The interglenoid tubercle of the atlas is ancestral to lissamphibians.

Lissamphibians, represented today by frogs, salamanders, and caecilians, diverged deep in the tetrapod tree of life. Extensive morphological adaptations to disparate lifestyles have made linking extant lissamphibians to one another and to their extinct relatives difficult and controversial. However, the discovery of a feature on the atlas of the frog Xenopus laevis, may add to the small set of osteological traits that unite lissamphibians. In this study, we combine our observations of atlas development in X. laevis with a deep examination of atlantal interglenoid tubercle (TI) occurrence in fossil taxa. The TI is shown herein to occur transiently on the ossifying atlas of roughly one-third of X. laevis tadpoles but is absent in adults of this species. In ancestral character state estimations (ACSE), within the evolutionary context of lissamphibians as dissorophoid temnospondyls, this feature is found to be ancestrally shared among lissamphibians, its presence is uncertain in stem batrachians, and then the TI is lost in extant caecilians and frogs. However, our data suggests apparent TI loss around the origin of frogs may be explained by its ontogenetically transient nature. The only nonamphibian tetrapods with a TI are "microsaurs," and this similarity is interpreted as one of many convergences that resulted from convergent evolutionary processes that occurred in the evolution of "microsaurs" and lissamphibians. The TI is thus interpreted to be ancestral to lissamphibians as it is found to be present in some form throughout each extant lissamphibian clade's history.

Normal development in Xenopus laevis: A complementary staging table for the skull based on cartilage and bone.

BACKGROUND: Xenopus laevis is a widely used model organism in the fields of genetics and development, and more recently evolution. At present, the most widely used staging table for X. laevis is based primarily on external features and does not describe the corresponding skull development in detail. Here, we describe skull development in X. laevis, complete with labeled figures, for each relevant stage in the most widely used staging table. RESULTS: We find skull development in X. laevis is, for the most part, distinct at each of the previously established stages based on external anatomy. However, variation does exist in the timing of onset of ossification of certain bones in the skull, which results in a range of stages where a skull element first ossifies. The overall sequence of ossification is less variable than the timing of ossification onset. CONCLUSIONS: While events in skull development vary somewhat between specimens, and in comparison, to external events, this staging table is useful in showing both when bones first appear and for documenting the range of temporal variance in X. laevis skull development more accurately than previously done. Furthermore, when only skull data are available, the approximate stage of a specimen can now be determined.

The histology of sutures in chicken skulls: Types, conservation, and ontogeny.

Sutures are fibrous joints that occur between bone elements in vertebrate skulls, where they play a variety of roles including facilitating skull growth and function. In addition, a variety of studies examining sutures from diverse perspectives in many taxa have enabled the determination of anatomical homologs. Surprisingly, one important aspect of sutures-histology-remains unknown in the key model organism of the chicken. To fill this gap in our knowledge, we generated histological sections of six different cranial sutures across a range of developmental stages in embryonic chicken. Despite having a skull that is largely co-ossified or fused as an adult, we found that the types, components, and ontogeny of sutures in chicken skulls are very similar to sutures in other vertebrates. We did, however, find a new transient stage in the ontogeny of sutures between endochondral bone elements, in which one element has ossified and one was still cartilaginous. Moreover, to better understand the morphogenetic events at the onset of suture formation, we compared the developmental histology of six sutures with that of the space between the two ossification centers of the frontal-a location expected to be void of suture structures. We found that the mesenchymal cells in sutures condense and form a middle vascular layer. This was not found to be the case in the space between the two ossifications of the frontal, where instead only osteoid occurs.

Normal development in Ambystoma mexicanum: A complementary staging table for the skull based on Alizarin red S staining.

BACKGROUND: As the role of Ambystoma mexicanum, or the Mexican axolotl, expands in research applications beyond its traditional use in studies of limb regeneration, a staging table that is more anatomically extensive is required. Here, we describe axolotl skull development as it relates to previously established developmental stages that were based on limb development. RESULTS: We find that most key developmental events in the skull correspond to these previously established stages, creating easily recognizable stages of axolotl throughout skull morphogenesis. CONCLUSIONS: With this complementary staging table in hand, researchers can stage axolotl larvae when limb data are missing or incomplete, or when cranial data alone is available.

Computed tomography analysis of the cranium of Champsosaurus lindoei and implications for the choristoderan neomorphic ossification.

Choristoderes are extinct neodiapsid reptiles that are well known for their unusual cranial anatomy, possessing an elongated snout and expanded temporal arches. Although choristodere skulls are well described externally, their internal anatomy remains unknown. An internal description was needed to shed light on peculiarities of the choristodere skull, such as paired gaps on the ventral surface of the skull that may pertain to the fenestra ovalis, and a putative neomorphic ossification in the lateral wall of the braincase. Our goals were: (i) to describe the cranial elements of Champsosaurus lindoei in three dimensions; (ii) to describe paired gaps on the ventral surface of the skull to determine if these are indeed the fenestrae ovales; (iii) to illustrate the morphology of the putative neomorphic bone; and (iv) to consider the possible developmental and functional origins of the neomorph. We examined the cranial anatomy of the choristodere Champsosaurus lindoei (CMN 8920) using high-resolution micro-computed tomography scanning. We found that the paired gaps on the ventral surface of the skull do pertain to the fenestrae ovales, an unusual arrangement that may be convergent with some plesiosaurs, some aistopods, and some urodeles. The implications of this morphology in Champsosaurus are unknown and will be the subject of future work. We found that the neomorphic bone is a distinct ossification, but is not part of the wall of the brain cavity or the auditory capsule. Variation in the developmental pathways of cranial bones in living amniotes was surveyed to determine how the neomorphic bone may have developed. We found that the chondrocranium and splanchnocranium show little to no variation across amniotes, and the neomorphic bone is therefore most likely to have developed from the dermatocranium; however, the stapes is a pre-existing cranial element that is undescribed in choristoderes and may be homologous with the neomorphic bone. If the neomorphic bone is not homologous with the stapes, the neomorph likely developed from the dermatocranium through incomplete fusion of ossification centres from a pre-existing bone, most likely the parietal. Based on the apparent morphology of the neomorph in Coeruleodraco, the neomorph was probably too small to play a significant structural role in the skull of early choristoderes and it may have arisen through non-adaptive means. In neochoristoderes, such as Champsosaurus, the neomorph was likely recruited to support the expanded temporal arches.

Influence of fossoriality on inner ear morphology: insights from caecilian amphibians.

It is widely accepted that a relationship exists between inner ear morphology and functional aspects of an animal's biology, such as locomotor behaviour. Animals that engage in agile and spatially complex behaviours possess semicircular canals that morphologically maximise sensitivity to correspondingly complex physical stimuli. Stemming from the prediction that fossorial tetrapods require a well-developed sense of spatial awareness, we investigate the hypothesis that fossoriality leads to inner ear morphology that is convergent with other spatially adept tetrapods. We apply morphometrics to otic capsule endocasts of 26 caecilian species to quantify aspects of inner ear shape, and compare these with a sample of frog and salamander species. Our results reveal caecilians (and also frogs) possess strongly curved canals, a feature in common with spatially adept species. However, significantly shorter canals in caecilians suggest reduced sensitivity, possibly associated with reduced reliance on vestibulo-ocular reflexes in this group of visually degenerate tetrapods. An elaboration of the sacculus of caecilians is interpreted as a unique adaptation among amphibians to increase sensitivity to substrate-borne vibrations transmitted through the head. This study represents the first quantitative analyses of inner ear morphology of limbless fossorial tetrapods, and identifies features within a new behavioural context that will contribute to our understanding of the biological consequences of physical stimuli on sensory function and associated morphological evolution.

Is solid always best? Cranial performance in solid and fenestrated caecilian skulls.

Caecilians (Lissamphibia: Gymnophiona) are characterized by a fossorial lifestyle that appears to play a role in the many anatomical specializations in the group. The skull, in particular, has been the focus of previous studies because it is driven into the substrate for burrowing. There are two different types of skulls in caecilians: (1) stegokrotaphic, where the squamosal completely covers the temporal region and the jaw closing muscles, and (2) zygokrotaphic, with incomplete coverage of the temporal region by the squamosal. We used 3-D imaging and modeling techniques to explore the functional consequences of these skull types in an evolutionary context. We digitally converted stegokrotaphic skulls into zygokrotaphic skulls and vice versa. We also generated a third, akinetic skull type that was presumably present in extinct caecilian ancestors. We explored the benefits and costs of the different skull types under frontal loading at different head angles with finite element analysis (FEA). Surprisingly, the differences in stress distributions and bending between the three tested skull types were minimal and not significant. This suggests that the open temporal region in zygokrotaphic skulls does not lead to poorer performance during burrowing. However, the results of the FEA suggest a strong relationship between the head angle and skull performance, implying there is an optimal head angle during burrowing.

Snake-like limb loss in a Carboniferous amniote.

Among living tetrapods, many lineages have converged on a snake-like body plan, where extreme axial elongation is accompanied by reduction or loss of paired limbs. However, when and how this adaptive body plan first evolved in amniotes remains poorly understood. Here, we provide insights into this question by reporting on a new taxon of molgophid recumbirostran, Nagini mazonense gen. et sp. nov., from the Francis Creek Shale (309-307 million years ago) of Illinois, United States, that exhibits extreme axial elongation and corresponding limb reduction. The molgophid lacks entirely the forelimb and pectoral girdle, thus representing the earliest occurrence of complete loss of a limb in a taxon recovered phylogenetically within amniotes. This forelimb-first limb reduction is consistent with the pattern of limb reduction that is seen in modern snakes and contrasts with the hindlimb-first reduction process found in many other tetrapod groups. Our findings suggest that a snake-like limb-reduction mechanism may be operating more broadly across the amniote tree.

Anatomy and development of skull-neck boundary structures in the skeleton of the extant crocodylian Alligator mississippiensis.

A system-by-system approach dominates morphological and evolutionary study; however, some structures that are better understood within the context of an interface between two systems or traditional units remain less well understood. As part of a larger goal to clarify aspects of skull-neck boundary evolution, we herein describe the morphology and development of the occiput and atlas-axis complex in the crocodylian Alligator mississippiensis. We apply micro-computed tomography scanning, clearing and double staining, and histological analyses to skull-neck boundary structures at three stages of development (embryonic stage 22, 23, and hatchling). Regions of ossification that could possibly pertain to a postparietal were found adjacent to the parietal bone and supraoccipital; however, these were not deemed convincing and are considered part of the supraoccipital. Within the atlas-axis complex, the proatlas appears as two discrete cartilaginous elements in Stage 22 that ossify together at Stage 23. Posterior to the proatlas, the atlas-axis complex is composed of two centra, each with cervical ribs ventrally and neural arches dorsally that begin ossifying at Stage 23. Histology and clearing and staining of Stages 22 and 23 embryos reveal a discrete atlas intercentrum applied to the ventral part of the occipital condyle of the skull. Posterior to this is a cartilage that appears to be a co-chondrified atlas pleurocentrum, axis intercentrum, and axis pleurocentrum. Ossification of this cartilaginous structure produces discrete atlas inter- and pleurocentra, as well as a singular axis centrum. Together these data are discussed with reference to clarifying historical discrepancies concerning elements at the crocodylian skull-neck boundary.

Joermungandr bolti, an exceptionally preserved 'microsaur' from the Mazon Creek Lagerstätte reveals patterns of integumentary evolution in Recumbirostra.

The Carboniferous Pennsylvanian-aged (309-307 Ma) Mazon Creek Lagerstätte produces some of the earliest fossils of major Palaeozoic tetrapod lineages. Recently, several new tetrapod specimens collected from Mazon Creek have come to light, including the earliest fossorially adapted recumbirostrans. Here, we describe a new long-bodied recumbirostran, Joermungandr bolti gen. et sp. nov., known from a single part and counterpart concretion bearing a virtually complete skeleton. Uniquely, Joermungandr preserves a full suite of dorsal, flank and ventral dermal scales, together with a series of thinned and reduced gastralia. Investigation of these scales using scanning electron microscopy reveals ultrastructural ridge and pit morphologies, revealing complexities comparable to the scale ultrastructure of extant snakes and fossorial reptiles, which have scales modified for body-based propulsion and shedding substrate. Our new taxon also represents an important early record of an elongate recumbirostran bauplan, wherein several features linked to fossoriality, including a characteristic recumbent snout, are present. We used parsimony phylogenetic methods to conduct phylogenetic analysis using the most recent recumbirostran-focused matrix. The analysis recovers Joermungandr within Recumbirostra with likely affinities to the sister clades Molgophidae and Brachystelechidae. Finally, we review integumentary patterns in Recumbirostra, noting reductions and losses of gastralia and osteoderms associated with body elongation and, thus, probably also associated with increased fossoriality.

Development of the chicken skull: A complement to the external staging table of Hamburger and Hamilton.

Embryonic staging tables provide a standard of developmental stages that can be used by individual investigators and provide approximate time points for the study of developmental phenomena. Surprisingly, despite the presence of a plethora of studies on the chicken skull and its role as a model species in developmental research, a staging table of the development of the chicken skull remains lacking. A detailed photographic staging table of the osseous portion of the chicken skull is thus presented here based on cleared and stained HH stages spanning HH 35 (first appearance of skull ossification) to the final stage before hatching (HH 45). This table documents the development of most of the cranial elements in the skull from the start of ossification until the element takes its final shape. The table shows that the elements of the lower jaw and ventral side of the skull begin ossifying before the skull roof and that most elements take roughly 5 days to reach their final shape, whereas others take up to 9 days (e.g., the frontal). The obtained results lead to several hypotheses about chicken skull development and provide a timeframe for future studies on chicken skull development.

Evolutionary development of the neurocranium in Dissorophoidea (Tetrapoda: Temnospondyli), an integrative approach.

Ontogenetic data can play a prominent role in addressing questions in tetrapod evolution, but such evidence from the fossil record is often incompletely considered because it is limited to initiation of ossification, or allometric changes with increasing size. In the present study, specimens of a new species of an archaic amphibian (280 Myr old), Acheloma n. sp., a member of the temnospondyl superfamily Dissorophoidea and the sister group to Amphibamidae, which is thought to include at least two of our modern amphibian clades, anurans and caudatans (Batrachia), provides us with new developmental data. We identify five ontogenetic events, enabling us to construct a partial ontogenetic trajectory (integration of developmental and transformation sequence data) related to the relative timing of completion of neurocranial structures. Comparison of the adult amphibamid morphology with this partial ontogeny identifies a heterochronic event that occurred within the neurocranium at some point in time between the two taxa, which is consistent with the predictions of miniaturization in amphibamids, providing the first insights into the influence of miniaturization on the neurocranium in a fossil tetrapod group. This study refines hypotheses of large-scale evolutionary trends within Dissorophoidea that may have facilitated the radiation of amphibamids and, projected forward, the origin of the generalized batrachian skull. Most importantly, this study highlights the importance of integrating developmental and transformation sequence data, instead of onset of ossification alone, into investigations of major events in tetrapod evolution using evidence provided by the fossil record, and highlights the value of even highly incomplete growth series comprised of relatively late-stage individuals.

Varanopid from the Carboniferous of Nova Scotia reveals evidence of parental care in amniotes.

Here we report on a fossil synapsid, Dendromaia unamakiensis gen. et sp. nov., from the Carboniferous period of Nova Scotia that displays evidence of parental care-approximately 40 million years earlier than the previous earliest record based on a varanopid from the Guadalupian (middle Permian) period of South Africa. The specimen, consisting of an adult and associated conspecific juvenile, is also identified as a varanopid suggesting parental care is more deeply rooted within this clade and evolved very close to the origin of Synapsida and Amniota in general. This specimen adds to growing evidence that parental care was more widespread among Palaeozoic synapsids than previously thought and further provides data permitting the identification of potential ontogeny-dependent traits within varanopids, the implications of which impact recent competing hypotheses of the phylogenetic affinities of the group.

Development and evolution of the tetrapod skull-neck boundary.

The origin and evolution of the vertebrate skull have been topics of intense study for more than two centuries. Whereas early theories of skull origin, such as the influential vertebral theory, have been largely refuted with respect to the anterior (pre-otic) region of the skull, the posterior (post-otic) region is known to be derived from the anteriormost paraxial segments, i.e. the somites. Here we review the morphology and development of the occiput in both living and extinct tetrapods, taking into account revised knowledge of skull development by augmenting historical accounts with recent data. When occipital composition is evaluated relative to its position along the neural axis, and specifically to the hypoglossal nerve complex, much of the apparent interspecific variation in the location of the skull-neck boundary stabilizes in a phylogenetically informative way. Based on this criterion, three distinct conditions are identified in (i) frogs, (ii) salamanders and caecilians, and (iii) amniotes. The position of the posteriormost occipital segment relative to the hypoglossal nerve is key to understanding the evolution of the posterior limit of the skull. By using cranial foramina as osteological proxies of the hypoglossal nerve, a survey of fossil taxa reveals the amniote condition to be present at the base of Tetrapoda. This result challenges traditional theories of cranial evolution, which posit translocation of the occiput to a more posterior location in amniotes relative to lissamphibians (frogs, salamanders, caecilians), and instead supports the largely overlooked hypothesis that the reduced occiput in lissamphibians is secondarily derived. Recent advances in our understanding of the genetic basis of axial patterning and its regulation in amniotes support the hypothesis that the lissamphibian occipital form may have arisen as the product of a homeotic shift in segment fate from an amniote-like condition.

Correction to 'Carbonodraco lundi gen et sp. nov., the oldest parareptile, from Linton, Ohio, and new insights into the early radiation of reptiles'.

[This corrects the article DOI: 10.1098/rsos.191191.].

The internal cranial anatomy of Champsosaurus (Choristodera: Champsosauridae): Implications for neurosensory function.

Although isolated Champsosaurus remains are common in Upper Cretaceous sediments of North America, the braincase of these animals is enigmatic due to the fragility of their skulls. Here, two well-preserved specimens of Champsosaurus (CMN 8920 and CMN 8919) are CT scanned to describe their neurosensory structures and infer sensory capability. The anterior portion of the braincase was poorly ossified and thus does not permit visualization of a complete endocast; however, impressions of the olfactory stalks indicate that they were elongate and likely facilitated good olfaction. The posterior portion of the braincase is ossified and morphologically similar to that of other extinct diapsids. The absence of an otic notch and an expansion of the pars inferior of the inner ear suggests Champsosaurus was limited to detecting low frequency sounds. Comparison of the shapes of semicircular canals with lepidosaurs and archosauromorphs demonstrates that the semicircular canals of Champsosaurus are most similar to those of aquatic reptiles, suggesting that Champsosaurus was well adapted for sensing movement in an aquatic environment. This analysis also demonstrates that birds, non-avian archosauromorphs, and lepidosaurs possess significantly different canal morphologies, and represents the first morphometric analysis of semicircular canals across Diapsida.

Early Jurassic dinosaur fetal dental development and its significance for the evolution of sauropod dentition.

Rare occurrences of dinosaurian embryos are punctuated by even rarer preservation of their development. Here we report on dental development in multiple embryos of the Early Jurassic Lufengosaurus from China, and compare these to patterns in a hatchling and adults. Histology and CT data show that dental formation and development occurred early in ontogeny, with several cycles of tooth development without root resorption occurring within a common crypt prior to hatching. This differs from the condition in hatchling and adult teeth of Lufengosaurus, and is reminiscent of the complex dentitions of some adult sauropods, suggesting that their derived dental systems likely evolved through paedomorphosis. Ontogenetic changes in successive generations of embryonic teeth of Lufengosaurus suggest that the pencil-like teeth in many sauropods also evolved via paedomorphosis, providing a mechanism for the convergent evolution of small, structurally simple teeth in giant diplodocoids and titanosaurids. Therefore, such developmental perturbations, more commonly associated with small vertebrates, were likely also essential events in sauropod evolution.

The anatomy and development of the claws of Xenopus laevis (Lissamphibia: Anura) reveal alternate pathways of structural evolution in the integument of tetrapods.

Digital end organs composed of hard, modified epidermis, generally referred to as claws, are present in mammals and reptiles as well as in several non-amniote taxa such as clawed salamanders and frogs, including Xenopus laevis. So far, only the claws and nails of mammals have been characterized extensively and the question of whether claws were present in the common ancestor of all extant tetrapods is as yet unresolved. To provide a basis for comparisons between amniote and non-amniote claws, we investigated the development, growth and ultrastructure of the epidermal component of the claws of X. laevis. Histological examination of developing claws of X. laevis shows that claw formation is initiated at the tip of the toe by the appearance of superficial cornified cells that are dark brown. Subsequent accumulation of new, proximally extended claw sheath corneocyte layers increases the length of the claw. Histological studies of adult claws show that proliferation of cornifying claw sheath cells occurs along the entire length of the claw-forming epidermis. Living epidermal cells that are converting into the cornified claw sheath corneocytes undergo a form of programmed cell death that is accompanied by degradation of nuclear DNA. Subsequently, the cytoplasm and the nuclear remnants acquire a brown colour by an as-yet unknown mechanism that is likely homologous to the colouration mechanism that occurs in other hard, cornified structures of amphibians such as nuptial pads and tadpole beaks. Transmission electron microscopy revealed that the cornified claw sheath consists of parallel layers of corneocytes with interdigitations being confined to intra-layer contacts and a cementing substance filling the intercorneocyte spaces. Together with recent reports that showed the main molecular components of amniote claws are absent in Xenopus, our data support the hypothesis that claws of amphibians likely represent clade-specific innovations, non-homologous to amniote claws.

Braincase simplification and the origin of lissamphibians.

Dissorophoidea, a group of temnospondyl tetrapods that first appear in the Late Carboniferous, is made up of two clades ⎼ Olsoniformes and Amphibamiformes (Branchiosauridae and Amphibamidae) ⎼ the latter of which is widely thought to have given rise to living amphibians (i.e., Lissamphibia). The lissamphibian braincase has a highly derived morphology with several secondarily lost elements; however, these losses have never been incorporated into phylogenetic analyses and thus the timing and nature of these evolutionary events remain unknown. Hindering research into this problem has been the lack of phylogenetic analyses of Dissorophoidea that includes both taxonomically dense sampling and specific characters to document changes in the braincase in the lineage leading to Lissamphibia. Here we build on a recent, broadly sampled dissorophoid phylogenetic analysis to visualize key events in the evolution of the lissamphibian braincase. Our ancestral character state reconstructions show a clear, step-wise trend towards reduction of braincase ossification leading to lissamphibians, including reduction of the sphenethmoid, loss of the basioccipital at the Amphibamiformes node, and further loss of both the basisphenoid and the hypoglossal nerve foramina at the Lissamphibia node. Our analysis confirms that the highly derived condition of the lissamphibian braincase is characterized by overall simplification in terms of the number and extent of chondrocranial ossifications.

Carbonodraco lundi gen et sp. nov., the oldest parareptile, from Linton, Ohio, and new insights into the early radiation of reptiles.

Redescription of the holotype specimen of Cephalerpeton ventriarmatum Moodie, 1912, from the Middle Pennsylvanian (Moscovian) Francis Creek Shale of Mazon Creek, Illinois, confirms that it is a basal eureptile with close postcranial similarities to other protorothyridids, such as Anthracodromeus and Paleothyris. The skull is long and lightly built, with large orbits and a dorsoventrally short mandible similar to most basal eureptiles. Two specimens referred previously to Cephalerpeton cf. C. ventriarmatum from the approximately coeval Linton, Ohio, locality differ significantly from the holotype in cranial and mandibular proportions and tooth morphology. This material and an additional Linton specimen compare favourably to 'short-faced' parareptiles, such as Colobomycter and Acleistorhinus, and justify recognition of an acleistorhinid parareptile in the Linton assemblage. The new binomen is thus the oldest known parareptile.