The origin of squamates revealed by a Middle Triassic lizard from the Italian Alps.

Modern squamates (lizards, snakes and amphisbaenians) are the world's most diverse group of tetrapods along with birds 1 and have a long evolutionary history, with the oldest known fossils dating from the Middle Jurassic period-168 million years ago2-4. The evolutionary origin of squamates is contentious because of several issues: (1) a fossil gap of approximately 70 million years exists between the oldest known fossils and their estimated origin5-7; (2) limited sampling of squamates in reptile phylogenies; and (3) conflicts between morphological and molecular hypotheses regarding the origin of crown squamates6,8,9. Here we shed light on these problems by using high-resolution microfocus X-ray computed tomography data from the articulated fossil reptile Megachirella wachtleri (Middle Triassic period, Italian Alps 10 ). We also present a phylogenetic dataset, combining fossils and extant taxa, and morphological and molecular data. We analysed this dataset under different optimality criteria to assess diapsid reptile relationships and the origins of squamates. Our results re-shape the diapsid phylogeny and present evidence that M. wachtleri is the oldest known stem squamate. Megachirella is 75 million years older than the previously known oldest squamate fossils, partially filling the fossil gap in the origin of lizards, and indicates a more gradual acquisition of squamatan features in diapsid evolution than previously thought. For the first time, to our knowledge, morphological and molecular data are in agreement regarding early squamate evolution, with geckoes-and not iguanians-as the earliest crown clade squamates. Divergence time estimates using relaxed combined morphological and molecular clocks show that lepidosaurs and most other diapsids originated before the Permian/Triassic extinction event, indicating that the Triassic was a period of radiation, not origin, for several diapsid lineages.

Plicidentine and the repeated origins of snake venom fangs.

Snake fangs are an iconic exemplar of a complex adaptation, but despite striking developmental and morphological similarities, they probably evolved independently in several lineages of venomous snakes. How snakes could, uniquely among vertebrates, repeatedly evolve their complex venom delivery apparatus is an intriguing question. Here we shed light on the repeated evolution of snake venom fangs using histology, high-resolution computed tomography (microCT) and biomechanical modelling. Our examination of venomous and non-venomous species reveals that most snakes have dentine infoldings at the bases of their teeth, known as plicidentine, and that in venomous species, one of these infoldings was repurposed to form a longitudinal groove for venom delivery. Like plicidentine, venom grooves originate from infoldings of the developing dental epithelium prior to the formation of the tooth hard tissues. Derivation of the venom groove from a large plicidentine fold that develops early in tooth ontogeny reveals how snake venom fangs could originate repeatedly through the co-option of a pre-existing dental feature even without close association to a venom duct. We also show that, contrary to previous assumptions, dentine infoldings do not improve compression or bending resistance of snake teeth during biting; plicidentine may instead have a role in tooth attachment.

Insights into skull evolution in fossorial snakes, as revealed by the cranial morphology of Atractaspis irregularis (Serpentes: Colubroidea).

Comparative osteological analyses of extant organisms provide key insight into major evolutionary transitions and phylogenetic hypotheses. This is especially true for snakes, given their unique morphology relative to other squamates and the persistent controversy regarding their evolutionary origins. However, the osteology of several major snake groups remains undescribed, thus hindering efforts to accurately reconstruct the phylogeny of snakes. One such group is the Atractaspididae, a family of fossorial colubroids. We herein present the first detailed description of the atractaspidid skull, based on fully segmented micro-computed tomography (micro-CT) scans of Atractaspis irregularis. The skull of Atractaspis presents a highly unique morphology influenced by both fossoriality and paedomorphosis. This paedomorphosis is especially evident in the jaws, palate, and suspensorium, the major elements associated with macrostomy (large-gaped feeding in snakes). Comparison to scolecophidians-a group of blind, fossorial, miniaturized snakes-in turn sheds light on current hypotheses of snake phylogeny. Features of both the naso-frontal joint and the morphofunctional system related to macrostomy refute the traditional notion that scolecophidians are fundamentally different from alethinophidians (all other extant snakes). Instead, these features support the controversial hypothesis of scolecophidians as "regressed alethinophidians," in contrast to their traditional placement as the earliest-diverging snake lineage. We propose that Atractaspis and scolecophidians fall along a morphological continuum, characterized by differing degrees of paedomorphosis. Altogether, a combination of heterochrony and miniaturization provides a mechanism for the derivation of the scolecophidian skull from an ancestral fossorial alethinophidian morphotype, exemplified by the nonminiaturized and less extreme paedomorph Atractaspis.

Tooth attachment and pleurodont implantation in lizards: Histology, development, and evolution.

Squamates present a unique challenge to the homology and evolution of tooth attachment tissues. Their stereotypically pleurodont teeth are fused in place by a single "bone of attachment", with seemingly dubious homology to the three-part tooth attachment system of mammals and crocodilians. Despite extensive debate over the interpretations of squamate pleurodonty, its phylogenetic significance, and the growing evidence from fossil amniotes for the homology of tooth attachment tissues, few studies have defined pleurodonty on histological grounds. Using a sample of extant squamate teeth that we organize into three broad categories of implantation, we investigate the histological and developmental properties of their dental tissues in multiple planes of section. We use these data to demonstrate the specific soft- and hard-tissue features of squamate teeth that produce their disparate tooth implantation modes. In addition, we describe cementum, periodontal ligaments, and alveolar bone in pleurodont squamates, dental tissues that were historically thought to be restricted to extant mammals and crocodilians. Moreover, we show how the differences between pleurodonty and thecodonty do not relate to the identity of the tooth attachment tissues, but rather the arrangements of homologous tissues around the teeth.

Sphenodontian phylogeny and the impact of model choice in Bayesian morphological clock estimates of divergence times and evolutionary rates.

BACKGROUND: The vast majority of all life that ever existed on earth is now extinct and several aspects of their evolutionary history can only be assessed by using morphological data from the fossil record. Sphenodontian reptiles are a classic example, having an evolutionary history of at least 230 million years, but currently represented by a single living species (Sphenodon punctatus). Hence, it is imperative to improve the development and implementation of probabilistic models to estimate evolutionary trees from morphological data (e.g., morphological clocks), which has direct benefits to understanding relationships and evolutionary patterns for both fossil and living species. However, the impact of model choice on morphology-only datasets has been poorly explored. RESULTS: Here, we investigate the impact of a wide array of model choices on the inference of evolutionary trees and macroevolutionary parameters (divergence times and evolutionary rates) using a new data matrix on sphenodontian reptiles. Specifically, we tested different clock models, clock partitioning, taxon sampling strategies, sampling for ancestors, and variations on the fossilized birth-death (FBD) tree model parameters through time. We find a strong impact on divergence times and background evolutionary rates when applying widely utilized approaches, such as allowing for ancestors in the tree and the inappropriate assumption of diversification parameters being constant through time. We compare those results with previous studies on the impact of model choice to molecular data analysis and provide suggestions for improving the implementation of morphological clocks. Optimal model combinations find the radiation of most major lineages of sphenodontians to be in the Triassic and a gradual but continuous drop in morphological rates of evolution across distinct regions of the phenotype throughout the history of the group. CONCLUSIONS: We provide a new hypothesis of sphenodontian classification, along with detailed macroevolutionary patterns in the evolutionary history of the group. Importantly, we provide suggestions to avoid overestimated divergence times and biased parameter estimates using morphological clocks. Partitioning relaxed clocks offers methodological limitations, but those can be at least partially circumvented to reveal a detailed assessment of rates of evolution across the phenotype and tests of evolutionary mosaicism.

The iliosacral joint in lizards: an osteological and histological analysis.

The development of the iliosacral joint (ISJ) in tetrapods represented a crucial step in the evolution of terrestrial locomotion. This structure is responsible for transferring forces between the vertebral column and appendicular skeleton, thus supporting the bodyweight on land. However, most research dealing with the water-to-land transition and biomechanical studies in general has focused exclusively on the articulation between the pelvic girdle and femur. Our knowledge about the contact between the pelvic girdle and vertebral column (i.e. the ISJ) at a tissue level is restricted so far to human anatomy, with little to no information available on other tetrapods. This lack of data limits our understanding of the development and evolution of such a key structure, and thus on the pattern and processes of the evolution of terrestrial locomotion. Therefore, we investigated the macro- and microanatomy of the ISJ in limb-bearing squamates that, similar to most non-mammalian, non-avian tetrapods, possess only two sacral ribs articulating with the posterior process of the ilium. Using a combination of osteology, micro-computed tomography and histology, we collected data on the ISJ apparatus of numerous specimens, sampling different taxa and different ontogenetic stages. Osteologically, we recorded consistent variability in all three processes of the ilium (preacetabular, supracetabular and posterior) and sacral ribs that correlate with posture and locomotion. The presence of a cavity between the ilium and sacral ribs, abundant articular cartilage and fibrocartilage, and a surrounding membrane of dense fibrous connective tissue allowed us to define this contact as a synovial joint. By comparison, the two sacral ribs are connected to each other mostly by dense fibrous tissue, with some cartilage found more distally along the margins of the two ribs, defining this joint as a combination of a syndesmosis and synchondrosis. Considering the intermediary position of the ISJ between the axial and appendicular skeletons, the shape of the articular surfaces of the sacral ribs and ilium, and the characteristics of the muscles associated with this structure, we argue that the mobility of the ISJ is primarily driven by the movements of the hindlimb during locomotion. We hypothesize that limited torsion of the ilium at the ISJ happens when the hip is abducted, and the joint is likely able to absorb the compressional and extensional forces related to the protraction and retraction of the femur. The mix of fibres and cartilage between the two sacral ribs instead serves primarily as a shock absorber, with the potential for limited vertical translation during locomotion.

The morphological diversity of the quadrate bone in squamate reptiles as revealed by high-resolution computed tomography and geometric morphometrics.

We examined the morphological diversity of the quadrate bone in squamate reptiles (i.e. lizards, snakes, amphisbaenians). The quadrate is the principal splanchnocranial element involved in suspending the lower jaw from the skull, and its shape is of particular interest because it is potentially affected by several factors, such as phylogenetic history, allometry, ecology, skull kinesis and hearing capabilities (e.g. presence or absence of a tympanic ear). Due to its complexity, the quadrate bone is also considered one of the most diagnostic elements in fragmentary fossil taxa. We describe quadrates from 38 species spread across all major squamate clades, using qualitative and quantitative (e.g. geometric morphometrics) methods. We test for possible correlations between shape variation and factors such as phylogeny, size, ecology and presence/absence of a tympanum. Our results show that the shape of the quadrate is highly evolutionarily plastic, with very little of the diversity explained by phylogenetic history. Size variation (allometric scaling) is similarly unable to explain much shape diversity in the squamate quadrate. Ecology (terrestrial/fossorial/aquatic) and presence of a tympanic ear are more significant, but together explain only about 20% of the diversity observed. Other unexplored and more analytically complex factors, such as skull biomechanics, likely play additional major roles in shaping the quadrates of lizards and snakes.

Giant taxon-character matrices II: a response to Laing et al. (2017).

The trend towards big data analyses in evolutionary biology has been observed in phylogenetics via the assembly of giant datasets composed of genomic and phenotypic data. We recently (Simões et al., 2017. Giant taxon-character matrices: Quality of character constructions remains critical regardless of size. Cladistics 33, 198-219) presented a critique of the phylogenetic character concepts used in current morphological datasets, with the caution that giant datasets did not obviate the empirical requirement of rigor in character construction. Laing et al. (2017. Giant taxon-character matrices: The future of morphological systematics. Cladistics, https://doi.org/10.1111/cla.12197) have since argued that we had 'suggested' that large datasets inherently contain flawed characters, and that we had presented a substandard methodology of phylogenetic analysis. Laing et al. concluded by discussing their approach to phylogenetic signal, total evidence and the inevitability of large datasets. We here reply to Laing et al. by reviewing what we actually wrote regarding dataset size, characters and methodology. We show that Laing et al.'s. central premise is unsupported, thus characterizing a Straw Man argument, and deeply misrepresents our original study. In part two, we discuss total evidence and phylogenetic signal issues raised by Laing et al. that are of major consequence to the appropriate construction of large morphological datasets.

Mosasaurs and snakes have a periodontal ligament: timing and extent of calcification, not tissue complexity, determines tooth attachment mode in reptiles.

Squamates present a unique challenge to our understanding of dental evolution in amniotes because they are the only extant tooth-bearing group for which a ligamentous tooth attachment is considered to be absent. This has led to the assumption that mammals and crocodilians have convergently evolved a ligamentous tooth attachment, composed of root cementum, periodontal ligament, and alveolar bone, whereas squamates are thought to possess a single bone of attachment tissue that fuses teeth to the jaws. The identity and homology of tooth attachment tissues between squamates, crocodilians, and mammals have thus been a focal point of debate for decades. We provide a novel interpretation of the mineralized attachment tissues in two focal taxa in this debate, mosasaurids and snakes, and compare dental tissue histology with that of the extant crocodilian Caiman sclerops. We identify a periodontal ligament in these squamates that usually exists temporarily as a soft connective tissue anchoring each tooth to the alveolar bone. We also identify two instances where complete calcification of the periodontal ligament does not occur: in a durophagous mosasaur, and in the hinged teeth of fossil and modern snakes. We propose that the periodontal ligament rapidly calcifies in the majority of mosasaurids and snakes, ankylosing the tooth to the jaw. This gives the appearance of a single, bone-like tissue fusing the tooth to the jaw in ankylosed teeth, but is simply the end stage of dental tissue ontogeny in most snakes and mosasaurids.

Giant taxon-character matrices: quality of character constructions remains critical regardless of size.

Giant morphological data matrices are increasingly common in cladistic analyses of vertebrate phylogeny, reporting numbers of characters never seen or expected before. However, the concern for size is usually not followed by an equivalent, if any, concern for character construction/selection criteria. Therefore, the question of whether quantity parallels quality for such influential works remains open. Here, we provide the largest compilation known to us of character construction methods and criteria, as derived from previous studies, and from our own de novo conceptualizations. Problematic character constructions inhibit the capacity of phylogenetic analyses to recover meaningful homology hypotheses and thus accurate clade structures. Upon a revision of two of the currently largest morphological datasets used to test squamate phylogeny, more than one-third of the almost 1000 characters analysed were classified within at least one of our categories of "types" of characters that should be avoided in cladistic investigations. These characters were removed or recoded, and the data matrices re-analysed, resulting in substantial changes in the sister group relationships for squamates, as compared to the original studies. Our results urge caution against certain types of character choices and constructions, also providing a methodological basis upon which problematic characters might be avoided.

The affinities of the Late Triassic Cryptovaranoides and the age of crown squamates.

Most living reptile diversity is concentrated in Squamata (lizards, including snakes), which have poorly known origins in space and time. Recently, †Cryptovaranoides microlanius from the Late Triassic of the United Kingdom was described as the oldest crown squamate. If true, this result would push back the origin of all major lizard clades by 30-65 Myr and suggest that divergence times for reptile clades estimated using genomic and morphological data are grossly inaccurate. Here, we use computed tomography scans and expanded phylogenetic datasets to re-evaluate the phylogenetic affinities of †Cryptovaranoides and other putative early squamates. We robustly reject the crown squamate affinities of †Cryptovaranoides, and instead resolve †Cryptovaranoides as a potential member of the bird and crocodylian total clade, Archosauromorpha. Bayesian total evidence dating supports a Jurassic origin of crown squamates, not Triassic as recently suggested. We highlight how features traditionally linked to lepidosaurs are in fact widespread across Triassic reptiles. Our study reaffirms the importance of critically choosing and constructing morphological datasets and appropriate taxon sampling to test the phylogenetic affinities of problematic fossils and calibrate the Tree of Life.

Cranial anatomy of the Galápagos marine iguana Amblyrhynchus cristatus (Squamata: Iguanidae).

Amblyrhynchus cristatus, the marine iguana, is unique among the ~7,000 species of living limbed lizards as it has successfully evolved adaptations that allow it to live in both terrestrial and marine environments. This species is endemic to the Galápagos Archipelago and has evolved a specialized feeding behavior, consuming primarily the algae that grow on the rocky seafloor. The intriguing questions arising around the evolution of the marine iguana concerns the use of exaptations of terrestrial features for aquatic and specifically marine adaptations. However, the lack of fundamental information about its anatomy currently prevents us from understanding how it became adapted to such a peculiar lifestyle in comparison to all other iguanids. The goal of this study is to provide the first ever description of the skull, mandible, and hyoid of Amblyrhynchus. We examined several specimens of marine iguana, including skeletal, wet, and ct-scanned material, and individuals at different ontogenetic stages. We also analyzed specimens of all other modern iguanid genera (Conolophus, Iguana, Ctenosaura, Cyclura, Dipsosaurus, Brachylophus, Sauromalus) in order to make comparisons between Amblyrhynchus and its closest relatives. We were able to identify several autapomorphic features that distinguish the marine iguana from all other iguanids. These unique morphologies are mostly associated with the modified configuration of the snout (nasal chamber), increased muscle attachments in the temporal-postorbital region of the skull, and dentition. Since Amblyrhynchus is the only nonophidian squamate currently able to exploit the ocean at least for some vital functions (i.e., feeding), we used comparisons to fossil marine lizards (e.g., mosasaurids) to discuss some of these unique traits. The new cranial features described for Amblyrhynchus may represent a source of novel morphological characters for use in future phylogenetic analyses of iguanian (or squamate) relationships, which will then serve as the foundation for the exploration of evolutionary patterns and processes that led to the development of such unique adaptations.

Convergence, divergence, and macroevolutionary constraint as revealed by anatomical network analysis of the squamate skull, with an emphasis on snakes.

Traditionally considered the earliest-diverging group of snakes, scolecophidians are central to major evolutionary paradigms regarding squamate feeding mechanisms and the ecological origins of snakes. However, quantitative analyses of these phenomena remain scarce. Herein, we therefore assess skull modularity in squamates via anatomical network analysis, focusing on the interplay between 'microstomy' (small-gaped feeding), fossoriality, and miniaturization in scolecophidians. Our analyses reveal distinctive patterns of jaw connectivity across purported 'microstomatans', thus supporting a more complex scenario of jaw evolution than traditionally portrayed. We also find that fossoriality and miniaturization each define a similar region of topospace (i.e., connectivity-based morphospace), with their combined influence imposing further evolutionary constraint on skull architecture. These results ultimately indicate convergence among scolecophidians, refuting widespread perspectives of these snakes as fundamentally plesiomorphic and morphologically homogeneous. This network-based examination of skull modularity-the first of its kind for snakes, and one of the first to analyze squamates-thus provides key insights into macroevolutionary trends among squamates, with particular implications for snake origins and evolution.

Successive climate crises in the deep past drove the early evolution and radiation of reptiles.

Climate change-induced mass extinctions provide unique opportunities to explore the impacts of global environmental disturbances on organismal evolution. However, their influence on terrestrial ecosystems remains poorly understood. Here, we provide a new time tree for the early evolution of reptiles and their closest relatives to reconstruct how the Permian-Triassic climatic crises shaped their long-term evolutionary trajectory. By combining rates of phenotypic evolution, mode of selection, body size, and global temperature data, we reveal an intimate association between reptile evolutionary dynamics and climate change in the deep past. We show that the origin and phenotypic radiation of reptiles was not solely driven by ecological opportunity following the end-Permian extinction as previously thought but also the result of multiple adaptive responses to climatic shifts spanning 57 million years.

Deconstructing the Gestalt: New concepts and tests of homology, as exemplified by a re-conceptualization of "microstomy" in squamates.

Snakes-a subset of lizards-have traditionally been divided into two major groups based on feeding mechanics: "macrostomy," involving the ingestion of proportionally large prey items; and "microstomy," the lack of this ability. "Microstomy"-considered present in scolecophidian and early-diverging alethinophidian snakes-is generally viewed as a symplesiomorphy shared with non-snake lizards. However, this perspective of "microstomy" as plesiomorphic and morphologically homogenous fails to recognize the complexity of this condition and its evolution across "microstomatan" squamates. To challenge this problematic paradigm, we formalize a new framework for conceptualizing and testing the homology of overall character complexes, or "morphotypes," which underlies our re-assessment of "microstomy." Using micro-computed tomography (micro-CT) scans, we analyze the morphology of the jaws and suspensorium across purported "microstomatan" squamates (scolecophidians, early-diverging alethinophidians, and non-snake lizards) and demonstrate that key components of the jaw complex are not homologous at the level of primary character state identity across these taxa. Therefore, rather than treating "microstomy" as a uniform condition, we instead propose that non-snake lizards, early-diverging alethinophidians, anomalepidids, leptotyphlopids, and typhlopoids each exhibit a unique and nonhomologous jaw morphotype: "minimal-kinesis microstomy," "snout-shifting," "axle-brace maxillary raking," "mandibular raking," and "single-axle maxillary raking," respectively. The lack of synapomorphy among scolecophidians is inconsistent with the notion of scolecophidians representing an ancestral snake condition, and instead reflects a hypothesis of the independent evolution of fossoriality, miniaturization, and "microstomy" in each scolecophidian lineage. We ultimately emphasize that a rigorous approach to comparative anatomy is necessary in constructing evolutionary hypotheses that accurately reflect biological reality.

Unique Tooth Morphology and Prismatic Enamel in Late Cretaceous Sphenodontians from Argentina.

Mammals and reptiles have evolved divergent adaptations for processing abrasive foods. Mammals have occluding, diphyodont dentitions with taller teeth (hypsodonty), more complex occlusal surfaces, continuous tooth eruption, and forms of prismatic enamel that prolong the functional life of each tooth [1, 2]. The evolution of prismatic enamel in particular was a key innovation that made individual teeth more resilient to abrasion in early mammals [2-4]. In contrast, reptiles typically have thin, non-prismatic enamel, and shearing, polyphyodont dentitions with multi-cusped or serrated tooth crowns, multiple tooth rows, rapid tooth replacement rates, or batteries made of hundreds of teeth [5-9]. However, there are rare cases where reptiles have evolved alternative solutions to cope with abrasive diets. Here, we show that the combined effects of herbivory and an ancestral loss of tooth replacement in a lineage of extinct herbivorous sphenodontians, distant relatives of the modern tuatara (Sphenodon punctatus) [10], are associated with the evolution of wear-resistant and highly complex teeth. Priosphenodon avelasi, an extinct sphenodontian from the Cretaceous of Argentina, possesses a unique cone-in-cone dentition with overlapping generations of teeth forming a densely packed tooth file. Each tooth is anchored to its predecessor via a rearrangement of dental tissues that results in a novel enamel-to-bone tooth attachment. Furthermore, the compound occlusal surfaces, thickened enamel, and the first report of prismatic enamel in a sphenodontian are convergent strategies with those in some mammals, challenging the perceived simplicity of acrodont dentitions [11-15] and showcasing the reptilian capacity to produce complex and unusual dentitions.

Megaevolutionary dynamics and the timing of evolutionary innovation in reptiles.

The origin of phenotypic diversity among higher clades is one of the most fundamental topics in evolutionary biology. However, due to methodological challenges, few studies have assessed rates of evolution and phenotypic disparity across broad scales of time to understand the evolutionary dynamics behind the origin and early evolution of new clades. Here, we provide a total-evidence dating approach to this problem in diapsid reptiles. We find major chronological gaps between periods of high evolutionary rates (phenotypic and molecular) and expansion in phenotypic disparity in reptile evolution. Importantly, many instances of accelerated phenotypic evolution are detected at the origin of major clades and body plans, but not concurrent with previously proposed periods of adaptive radiation. Furthermore, strongly heterogenic rates of evolution mark the acquisition of similarly adapted functional types, and the origin of snakes is marked by the highest rates of phenotypic evolution in diapsid history.

Skin of the Cretaceous mosasaur Plotosaurus: implications for aquatic adaptations in giant marine reptiles.

The physical nature of water and the environment it presents to an organism have long been recognized as important constraints on aquatic adaptation and evolution. Little is known about the dermal cover of mosasauroids (a group of secondarily aquatic reptiles that occupied a wide array of predatory niches in the Cretaceous marine ecosystems 92-65 Myr ago), a lack of information that has hindered inferences about the nature and level of their aquatic adaptations. A newly discovered Plotosaurus skeleton with integument preserved in three dimensions represents not only the first documented squamation in a mosasaurine mosasaur but also the first record of skin in an advanced member of the Mosasauroidea. The dermal cover comprises keeled and possibly osteoderm-reinforced scales that presumably contributed to an anterior-posterior channelling of the water flow and a reduction of microturbulent burst activities along the surface of the skin. Thus, hydrodynamic requirements of life in the water might have influenced the evolution of multiple-keeled body scales in advanced mosasauroids.

Cranial ontogeny of Thamnophis radix (Serpentes: Colubroidea) with a re-evaluation of current paradigms of snake skull evolution.

Accurate knowledge of skeletal ontogeny in extant organisms is crucial in understanding important morpho-functional systems and in enabling inferences of the ontogenetic stage of fossil specimens. However, detailed knowledge of skeletal ontogeny is lacking for most squamates, including snakes. Very few studies have discussed postnatal development in snakes, with none incorporating data from all three major ontogenetic stages-embryonic, juvenile and adult. Here, we provide the first analysis encompassing these three ontogenetic stages for any squamate, using the first complete micro-computed tomography (micro-CT)-based segmentations of any non-adult snake, based on fresh specimens of Thamnophis radix. The most significant ontogenetic changes involve the feeding apparatus, with major elongation of the tooth-bearing elements and jaw suspensorium causing a posterior shift in the jaw articulation. This shift enables macrostomy (large-gaped feeding in snakes) and occurs in T. radix via a different developmental trajectory than in most other macrostomatans, indicating that the evolution of macrostomy is more complex than previously thought. The braincase of T. radix is also evolutionarily unique among derived snakes in lacking a crista circumfenestralis, a phenomenon considered herein to represent paedomorphic retention of the embryonic condition. We thus present numerous important challenges to current paradigms regarding snake cranial evolution.