The anatomy of the head muscles in caecilians (Amphibia: Gymnophiona): Variation in relation to phylogeny and ecology?

In limbless fossorial vertebrates such as caecilians (Gymnophiona), head-first burrowing imposes severe constraints on the morphology and overall size of the head. As such, caecilians developed a unique jaw-closing system involving the large and well-developed m. interhyoideus posterior, which is positioned in such a way that it does not significantly increase head diameter. Caecilians also possess unique muscles among amphibians. Understanding the diversity in the architecture and size of the cranial muscles may provide insights into how a typical amphibian system was adapted for a head-first burrowing lifestyle. In this study, we use dissection and non-destructive contrast-enhanced micro-computed tomography (µCT) scanning to describe and compare the cranial musculature of 13 species of caecilians. Our results show that the general organization of the head musculature is rather constant across extant caecilians. However, the early-diverging Rhinatrema bivittatum mainly relies on the 'ancestral' amphibian jaw-closing mechanism dominated by the m. adductores mandibulae, whereas other caecilians switched to the use of the derived dual jaw-closing mechanism involving the additional recruitment of the m. interhyoideus posterior. Additionally, the aquatic Typhlonectes show a greater investment in hyoid musculature than terrestrial caecilians, which is likely related to greater demands for ventilating their large lungs, and perhaps also an increased use of suction feeding. In addition to three-dimensional interactive models, our study provides the required quantitative data to permit the generation of accurate biomechanical models allowing the testing of further functional hypotheses.

Is vertebral shape variability in caecilians (Amphibia: Gymnophiona) constrained by forces experienced during burrowing?

Caecilians are predominantly burrowing, elongate, limbless amphibians that have been relatively poorly studied. Although it has been suggested that the sturdy and compact skulls of caecilians are an adaptation to their head-first burrowing habits, no clear relationship between skull shape and burrowing performance appears to exist. However, the external forces encountered during burrowing are transmitted by the skull to the vertebral column, and, as such, may impact vertebral shape. Additionally, the muscles that generate the burrowing forces attach onto the vertebral column and consequently may impact vertebral shape that way as well. Here, we explored the relationships between vertebral shape and maximal in vivo push forces in 13 species of caecilian amphibians. Our results show that the shape of the two most anterior vertebrae, as well as the shape of the vertebrae at 90% of the total body length, is not correlated with peak push forces. Conversely, the shape of the third vertebrae, and the vertebrae at 20% and 60% of the total body length, does show a relationship to push forces measured in vivo. Whether these relationships are indirect (external forces constraining shape variation) or direct (muscle forces constraining shape variation) remains unclear and will require quantitative studies of the axial musculature. Importantly, our data suggest that mid-body vertebrae may potentially be used as proxies to infer burrowing capacity in fossil representatives.

Regional differences in vertebral shape along the axial skeleton in caecilians (Amphibia: Gymnophiona).

Caecilians are elongate, limbless and annulated amphibians that, as far as is known, all have an at least partly fossorial lifestyle. It has been suggested that elongate limbless vertebrates show little morphological differentiation throughout the postcranial skeleton. However, relatively few studies have explored the axial skeleton in limbless tetrapods. In this study, we used µCT data and three-dimensional geometric morphometrics to explore regional differences in vertebral shape across a broad range of caecilian species. Our results highlight substantial differences in vertebral shape along the axial skeleton, with anterior vertebrae being short and bulky, whereas posterior vertebrae are more elongated. This study shows that despite being limbless, elongate tetrapods such as caecilians still show regional heterogeneity in the shape of individual vertebrae along the vertebral column. Further studies are needed, however, to understand the possible causes and functional consequences of the observed variation in vertebral shape in caecilians.

The relationship between head shape, head musculature and bite force in caecilians (Amphibia: Gymnophiona).

Caecilians are enigmatic limbless amphibians that, with a few exceptions, all have an at least partly burrowing lifestyle. Although it has been suggested that caecilian evolution resulted in sturdy and compact skulls as an adaptation to their head-first burrowing habits, no relationship between skull shape and burrowing performance has been demonstrated to date. However, the unique dual jaw-closing mechanism and the osteological variability of their temporal region suggest a potential relationship between skull shape and feeding mechanics. Here, we explored the relationships between skull shape, head musculature and in vivo bite forces. Although there is a correlation between bite force and external head shape, no relationship between bite force and skull shape could be detected. Whereas our data suggest that muscles are the principal drivers of variation in bite force, the shape of the skull is constrained by factors other than demands for bite force generation. However, a strong covariation between the cranium and mandible exists. Moreover, both cranium and mandible shape covary with jaw muscle architecture. Caecilians show a gradient between species with a long retroarticular process associated with a large and pennate-fibered m. interhyoideus posterior and species with a short process but long and parallel-fibered jaw adductors. Our results demonstrate the complexity of the relationship between form and function of this jaw system. Further studies that focus on factors such as gape distance or jaw velocity will be needed in order to fully understand the evolution of feeding mechanics in caecilians.

Under pressure: the relationship between cranial shape and burrowing force in caecilians (Gymnophiona).

Caecilians are elongate, limbless and annulated amphibians that, with the exception of one aquatic family, all have an at least partly fossorial lifestyle. It has been suggested that caecilian evolution resulted in sturdy and compact skulls with fused bones and tight sutures, as an adaptation to their head-first burrowing habits. However, although their cranial osteology is well described, relationships between form and function remain poorly understood. In the present study, we explored the relationship between cranial shape and in vivo burrowing forces. Using micro-computed tomography (µCT) data, we performed 3D geometric morphometrics to explore whether cranial and mandibular shapes reflected patterns that might be associated with maximal push forces. The results highlight important differences in maximal push forces, with the aquatic Typhlonectes producing a lower force for a given size compared with other species. Despite substantial differences in head morphology across species, no relationship between overall skull shape and push force could be detected. Although a strong phylogenetic signal may partly obscure the results, our conclusions confirm previous studies using biomechanical models and suggest that differences in the degree of fossoriality do not appear to be driving the evolution of head shape.

Burrowing in blindsnakes: A preliminary analysis of burrowing forces and consequences for the evolution of morphology.

Burrowing is a common behavior in vertebrates. An underground life-style offers many advantages but also poses important challenges including the high energetic cost of burrowing. Scolecophidians are a group of morphologically derived subterranean snakes that show great diversity in form and function. Although it has been suggested that leptotyphlopids and anomalepidids mostly use existing underground passageways, typhlopids are thought to create their own burrows. However, the mechanisms used to create burrows and the associated forces that animals may be able to generate remain unknown. Here, we provide the first data on push forces in scolecophidians and compare them with those in some burrowing alethinophidian snakes. Our results show that typhlopids are capable of generating higher forces for a given size than other snakes. The observed differences are not due to variation in body diameter or length, suggesting fundamental differences in the mechanics of burrowing or the way in which axial muscles are used. Qualitative observations of skull and vertebral shape suggest that the higher forces exerted by typhlopids may have impacted the evolution of their anatomy. Our results provide the basis for future studies exploring the diversity of form and function in this fascinating group of animals. Quantitative comparisons of the cranial and vertebral shape in addition to collecting functional and ecological data on a wider array of species would be particularly important to test the patterns described here.

Are endocasts good proxies for brain size and shape in archosaurs throughout ontogeny?

Cranial endocasts, or the internal molds of the braincase, are a crucial correlate for investigating the neuroanatomy of extinct vertebrates and tracking brain evolution through deep time. Nevertheless, the validity of such studies pivots on the reliability of endocasts as a proxy for brain morphology. Here, we employ micro-computed tomography imaging, including diffusible iodine-based contrast-enhanced CT, and a three-dimensional geometric morphometric framework to examine both size and shape differences between brains and endocasts of two exemplar archosaur taxa - the American alligator (Alligator mississippiensis) and the domestic chicken (Gallus gallus). With ontogenetic sampling, we quantitatively evaluate how endocasts differ from brains and whether this deviation changes during development. We find strong size and shape correlations between brains and endocasts, divergent ontogenetic trends in the brain-to-endocast correspondence between alligators and chickens, and a comparable magnitude between brain-endocast shape differences and intraspecific neuroanatomical variation. The results have important implications for paleoneurological studies in archosaurs. Notably, we demonstrate that the pattern of endocranial shape variation closely reflects brain shape variation. Therefore, analyses of endocranial morphology are unlikely to generate spurious conclusions about large-scale trends in brain size and shape. To mitigate any artifacts, however, paleoneurological studies should consider the lower brain-endocast correspondence in the hindbrain relative to the forebrain; higher size and shape correspondences in chickens than alligators throughout postnatal ontogeny; artificially 'pedomorphic' shape of endocasts relative to their corresponding brains; and potential biases in both size and shape data due to the lack of control for ontogenetic stages in endocranial sampling.

The Utility of DiceCT Imaging for High-Throughput Comparative Neuroanatomical Studies.

Advancements in imaging techniques have drastically improved our ability to visualize, study, and digitally share complex, often minute, anatomical relationships. The recent adoption of soft-tissue X-ray imaging techniques, such as diffusible iodine-based contrast-enhanced computed tomography (diceCT), is beginning to offer previously unattainable insights into the detailed configurations of soft- tissue complexes across Metazoa. As a contrast agent, dissolved iodine diffuses deeply throughout preserved specimens to bind fats and carbohydrates that are natural ly present within metazoan soft tissues, increasing the radiodensities of these tissues in predictable ways. Like the current "gold standard" of magnetic resonance imaging, diceCT does not require physical dissection and can differentiate between the lipid content of myelinated versus nonmyelinated tissues, thereby offering great potential for neuroanatomical studies. Within the brain, for example, diceCT distinguishes myelinated fiber tracts from unmyelinated cortices, nuclei, and ganglia and allows three-dimensional visualization of their anatomical interrelationships at previously unrealized spatial scales. In this study, we illustrate the utility of diceCT for the rapid visualization of both external and internal brain anatomy in vertebrates - alongside the intact bones of the skull and the complete, undisturbed pathways of peripheral nerves, up to and including the target organs that they innervate. We demonstrate the transformative potential of this technique for developing high-resolution neuroanatomical datasets and describe best practices for imaging large numbers of specimens for broad evolutionary studies across vertebrates.

Diffusible iodine-based contrast-enhanced computed tomography (diceCT): an emerging tool for rapid, high-resolution, 3-D imaging of metazoan soft tissues.

Morphologists have historically had to rely on destructive procedures to visualize the three-dimensional (3-D) anatomy of animals. More recently, however, non-destructive techniques have come to the forefront. These include X-ray computed tomography (CT), which has been used most commonly to examine the mineralized, hard-tissue anatomy of living and fossil metazoans. One relatively new and potentially transformative aspect of current CT-based research is the use of chemical agents to render visible, and differentiate between, soft-tissue structures in X-ray images. Specifically, iodine has emerged as one of the most widely used of these contrast agents among animal morphologists due to its ease of handling, cost effectiveness, and differential affinities for major types of soft tissues. The rapid adoption of iodine-based contrast agents has resulted in a proliferation of distinct specimen preparations and scanning parameter choices, as well as an increasing variety of imaging hardware and software preferences. Here we provide a critical review of the recent contributions to iodine-based, contrast-enhanced CT research to enable researchers just beginning to employ contrast enhancement to make sense of this complex new landscape of methodologies. We provide a detailed summary of recent case studies, assess factors that govern success at each step of the specimen storage, preparation, and imaging processes, and make recommendations for standardizing both techniques and reporting practices. Finally, we discuss potential cutting-edge applications of diffusible iodine-based contrast-enhanced computed tomography (diceCT) and the issues that must still be overcome to facilitate the broader adoption of diceCT going forward.

Iodine-enhanced micro-CT imaging: methodological refinements for the study of the soft-tissue anatomy of post-embryonic vertebrates.

The now widespread use of non-destructive X-ray computed tomography (CT) and micro-CT (µCT) has greatly augmented our ability to comprehensively detail and quantify the internal hard-tissue anatomy of vertebrates. However, the utility of X-ray imaging for gaining similar insights into vertebrate soft-tissue anatomy has yet to be fully realized due to the naturally low X-ray absorption of non-mineralized tissues. In this study, we show how a wide diversity of soft-tissue structures within the vertebrate head-including muscles, glands, fat deposits, perichondria, dural venous sinuses, white and gray matter of the brain, as well as cranial nerves and associated ganglia-can be rapidly visualized in their natural relationships with extraordinary levels of detail using iodine-enhanced (i-e) µCT imaging. To date, Lugol's iodine solution (I2 KI) has been used as a contrast agent for µCT imaging of small invertebrates, vertebrate embryos, and certain isolated parts of larger, post-embryonic vertebrates. These previous studies have all yielded promising results, but visualization of soft tissues in smaller invertebrate and embryonic vertebrate specimens has generally been more complete than that for larger, post-embryonic vertebrates. Our research builds on these previous studies by using high-energy µCT together with more highly concentrated I2 KI solutions and longer staining times to optimize the imaging and differentiation of soft tissues within the heads of post-embryonic archosaurs (Alligator mississippiensis and Dromaius novaehollandiae). We systematically quantify the intensities of tissue staining, demonstrate the range of anatomical structures that can be visualized, and generate a partial three-dimensional reconstruction of alligator cephalic soft-tissue anatomy.

Drinking in snakes: resolving a biomechanical puzzle.

Snakes have long been thought to drink with a two-phase buccal-pump mechanism, but observations that some snakes can drink without sealing the margins of their mouths suggest that buccal pumping may not be the only drinking mechanism used by snakes. Here, we report that some snakes appear to drink using sponge-like qualities of specific regions of the oropharyngeal and esophageal mucosa and sponge-compressing functions of certain muscles and bones of the head. The resulting mechanism allows them to transport water upward against the effects of gravity using movements much slower than those of many other vertebrates. To arrive at this model, drinking was examined in three snake species using synchronized ciné and electromyographic recordings of muscle activity and in a fourth species using synchronized video and pressure recordings. Functional data were correlated with a variety of anatomical features to test specific predictions of the buccal-pump model. The anatomical data suggest explanations for the lack of conformity between a buccal-pump model of drinking and the performance of the drinking apparatus in many species. Electromyographic data show that many muscles with major functions in feeding play minor roles in drinking and, conversely, some muscles with minor roles in feeding play major roles in drinking. Mouth sealing by either the tongue or mental scale, previously considered critical to drinking in snakes, is incidental to drinking performance in some species. The sponge mechanism of drinking may represent a macrostomatan exaptation of mucosal folds, the evolution of which was driven primarily by the demands of feeding.

Effects of precaudal elongation on visceral topography in a basal clade of ray-finned fishes.

Elongate body forms have evolved numerous times independently within Vertebrata. Such body forms have evolved in large part via changes to the vertebral column, either through addition or lengthening of vertebrae. Previous studies have shown that body elongation in fishes has evolved most frequently through the addition of caudal vertebrae. In contrast, however, body elongation in Polypteriformes, a basal clade of ray-finned fishes (Actinopterygii), has evolved through the addition of precaudal vertebrae; one genus, Erpetoichthys, has approximately twice as many precaudal vertebrae as do members of its sister genus, Polypterus. Thus, polypteriform fishes provide an excellent opportunity to study the effects of precaudal elongation on the gross morphology and organization of visceral organs contained within the body cavity. In this study, we document the anteroposterior positions of most major visceral organs in representative species of both genera (E. calabaricus and P. palmas), relative to both vertebral number and percent pre-anal length. We found that, whereas the positions of the anterior and posterior borders of the visceral organs relative to percent pre-anal length were generally similar between the two species, most visceral organs were positioned further posteriorly in E. calabaricus than in P. palmas with respect to vertebral number. Based on previous determinations of the molecular control of anteroposterior patterning of the visceral organs, we discuss which possible changes in gene expression may have led to the anatomical modifications seen in the visceral morphology of Erpetoichthys.

Morphology of the skull of the white-nosed blindsnake, Liotyphlops albirostris (Scolecophidia: Anomalepididae).

This article presents a detailed description and illustration of the skull of Liotyphlops albirostris in comparison to the skulls of Typhlophis squamosus, Leptotyphlops dulcis, and Typhlops jamaicensis, based on high-resolution X-ray computed tomography (HRXCT). The skull of T. squamosus is illustrated and discussed in detail for the first time. A number of uniquely shared derived characters is identified that support the monophyly of the clade Anomalepididae. Anomalepidids retain some features that are plesiomorphic relative to other scolecophidians, such as the presence of a supratemporal (except in Anomalepis) and ectopterygoid. The homology of the element located posteroventral to the eyeball in anomalepidids and variably referred to as a jugal or postorbital (or a fusion of both in Anomalepis) remains unknown. Scolecophidians exhibit a highly derived skull morphology adapted to head-first burrowing. Both anomalepidids and typhlopids evolved a condition known as an "outer shell design," but did so in different ways. Leptotyphlopids combine elements of both the anomalepidid and typhlopid snout morphologies.

Are ontogenetic shifts in diet linked to shifts in feeding mechanics? Scaling of the feeding apparatus in the banded watersnake Nerodia fasciata.

The effects of size on animal behaviour, ecology, and physiology are widespread. Theoretical models have been developed to predict how animal form, function, and performance should change with increasing size. Yet, numerous animals undergo dramatic shifts in ecology (e.g. habitat use, diet) that may directly influence the functioning and presumably the scaling of the musculoskeletal system. For example, previous studies have shown that banded watersnakes (Nerodia fasciata) switch from fish prey as juveniles to frog prey as adults, and that fish and frogs represent functionally distinct prey types to watersnakes. We therefore tested whether this ontogenetic shift in diet was coupled to changes in the scaling patterns of the cranial musculoskeletal system in an ontogenetic size series (70-600 mm snout-vent length) of banded watersnakes. We found that all cranial bones and gape size exhibited significant negative allometry, whereas the muscle physiological cross-sectional area (pCSAs) scaled either isometrically or with positive allometry against snout-vent length. By contrast, we found that gape size, most cranial bones, and muscle pCSAs exhibited highly significant positive allometry against head length. Furthermore, the mechanical advantage of the jaw-closing lever system remained constant over ontogeny. Overall, these cranial allometries should enable watersnakes to meet the functional requirements of switching from fusiform fish to bulky frog prey. However, recent studies have reported highly similar allometries in a wide diversity of vertebrate taxa, suggesting that positive allometry within the cranial musculoskeletal system may actually be a general characteristic of vertebrates.

Morphology of the lower jaw and suspensorium in the Texas blindsnake, Leptotyphlops dulcis (Scolecophidia: Leptotyphlopidae).

Slender blindsnakes (Leptotyphlopidae) are known to use a unique feeding mechanism that involves rapid flexions of the tooth-bearing lower jaw. However, the morphology of the leptotyphlopid jaw apparatus has remained poorly studied due to the extremely small size of these snakes. Here I present a detailed description of the bones, cartilages, and ligaments of the lower jaw and suspensorium in a representative leptotyphlopid, Leptotyphlops dulcis, based on microanatomical studies of nearly 30 specimens prepared and examined in a variety of ways. The leptotyphlopid mandible is found to exhibit a complex mixture of symplesiomorphies shared with nonophidian squamates ("lizards"), synapomorphies shared with other snakes, and autapomorphies unique to Leptotyphlopidae. Most autapomorphies are functional correlates of the mandibular raking mechanism used by Leptotyphlops, primarily involving specializations of the intramandibular joint and the linkage between the suspensorium and the skull. Most notably, the quadrates are suspended via sliding articulations with the stapedes and do not articulate directly with the braincase. Posterior translation of the suspensorium at this loose, sliding articulation during jaw retraction may account for approximately one-third of the distance that prey are transported during each cycle of jaw flexion. This primary quadratostapedial articulation is believed to be unique among gnathostomes. Several anatomical features of the jaw apparatus suggest that Leptotyphlops evolved from more typical snake-like ancestors that: 1) had already lost the firm symphysis between the distal tips of the mandibular rami; and 2) had already evolved a high degree of upper jaw mobility.

How snakes eat snakes: the biomechanical challenges of ophiophagy for the California kingsnake, Lampropeltis getula californiae (Serpentes: Colubridae).

In this study we investigated how ophiophagous snakes are able to ingest prey snakes that equal or exceed their own length. We used X-ray video, standard video, dissection, and still X-rays to document the process of ophiophagy in kingsnakes (Lampropeltis getula) feeding on corn snakes (Elaphe guttata). Most kingsnakes readily accepted the prey snakes, subdued them by constriction, and swallowed them head first. In agreement with previous observations of ophiophagy, we found that the predator snake forces the vertebral column of the prey snake to bend into waves. These waves shorten the prey's body axis and allow it to fit inside the gastrointestinal (GI) tract and body cavity of the predator. Dissection of a kingsnake immediately following ingestion revealed extensive longitudinal stretching of the anterior portion of the GI tract (oesophagus and stomach), and no visible incursion of the prey into the intestine. X-ray video of ingestion showed that the primary mechanism of prey transport was the pterygoid walk, with some contribution from concertina-like compression and extension cycles of the predator's vertebral column in two out of three observations. Complete digestion was observed in only one individual, as others regurgitated before digestion was finished. X-ray stills taken every 4 days following ingestion revealed that the corn snakes were about half digested within the first 4 days, and digestion was complete within 15 days.

Post-cranial prey transport mechanisms in the black pinesnake, Pituophis melanoleucus lodingi: an x-ray videographic study.

Most previous studies of snake feeding mechanisms have focused on the functional morphology of the highly specialized ophidian jaw apparatus. Although some of these studies have included observations of post-cranial movements during feeding, the functional roles of these movements have remained poorly understood. In this study, we used x-ray videography to examine post-cranial prey transport mechanisms in a colubrid snake, Pituophis melanoleucus lodingi. We found that prey transport in this species progresses through four distinct phases, three of which are characterized by either undulatory or concertina-like movements of the anterior portion of the trunk. In the first phase of transport (the oral phase), unilateral movements of the jaws are used to pull the head forward around the prey. In the second phase (the orocervical phase), unilateral jaw movements continue, but are augmented by concertina-like movements of the anterior portion of the trunk. In the third phase (the cervical phase), prey transport occurs exclusively through concertina-like movements of the neck. Finally, in the fourth phase (the thoracic phase), prey is transported to the stomach via undulatory movements of the trunk. Our observations of feeding behavior in a phylogenetically diverse sample of fourteen other snake species demonstrate that similar post-cranial transport mechanisms are used by a wide variety of alethinophidian snakes that feed on large, bulky prey.