially contribute to the overall power required for suction feeding. However, the function of the SH varies interspecifically. In largemouth bass (Micropterus salmoides) and several clariid catfishes, the SH functions similarly to a stiff ligament. In these species, the SH remains isometric and transmitts power from the hypaxial musculature to the hyoid apparatus during suction feeding. Alternatively, the SH can shorten and contribute muscle power during suction feeding, a condition observed in the bluegill sunfish (Lepomis macrochirus) and one clariid catfish. An emerging hypothesis centers on SH muscle size as a predictor of function: in fishes with a large SH, the SH shortens during suction feeding, whereas in fish with a smaller SH, the muscle may remain isometric. Here, we studied striped surfperch (Embiotoca lateralis), a species in which the SH is relatively large at 8.8% of axial muscle mass compared with 4.0% for L. macrochirus and 1.7% for M. salmoides, to determine whether the SH shortens during suction feeding and is, therefore, bifunctional-both transmitting and generating power-or remains isometric and only transmits power. We measured skeletal kinematics of the neurocranium, urohyal, and cleithrum with Video Reconstruction of Moving Morphology, along with muscle strain and shortening velocity in the SH and epaxial muscles, using a new method of 3D external marker tracking. We found mean SH shortening during suction feeding strikes (n = 22 strikes from four individual E. lateralis) was 7.2 ± 0.55% (±SEM) of initial muscle length. Mean peak speed of shortening was 4.9 ± 0.65 lengths s-1, and maximum shortening speed occurred right around peak gape when peak power is generated in suction feeding. The cleithrum of E. lateralis retracts and depresses but the urohyal retracts and depresses even more, a strong indicator of a bifunctional SH capable of not only generating its own power but also transmitting hypaxial power to the hyoid. While power production in E. lateralis is still likely dominated by the axial musculature, since even the relatively large SH of E. lateralis is only 8.8% of axial muscle mass, the SH may contribute a meaningful amount of power given its continual shortening just prior to peak gape across all strikes. These results support the finding from other groups of fishes that a large SH muscle, relative to axial muscle mass, is likely to both generate and transmit power during suction feeding.

Multiple Degrees of Freedom in the Fish Skull and Their Relation to Hydraulic Transport of Prey in Channel Catfish.

Fish perform many complex manipulation behaviors without hands or flexible muscular tongues, instead relying on more than 20 movable skeletal elements in their highly kinetic skulls. How fish use their skulls to accomplish these behaviors, however, remains unclear. Most previous mechanical models have represented the fish skull using one or more planar four-bar linkages, which have just a single degree of freedom (DoF). In contrast, truncated-cone hydrodynamic models have assumed up to five DoFs. In this study, we introduce and validate a 3D mechanical linkage model of a fish skull that incorporates the pectoral girdle and mandibular and hyoid arches. We validate this model using an in vivo motion dataset of suction feeding in channel catfish and then use this model to quantify the DoFs in the fish skull, to categorize the motion patterns of the cranial linkage during feeding, and to evaluate the association between these patterns and food motion. We find that the channel catfish skull functions as a 17-link, five-loop parallel mechanism. Despite having 19 potential DoFs, we find that seven DoFs are sufficient to describe most of the motion of the cranial linkage, consistent with the fish skull functioning as a multi-DoF, manipulation system. Channel catfish use this linkage to generate three different motion patterns (rostrocaudal wave, caudorostral wave, and compressive wave), each with its own associated food velocity profile. These results suggest that biomechanical manipulation systems must have a minimum number of DoFs to effectively control objects, whether in water or air.

Rib Motions Don't Completely Hinge on Joint Design: Costal Joint Anatomy and Ventilatory Kinematics in a Teiid Lizard, Salvator merianae.

Rib rotations contribute to lung ventilation in most extant amniotes. These rotations are typically described as bucket-handle rotation about a dorsoventral axis, caliper rotation about a craniocaudal axis, and pump-handle rotation about a mediolateral axis. A synapomorphy for Lepidosauria is single-headed costovertebral articulations derived from the ancestral double-headed articulations of most amniotes. With a single articular surface, the costovertebral joints of squamates have the potential to rotate with three degrees-of-freedom (DOFs), but considerable variation exists in joint shape. We compared the costovertebral morphology of the Argentine black and white tegu, Salvator merianae, with the green iguana, Iguana iguana, and found that the costovertebral articulations of I. iguana were hemispherical, while those of S. merianae were dorsoventrally elongated and hemiellipsoidal. We predicted that the elongate joints in S. merianae would permit bucket-handle rotations while restricting caliper and pump-handle rotations, relative to the rounded joints of I. iguana. We used X-ray reconstruction of moving morphology to quantify rib rotations during breathing in S. merianae for comparison with prior work in I. iguana. Consistent with our hypothesis, we found less caliper motion in S. merianae than in I. iguana, but unexpectedly found similar pump-handle magnitudes in each species. The dorsoventrally elongate costovertebral morphology of S. merianae may provide passive rib support to reduce the conflict between locomotion and ventilation. Moreover, the observation of multiple DOFs during rib rotations in both species suggests that permissive costovertebral morphology may be more related to the biological roles of ribs outside of ventilation and help explain the evolution of this trait.

Eye Movements in Frogs and Salamanders-Testing the Palatal Buccal Pump Hypothesis.

In frogs and salamanders, movements of the eyeballs in association with an open palate have often been proposed to play a functional role in lung breathing. In this "palatal buccal pump," the eyeballs are elevated during the lowering of the buccal floor to suck air in through the nares, and the eyeballs are lowered during elevation of the buccal floor to help press air into the lungs. Here, we used X-Ray Reconstruction of Moving Morphology to investigate eye movements during lung breathing and feeding in bullfrogs and axolotls. Our data do not show eye movements that would be in accordance with the palatal buccal pump. On the contrary, there is a small passive elevation of the eyeballs when the buccal floor is raised. Inward drawing of the eyeballs occurs only during body motion and for prey transport in bullfrogs, but this was not observed in axolotls. Each eye movement in bullfrogs has a vertical, a mediolateral, and an anteroposterior component. Considering the surprisingly weak posterior motion component of the eyeballs, their main role in prey transport might be fixing the prey by pressing it against the buccal floor. The retraction of the buccal floor would then contribute to the posterior push of the prey. Because our study provides no evidence for a palatal buccal pump in frogs and salamanders, there is also no experimental support for the idea of a palatal buccal pump in extinct temnospondyl amphibians, in contrast to earlier suggestions.

Movimientos oculares en ranas y salamandras: prueba de la hipótesis de la bomba bucal palatina (Eye movements in frogs and salamanders ­ testing the palatal buccal pump hypothesis) En ranas y salamandras, los movimientos oculares asociados con el paladar abierto a menudo se ha propuesto que desempeñan un papel funcional en la respiración pulmonar. En esta "bomba bucal palatina", los globos oculares se elevan durante la bajada del piso bucal para inhalar por las narinas, y los globos oculares se bajan durante la elevación del piso bucal para ayudar a presionar el aire hacia los pulmones. Aquí utilizamos la Reconstrucción de Rayos X de la Morfología en Movimiento para investigar los movimientos oculares durante la respiración pulmonar y la alimentación en ranas mugidoras y ajolotes. Nuestros datos no muestran movimientos oculares que estarían de acuerdo con la bomba bucal palatina. Por el contrario, hay una pequeña elevación pasiva de los globos oculares cuando se eleva el suelo bucal. La retracción interna de los globos oculares ocurre solo durante el movimiento del cuerpo y para el transporte de presas en las ranas mugidoras, pero esto no se observó en los ajolotes. Cada movimiento ocular en las ranas mugidoras tiene un componente vertical, mediolateral y anteroposterior. Considerando el componente de movimiento posterior sorprendentemente pequeño de los globos oculares, su función principal en el transporte de presas podría ser la fijación de la presa presionándola contra el suelo bucal. La retracción del suelo bucal contribuiría entonces al empuje posterior de la presa. Debido a que nuestro estudio no proporciona evidencia de una bomba bucal palatina en ranas y salamandras, tampoco hay apoyo experimental para la idea de una bomba bucal palatina en anfibios temnospóndilos extintos, en contraste con sugerencias anteriores. translated to Spanish by Y.E. Jimenez (yordano_jimenez@brown.edu).

Heterochrony in the evolution of Trinidadian guppy offspring size: maturation along a uniform ontogenetic trajectory.

The size and maturity of Trinidadian guppy (Poecilia reticulata) offspring vary among populations adapted to environments of differential predation. Guppy offspring born to low-predation, high-competition environments are larger and more mature than their high-predation ancestors. Here we ask: what specific changes in developmental or birth timing occur to produce the larger, more mature neonates? We collected specimens across the perinatal window of development from five populations and quantified musculoskeletal maturation. We found that all populations undergo similar ontogenetic trajectories in skeletal and muscle acquisition; the only difference among populations is when neonates emerge along the trajectory. The smallest neonates are born with 20% of their skeleton ossified, whereas the largest neonates are born with over 70% of their skeleton ossified. The area of the major jaw-closing muscle is relatively larger in larger offspring, scaling with length as L2.5 The size range over which offspring are birthed among populations sits along the steepest part of the size-maturity relationship, which provides a large marginal increase in fitness for the high-competition female. Because of the functional effects of producing more mature offspring at birth, offspring size may be the first and most critical life-history trait selected upon in highly competitive environments.

Morphological and functional maturity of the oral jaws covary with offspring size in Trinidadian guppies.

Large size of individual offspring is routinely selected for in highly competitive environments, such as in low-predation populations of the Trinidadian guppy (Poecilia reticulata). Large guppy offspring outcompete their smaller conspecifics, but the functional mechanisms underlying this advantage are unknown. We measured jaw kinematics during benthic feeding and cranial musculoskeletal morphologies in neonates and juveniles from five populations of Trinidadian guppy and found that both kinematics and morphologies vary substantially with neonatal size. Rotation at the intramandibular joint (IMJ), but not the quadratomandibular joint (QMJ), increases with size among guppy offspring, from 11.7° in the smallest neonates to 22.9° in the largest neonates. Ossification of the cranial skeleton varies from 20% in the smallest neonates to 90% in the largest. Relative to standard length (SL; jaw tip to caudal fin base distance), the surface area of jaw-closing musculature scales with positive allometry (SL2.72) indicating that muscle growth outpaces body growth. Maximum gape also scales with positive allometry (SL1.20), indicating that larger neonates are capable of greater jaw excursions. These findings indicate that size is not the sole adaptive benefit to producing larger offspring; maturation provides a potential functional mechanism underlying the competitive advantage of large offspring size among Trinidadian guppies.

X-ray motion analysis of the vertebral column during the startle response in striped bass, Morone saxatilis.

Whole-body stiffness has a substantial impact on propulsive wave speed during axial undulatory locomotion in fishes. The connective tissues of the vertebral column may contribute to body stiffness, but without mechanical and kinematic analysis it is unclear whether the in vivo range of motion of intervertebral joints (IVJs) is great enough to stress IVJ tissues, thus generating stiffness. The present study used 2D videoradiography and 3D X-ray reconstruction of moving morphology (XROMM) to quantify vertebral kinematics during the startle response in striped bass (Morone saxatilis). X-ray video revealed two distinct patterns of bending: pattern I begins in the abdominal region and then proceeds to maximum IVJ angles in the caudal region, whereas pattern II begins in the cervical region and proceeds to maximum IVJ angles in the abdominal and then the caudal joints. In pattern II bends, the cervical joints exhibit a greater in vivo range of motion than previously reported in other species. XROMM analysis of caudal IVJs suggests primarily lateral bending: mean axial and dorsoventral rotations were less than 2 deg and inconsistent across 51 sequences analyzed from five individuals, whereas mean maximum lateral bending angles were 10.4±3.57 deg. These angles, combined with previous investigations of mechanical properties, reveal that the maximum angles all occur within the neutral zone of bending, indicating that little stress is experienced about the joint. This suggests that the IVJs of striped bass are quite compliant and likely do not contribute significantly to whole-body stiffness or elastic recoil during swimming in vivo.

Regional variation in the mechanical properties of the vertebral column during lateral bending in Morone saxatilis.

Unlike mammalian, disc-shaped intervertebral joints (IVJs), the IVJs in fishes are biconid structures, filled with fluid and thought to act as hydrostatic hinge joints during swimming. However, it remains unclear which IVJ structures are dominant in mechanical resistance to forces in fishes, and whether variation in these tissues might impact the function of the vertebral column along its length. Here, we measured the dynamic mechanical behaviour of IVJs from striped bass, Morone saxatilis. During lateral bending, angular stiffness was significantly lower in the caudal and cervical regions, relative to the abdominal region. The neutral zone, defined as the range of motion (ROM) at bending moments less than 0.001 Nm, was longer in the caudal relative to the abdominal IVJs. Hysteresis was 30-40% in all regions, suggesting that IVJs may play a role in energy dissipation during swimming. Cutting the vertical septum had no statistically significant effect, but cutting the encapsulating tissues caused a sharp decline in angular stiffness and a substantial increase in ROM and hysteresis. We conclude that stiffness decreases and ROM increases from cranial to caudal in striped bass, and that the encapsulating tissues play a prominent role in mechanical variation along the length of the vertebral column.

Regional variation in morphology of vertebral centra and intervertebral joints in striped bass, Morone saxatilis.

The vertebral column of fishes has traditionally been divided into just two distinct regions, abdominal and caudal. Recently, however, developmental, morphological, and mechanical investigations have brought this traditional regionalization scheme into question. Alternative regionalization schema advocate the division of the abdominal vertebrae into cervical, abdominal, and in some cases, transitional regions. Here, we investigate regional variation at the level of the vertebrae and intervertebral joint (IVJ) tissues in the striped bass, Morone saxatilis. We use gross dissection, histology, and polarized light imaging to quantify vertebral height, width, length, IVJ length, IVJ tissue volume and cross-sectional area, and vertical septum fiber populations, and angles of insertion. Our results reveal regional differences between the first four (most rostral) abdominal vertebrae and IVJs and the next six abdominal vertebrae and IVJs, supporting the recognition of a distinct cervical region. We found significant variation in vertebral length, width, and height from cranial to caudal. In addition, we see a significant decline in the volume of notochordal cells and the cross-sectional area of the fibrous sheath from cranial to caudal. Further, polarized light imaging revealed four distinct fiber populations within the vertical septum in the cervical and abdominal regions in contrast with just one fiber population found in the caudal region. Measurement of the insertion angles of these fiber populations revealed significant differences between the cervical and abdominal regions. Differences in vertebral, IVJ, and vertical septum morphology all predict greater range of motion and decreased stiffness in the caudal region of the fish compared with the cervical and abdominal regions.

Twisting and bending: the functional role of salamander lateral hypaxial musculature during locomotion.

The function of the lateral hypaxial muscles during locomotion in tetrapods is controversial. Currently, there are two hypotheses of lateral hypaxial muscle function. The first, supported by electromyographic (EMG) data from a lizard (Iguana iguana) and a salamander (Dicamptodon ensatus), suggests that hypaxial muscles function to bend the body during swimming and to resist long-axis torsion during walking. The second, supported by EMG data from lizards during relatively high-speed locomotion, suggests that these muscles function primarily to bend the body during locomotion, not to resist torsional forces. To determine whether the results from D. ensatus hold for another salamander, we recorded lateral hypaxial muscle EMGs synchronized with body and limb kinematics in the tiger salamander Ambystoma tigrinum. In agreement with results from aquatic locomotion in D. ensatus, all four layers of lateral hypaxial musculature were found to show synchronous EMG activity during swimming in A. tigrinum. Our findings for terrestrial locomotion also agree with previous results from D. ensatus and support the torsion resistance hypothesis for terrestrial locomotion. We observed asynchronous EMG bursts of relatively high intensity in the lateral and medial pairs of hypaxial muscles during walking in tiger salamanders (we call these 'alpha-bursts'). We infer from this pattern that the more lateral two layers of oblique hypaxial musculature, Mm. obliquus externus superficialis (OES) and obliquus externus profundus (OEP), are active on the side towards which the trunk is bending, while the more medial two layers, Mm. obliquus internus (OI) and transversus abdominis (TA), are active on the opposite side. This result is consistent with the hypothesis proposed for D. ensatus that the OES and OEP generate torsional moments to counteract ground reaction forces generated by forelimb support, while the OI and TA generate torsional moments to counteract ground reaction forces from hindlimb support. However, unlike the EMG pattern reported for D. ensatus, a second, lower-intensity burst of EMG activity ('beta-burst') was sometimes recorded from the lateral hypaxial muscles in A. tigrinum. As seen in other muscle systems, these beta-bursts of hypaxial muscle coactivation may function to provide fine motor control during locomotion. The presence of asynchronous, relatively high-intensity alpha-bursts indicates that the lateral hypaxial muscles generate torsional moments during terrestrial locomotion, but it is possible that the balance of forces from both alpha- and beta-bursts may allow the lateral hypaxial muscles to contribute to lateral bending of the body as well.

Biomechanics of the movable pretarsal adhesive organ in ants and bees.

Hymenoptera attach to smooth surfaces with a flexible pad, the arolium, between the claws. Here we investigate its movement in Asian weaver ants (Oecophylla smaragdina) and honeybees (Apis mellifera). When ants run upside down on a smooth surface, the arolium is unfolded and folded back with each step. Its extension is strictly coupled with the retraction of the claws. Experimental pull on the claw-flexor tendon revealed that the claw-flexor muscle not only retracts the claws, but also moves the arolium. The elicited arolium movement comprises (i) about a 90 degrees rotation (extension) mediated by the interaction of the two rigid pretarsal sclerites arcus and manubrium and (ii) a lateral expansion and increase in volume. In severed legs of O. smaragdina ants, an increase in hemolymph pressure of 15 kPa was sufficient to inflate the arolium to its full size. Apart from being actively extended, an arolium in contact also can unfold passively when the leg is subject to a pull toward the body. We propose a combined mechanical-hydraulic model for arolium movement: (i) the arolium is engaged by the action of the unguitractor, which mechanically extends the arolium; (ii) compression of the arolium gland reservoir pumps liquid into the arolium; (iii) arolia partly in contact with the surface are unfolded passively when the legs are pulled toward the body; and (iv) the arolium deflates and moves back to its default position by elastic recoil of the cuticle.

Buccal oscillation and lung ventilation in a semi-aquatic turtle, Platysternon megacephalum.

Movements of the hyobranchial apparatus in reptiles and amphibians contribute to many behaviors including feeding, lung ventilation, buccopharyngeal respiration, thermoregulation, olfaction, defense and display. In a semi-aquatic turtle, Platysternon megacephalum, x-ray video and airflow measurements from blowhole pneumotachography show no evidence that above water hyobranchial movements contribute to lung inflation, as in the buccal or gular pump of amphibians and some lizards. Instead, hyobranchial movements produce symmetrical oscillations of air into and out of the buccal cavity. The mean tidal volume of these buccal oscillations is 7.8 times smaller than the mean tidal volume of lung ventilation (combined mean for four individuals). Airflow associated with buccal oscillation occurs in the sequence of inhalation followed by exhalation, distinguishing it from lung ventilation which occurs as exhalation followed by inhalation. No fixed temporal relationship between buccal oscillation and lung ventilation was observed. Periods of ventilation often occur without buccal oscillation and buccal oscillation sometimes occurs without lung ventilation. When the two behaviors occur together, the onset of lung ventilation often interrupts buccal oscillation. The initiation of lung ventilation was found to occur in all phases of the buccal oscillation cycle, suggesting that the neural control mechanisms of the two behaviors are not coupled. The pattern of occurrence of both buccal oscillation and lung ventilation was found to vary over time with no obvious effect of activity levels.

Patterns of genome size evolution in tetraodontiform fishes.

We used flow cytometry to measure genome size in 15 species from seven families and subfamilies of tetraodontiform fishes. Previous studies have found that smooth pufferfishes (Tetraodontidae) have the smallest genome of any vertebrate measured to date (0.7-1.0 picograms diploid). We found that spiny pufferfishes (Diodontidae, sister group to the smooth puffers) possess a genome that is about two times larger (1.6-1.8 pg). Mola mola, a member of the sister group to Diodontidae and Tetraodontidae, also has a relatively large genome (1.7 pg). Parsimony analysis of this pattern indicates that the plesiomorphic condition for Molidae (Diodontidae, Tetraodontidae) is a genome size of 1.6-1.8 pg, and that tiny genome size is a derived character unique to smooth puffers. However, an alternative explanation is that the ancestor of Tetraodontidae acquired a heritable tendency toward decreasing genome size, such as a new or modified deletion mechanism, and genome size in all of the tetraodontid lineages has been decreasing in parallel since the split from Diodontidae. Small genome size (1.1-1.3 pg) also appears to have evolved independently in some members of Balistoidea (triggerfishes and filefishes) within Tetraodontiformes.

Mechanics of lung ventilation in a post-metamorphic salamander, Ambystoma Tigrinum.

The mechanics of lung ventilation in frogs and aquatic salamanders has been well characterized, whereas lung ventilation in terrestrial-phase (post-metamorphic) salamanders has received little attention. We used electromyography (EMG), X-ray videography, standard videography and buccal and body cavity pressure measurements to characterize the ventilation mechanics of adult (post-metamorphic) tiger salamanders (Ambystoma tigrinum). Three results emerged: (i) under terrestrial conditions or when floating at the surface of the water, adult A. tigrinum breathed through their nares using a two-stroke buccal pump; (ii) in addition to this narial two-stroke pump, adult tiger salamanders also gulped air in through their mouths using a modified two-stroke buccal pump when in an aquatic environment; and (iii) exhalation in adult tiger salamanders is active during aquatic gulping breaths, whereas exhalation appears to be passive during terrestrial breathing at rest. Active exhalation in aquatic breaths is indicated by an increase in body cavity pressure during exhalation and associated EMG activity in the lateral hypaxial musculature, particularly the M. transversus abdominis. In terrestrial breathing, no EMG activity in the lateral hypaxial muscles is generally present, and body cavity pressure decreases during exhalation. In aquatic breaths, tidal volume is larger than in terrestrial breaths, and breathing frequency is much lower (approximately 1 breath 10 min(-)(1 )versus 4-6 breaths min(-)(1)). The use of hypaxial muscles to power active exhalation in the aquatic environment may result from the need for more complete exhalation and larger tidal volumes when breathing infrequently. This hypothesis is supported by previous findings that terrestrial frogs ventilate their lungs with small tidal volumes and exhale passively, whereas aquatic frogs and salamanders use large tidal volumes and and exhale actively.

Morphological variation of hypaxial musculature in salamanders (Lissamphibia: caudata).

Despite the acknowledged importance of the locomotory and respiratory functions associated with hypaxial musculature in salamanders, variation in gross morphology of this musculature has not been documented or evaluated within a phylogenetic or ecological context. In this study, we characterize and quantify the morphological variation of lateral hypaxial muscles using phylogenetically and ecologically diverse salamander species from eight families: Ambystomatidae (Ambystoma tigrinum), Amphiumidae (Amphiuma tridactylum), Cryptobranchidae (Cryptobranchus alleganiensis), Dicamptodontidae (Dicamptodon sp.), Plethodontidae (Gyrinophilus porphyriticus), Proteidae (Necturus maculosus), Salamandridae (Pachytriton sp.), and Sirenidae (Siren lacertina). For the lateral hypaxial musculature, we document 1) the presence or absence of muscle layers, 2) the muscle fiber angles of layers at mid-trunk, and 3) the relative dorsoventral positions and cross-sectional areas of muscle layers. Combinations of two, three, or four layers are observed. However, all species retain at least two layers with opposing fiber angles. The number of layers and the presence or absence of layers vary within species (Necturus maculosus and Siren lacertina), within genera (e.g., Triturus), and within families. No phylogenetic pattern in the number of layers can be detected with a family-level phylogeny. Fiber angle variation of hypaxial muscles is considerable: fiber angles of the M. obliquus externus range from 20-80 degrees; M. obliquus internus, 14-34 degrees; M. transversus abdominis, 58-80 degrees (acute angles measured relative to the horizontal septum). Hypaxial musculature comprises 17-37% of total trunk cross-sectional area. Aquatic salamanders show relatively larger total cross-sectional hypaxial area than salamanders that are primarily terrestrial.

Contribution of gular pumping to lung ventilation in monitor lizards.

A controversial hypothesis has proposed that lizards are subject to a speed-dependent axial constraint that prevents effective lung ventilation during moderate- and high-speed locomotion. This hypothesis has been challenged by results demonstrating that monitor lizards (genus Varanus) experience no axial constraint. Evidence presented here shows that, during locomotion, varanids use a positive pressure gular pump to assist lung ventilation. Disabling the gular pump reveals that the axial constraint is present in varanids but it is masked by gular pumping under normal conditions. These findings support the prediction that the axial constraint may be found in other tetrapods that breathe by costal aspiration and locomote with a lateral undulatory gait.

Mechanics of lung ventilation in a larval salamander Ambystoma tigrinum.

The larval stage of the tiger salamander Ambystoma tigrinum is entirely aquatic, but the larvae rely on their lungs for a large proportion of their oxygen uptake. X-ray video and pressure measurements from the buccal and body cavities demonstrate that the larvae inspire using a two-stroke buccal pump and exhale actively by contracting the hypaxial musculature to increase body pressure. Larvae begin a breath by expanding the buccal cavity to draw in air through the mouth, while simultaneously exhaling air from the lungs to mix with the fresh air in the buccal cavity. The mouth then closes, and the buccal cavity compresses to pump a portion of the mixture into the lungs. The remaining air in the buccal cavity is then released as bubbles from the mouth and gill slits. Ventilatory volumes estimated from X-ray video records indicate that approximately 80% of the air pumped into the lungs is fresh air and 20% is previously expired air. Exhalation in larval tiger salamanders is active, powered by contraction of all four layers of lateral hypaxial musculature. Electromyography indicates that the transverse abdominis (TA) muscle is active for the longest duration and shows the highest-amplitude activity, but the external oblique superficialis, the external oblique profundus and the internal oblique also show consistent, low-level activity. The finding that the TA muscle is active during exhalation in larval tiger salamanders contributes to a growing body of evidence that the use of the TA for exhalation is a primitive character for tetrapods.