Fossil-informed biogeographic analysis suggests Eurasian regionalization in crown Squamata during the early Jurassic.

Background: Squamata (lizards, snakes, and amphisbaenians) is a Triassic lineage with an extensive and complex biogeographic history, yet no large-scale study has reconstructed the ancestral range of early squamate lineages. The fossil record indicates a broadly Pangaean distribution by the end- Cretaceous, though many lineages (e.g., Paramacellodidae, Mosasauria, Polyglyphanodontia) subsequently went extinct. Thus, the origin and occupancy of extant radiations is unclear and may have been localized within Pangaea to specific plates, with potential regionalization to distinct Laurasian and Gondwanan landmasses during the Mesozoic in some groups. Methods: We used recent tectonic models to code extant and fossil squamate distributions occurring on nine discrete plates for 9,755 species, with Jurassic and Cretaceous fossil constraints from three extinct lineages. We modeled ancestral ranges for crown Squamata from an extant-only molecular phylogeny using a suite of biogeographic models accommodating different evolutionary processes and fossil-based node constraints from known Jurassic and Cretaceous localities. We hypothesized that the best-fit models would not support a full Pangaean distribution (i.e., including all areas) for the origin of crown Squamata, but would instead show regionalization to specific areas within the fragmenting supercontinent, likely in the Northern Hemisphere where most early squamate fossils have been found. Results: Incorporating fossil data reconstructs a localized origin within Pangaea, with early regionalization of extant lineages to Eurasia and Laurasia, while Gondwanan regionalization did not occur until the middle Cretaceous for Alethinophidia, Scolecophidia, and some crown Gekkotan lineages. While the Mesozoic history of extant squamate biogeography can be summarized as a Eurasian origin with dispersal out of Laurasia into Gondwana, their Cenozoic history is complex with multiple events (including secondary and tertiary recolonizations) in several directions. As noted by previous authors, squamates have likely utilized over-land range expansion, land-bridge colonization, and trans-oceanic dispersal. Tropical Gondwana and Eurasia hold more ancient lineages than the Holarctic (Rhineuridae being a major exception), and some asymmetries in colonization (e.g., to North America from Eurasia during the Cenozoic through Beringia) deserve additional study. Future studies that incorporate fossil branches, rather than as node constraints, into the reconstruction can be used to explore this history further.

Paedomorphosis and retention of juvenile diet lead speciation in a group of Neotropical snakes (Colubroides-Philodryadini).

Dipsadidae is one of the largest clades of extant reptiles, showing an impressive morphological and ecological diversity. Despite this fact, the developmental processes behind its diversity are still largely unknown. In this study, we used 3D reconstructions based on micro-CT data and geometric morphometrics to evaluate the skull morphology of Philodryas agassizii, a small, surface-dwelling dipsadid that consume spiders. Adult individuals of P. agassizii exhibit a cranial morphology frequently observed in juveniles of other surface-dwelling colubroideans, represented in our analysis by its close relative Philodryas patagoniensis. Large orbits, gibbous neurocranial roof and a relatively short jaw complex are features present in juveniles of the latter species. Furthermore, we performed an extensive survey about diet of P. patagoniensis in which we detected an ontogenetic dietary shift, indicating that arthropods are more frequently consumed by juveniles of this dietary generalist. Thus, we infer that P. agassizzii retained not only the ancestral juvenile skull morphology but also dietary preferences. This study reveals that morphological changes driven by heterochronic changes, specifically paedomorphosis, influenced the retention of ancestral life history traits in P. agassizii, and therefore promoted cladogenesis. In this way, we obtained first evidence that heterochronic processes lead speciation in the snake megadiverse clade Dipsadidae.

Comparative electron microscopy study of spermatozoa in snakes (Lepidosauria, Squamata).

The ultrastructure of snake sperm has received substantial attention primarily because snakes exhibit considerable variability in reproductive characteristics between species, with a wide range of mating systems and reproductive behaviors. Variability of sperm morphology among snake species may be associated with the reproductive strategies of each taxon, such as competition or sperm storage. We provide a detailed description of the sperm ultrastructure of nine snake species (Anilius scytale, Tropidophis paucisquamis, Bothrops jararaca, Oxyrhopus guibei, Dipsas mikanii, Micrurus corallinus, Xenopholis scalaris, Acrochordus javanicus, and Cylindrophis ruffus) and compared this with sperm data from the literature for the following taxa: Liotyphlops beui, Amerotyphlops reticulatus, Trilepida koppesi, Anilios waitii, Anilios endoterus, Aspidites melanochephalus, Boa constrictor amarali, Corallus hortulana, Epicrates cenchria, Boa constrictor occidentalis, Eryx jayakari, Micrurus corallinus, Micrurus surinamensis, Micrurus frontalis, Micrurus altirostris, Oxyuranus microlepidotus, Bothrops alternatus, Bothrops diporus, Crotalus durissus, Agkistrodon contortrix, Vipera aspis, Boiga irregularis, Zamenis schrenckii, Zamenis scalaris, Stegonotus cuculatus, Nerodia sipedon, Liodytes pygaea, and Myrrophis chinensis. We found twelve polymorphic characters in the ultrastructure of sperm among the described snakes. Our work supports the importance of ultrastructural analysis of sperm morphology to understand snake reproduction, and provides sperm-derived morphological characters for phylogenetic analysis.

Comparative morphology of oral glands in snakes of the family Homalopsidae reveals substantial variation and additional independent origins of salt glands within Serpentes.

Using diffusible iodine-based contrast-enhanced computed tomography (diceCT), we examined the morphology of the oral glands of 12 species of the family Homalopsidae. Snakes of this family exhibit substantial interspecific morphological variation in their oral glands. Particular variables are the venom glands, ranging from large (e.g., Subsessor bocourti) to small (e.g., Erpeton tentaculatum). The supra- and infralabial glands are more uniform in morphology, being the second most developed in almost all the sampled species. Premaxillary glands distinct from the supralabial glands were observed in five species (Myron richardsonii, Bitia hydroides, Cantoria violacea, Fordonia leucobalia, and Gerarda prevostiana), in addition to Cerberus rynchops, the only species in which this condition was previously documented associated with the excretion of salt. In the three species of the saltwater group of homalopsids (C. violacea, F. leucobalia, and G. prevostiana), the premaxillary glands also extend posteriorly, occupying a large area above the supralabial gland, a condition not observed in any other species of snake studied thus far. Character evolution analyses indicate that premaxillary glands differentiated from the supralabial gland and evolved independently three or four times in the family, always in lineages that invaded marine habitats. Our results suggest that the differentiated premaxillary glands are likely salt glands, as is the case in C. rynchops. If corroborated, this increases to six or seven the number of independent evolutionary origins of salt glands in snakes that have undergone an evolutionary transition to marine life.

Anatomy of the female reproductive tract organs of the brown anole (Anolis sagrei).

Female reproduction in squamate reptiles (lizards and snakes) is highly diverse and mode of reproduction, clutch size, and reproductive tract morphology all vary widely across this group of ~11,000 species. Recently, CRISPR genome editing techniques that require manipulation of the female reproductive anatomy have been developed in this group, making a more complete understanding of this anatomy essential. We describe the adult female reproductive anatomy of the model reptile the brown anole (Anolis sagrei). We show that the brown anole female reproductive tract has three distinct anterior-to-posterior regions, the infundibulum, the glandular uterus, and the nonglandular uterus. The infundibulum has a highly ciliated epithelial lip, a region where the epithelium is inverted so that cilia are present on the inside and outside of the tube. The glandular uterus has epithelial ducts that are patent with a lumen as well as acinar structures with a lumen. The nonglandular uterus has a heterogeneous morphology from anterior to posterior, with a highly folded, ciliated epithelium transitioning to a stratified squamous epithelium. This transition is accompanied by a loss of keratin-8 expression and together, these changes are similar to the morphological and gene expression changes that occur in the mammalian cervix. We recommend that description of the nonglandular uterus include the regional sub-specification of a "cervix" and "vagina" as this terminology change more accurately describes the morphology. Our data extend histological studies of reproductive organ morphology in reptiles and expand our understanding of the variation in reproductive system anatomy across squamates and vertebrates.

Embryonic development of the pelvic girdle and hindlimb skeletal elements in Anilius scytale (Linnaeus, 1758) (Serpentes: Aniliidae).

Anilius scytale is the sister lineage of all other alethinophidian snakes. Morphology of the hind limb complex in adult A. scytale (Aniliidae) has been documented. We herein, for the first time, describe the embryology of the skeletal elements of its hind limb and pelvic girdle and contextualize the evolution of these structures. We identified pregnant females of A. scytale in the Herpetology Collection of the Museu Paraense Emílio Goeldi and separated 40 embryos. The embryos were sequentially staged using external and internal anatomy, collectively comprising a developmental series representing six stages. We cleared-stained one specimen of stages 31, 34, 36, and 37. Using the embryological information gleaned from A. scytale, we reinterpret evidence relating to the ossification of the pelvis and hindlimbs. In A. scytale hindlimb buds develop as transient structures that developed before Stage 30 and regresses in subsequent stages. There is no external or internal evidence of the forelimb or scapular girdle. From Stage 31 onwards the ischium, pubis, ilium, femur and zeugopodial cartilages are visible. Pubis and femur ossify towards the end of embryonic life, and cloacal spurs do not develop in the embryo. Skeletal elements of the hindlimb and pelvic girdle develop initially in the ventral zone of the cloaca-tail region. In subsequent stages the hindlimb and pelvic girdle elements migrate dorsally, with the pubis/ischium positioned medial to the ribs. A similar process may be associated with the achievement of the condition of the pelvic girdle in adults of scolecophidians, pythonids and boids.

Foraging mode constrains the evolution of cephalic horns in lizards and snakes.

A phylogenetically diverse minority of snake and lizard species exhibit rostral and ocular appendages that substantially modify the shape of their heads. These cephalic horns have evolved multiple times in diverse squamate lineages, enabling comparative tests of hypotheses on the benefits and costs of these distinctive traits. Here, we demonstrate correlated evolution between the occurrence of horns and foraging mode. We argue that although horns may be beneficial for various functions (e.g. camouflage, defence) in animals that move infrequently, they make active foragers more conspicuous to prey and predators, and hence are maladaptive. We therefore expected horns to be more common in species that ambush prey (entailing low movement rates) rather than in actively searching (frequently moving) species. Consistent with that hypothesis, our phylogenetic comparative analysis of published data on 1939 species reveals that cephalic horns occur almost exclusively in sit-and-wait predators. This finding underlines how foraging mode constrains the morphology of squamates and provides a compelling starting point for similar studies in other animal groups.

Reconstructing the origin and early evolution of the snake brain.

Snakes represent one-eighth of terrestrial vertebrate diversity, encompassing various lifestyles, ecologies, and morphologies. However, the ecological origins and early evolution of snakes are controversial topics in biology. To address the paucity of well-preserved fossils and the caveats of osteological traits for reconstructing snake evolution, we applied a different ecomorphological hypothesis based on high-definition brain reconstructions of extant Squamata. Our predictive models revealed a burrowing lifestyle with opportunistic behavior at the origin of crown snakes, reflecting a complex ancestral mosaic brain pattern. These findings emphasize the importance of quantitatively tracking the phenotypic diversification of soft tissues-including the accurate definition of intact brain morphological traits such as the cerebellum-in understanding snake evolution and vertebrate paleobiology. Furthermore, our study highlights the power of combining extant and extinct species, soft tissue reconstructions, and osteological traits in tracing the deep evolution of not only snakes but also other groups where fossil data are scarce.

A conserved tooth resorption mechanism in modern and fossil snakes.

Whether snakes evolved their elongated, limbless bodies or their specialized skulls and teeth first is a central question in squamate evolution. Identifying features shared between extant and fossil snakes is therefore key to unraveling the early evolution of this iconic reptile group. One promising candidate is their unusual mode of tooth replacement, whereby teeth are replaced without signs of external tooth resorption. We reveal through histological analysis that the lack of resorption pits in snakes is due to the unusual action of odontoclasts, which resorb dentine from within the pulp of the tooth. Internal tooth resorption is widespread in extant snakes, differs from replacement in other reptiles, and is even detectable via non-destructive µCT scanning, providing a method for identifying fossil snakes. We then detected internal tooth resorption in the fossil snake Yurlunggur, and one of the oldest snake fossils, Portugalophis, suggesting that it is one of the earliest innovations in Pan-Serpentes, likely preceding limb loss.

Diet and habit explain head-shape convergences in natricine snakes.

The concept of ecomorphs, whereby species with similar ecologies have similar phenotypes regardless of their phylogenetic relatedness, is often central to discussions regarding the relationship between ecology and phenotype. However, some aspects of the concept have been questioned, and sometimes species have been grouped as ecomorphs based on phenotypic similarity without demonstrating ecological similarity. Within snakes, similar head shapes have convergently evolved in species living in comparable environments and/or with similar diets. Therefore, ecomorphs could exist in some snake lineages, but this assertion has rarely been tested for a wide-ranging group within a single framework. Natricine snakes (Natricinae) are ecomorphologically diverse and currently distributed in Asia, Africa, Europe and north-central America. They are primarily semiaquatic or ground-dwelling terrestrial snakes, but some are aquatic, burrowing or aquatic and burrowing in habit and may be generalist or specialist in diet. Thus, natricines present an interesting system to test whether snakes from different major habit categories represent ecomorphs. We quantify morphological similarity and disparity in head shape among 191 of the ca. 250 currently recognized natricine species and apply phylogenetic comparative methods to test for convergence. Natricine head shape is largely correlated with habit, but in some burrowers is better explained by dietary specialism. Convergence in head shape is especially strong for aquatic burrowing, semiaquatic and terrestrial ecomorphs and less strong for aquatic and burrowing ecomorphs. The ecomorph concept is useful for understanding natricine diversity and evolution, though would benefit from further refinement, especially for aquatic and burrowing taxa.

Perils of ingesting harmful prey by advanced snakes.

The advanced snakes (Alethinophidia) include the extant snakes with a highly evolved head morphology providing increased gape and jaw flexibility. Along with other physiological and morphological adaptations, this allows them to immobilize, ingest, and transport prey that may be disproportionately large or presents danger to the predator from bites, teeth, horns, or spines. Reported incidents of snakes failing to consume prey and being injured or killed during feeding mostly reflect information in the form of natural-history notes. Here we provide the first extensive review of such incidents, including 101 publications describing at least 143 cases of mortality (including six of 'multiple individuals') caused by ingestion or attempted consumption of injurious prey. We also report on 15 previously unpublished injurious feeding incidents from the USA, Austria, and Bulgaria, including mortality of five juvenile piscivorous dice snakes (Natrix tessellata) from a single location. Occurrences are spread across taxa, with mortality documented for at least 73 species from eight families and 45 genera. Incidents were generally well represented within each of three major categories: oversized prey (40.6%), potentially harmful prey (40.6%), and predator's behavioural/mechanical errors (18.9%). Reptile (33%) and fish (26%) prey caused disproportionately high mortality compared to mammals (16%). Feeding can be dangerous throughout a snake's life, with the later stages of feeding likely being more perilous. The number of reports has increased over time, and the data seem biased towards localities with a higher number of field-working herpetologists. We propose a standardized framework, comprising a set of basic information that should ideally be collected and published, and which could be useful as a template for future data collection, reporting, and analyses. We conclude that incidents of mortality during feeding are likely to be more common than previously assumed, and this hypothesis has implications for the ecology of persistence where populations are impacted by changing trophic environments.

Phylogeography of cliff racer (Platyceps rhodorachis Jan, 1865) from Punjab, Pakistan / Filografia de penhasco (Platyceps rhodorachis Jan, 1865) de Punjab, Paquistão

The present study reports the existence of cliff racer, Platyceps rhodorachis from the plains of Punjab, Pakistan. A total of 10 specimens were captured during the field surveys from June to September, 2018 from different sites of Punjab. Platyceps rhodorachis was identify on the basis of morphology and confirmed through COI gene sequences. The obtained DNA sequences have shown reliable and exact species identification. Newly produced DNA sequences of Platyceps rhodorachis were submitted to GenBank and accession numbers were obtained (MK936174.1, MK941839.1 and MT790210.1). N-J tree based on COI sequences of Platyceps rhodorachis clearly separated as out-group with other members of family Colubridae based on p-distance. The intra-specific genetic variation ranges from 12% to 18%. The DNA sequences of Platyceps rhodorachis kashmirensis, Platyceps rhodorachis ladacensis, Platyceps ventromaculatus, Platyceps ventromaculatus bengalensis and Platyceps ventromaculatus indusai are not available at NCBI to validate their taxonomic positions. In our recommendations, a large scale molecular based identification of Pakistan’s herpetofauna is required to report more new or subspecies from country.

O presente estudo relata a existência de um corredor de penhasco, Platyceps rhodorachis, das planícies de Punjab, Paquistão. Um total de 10 espécimes foi capturado durante os levantamentos de campo de junho a setembro de 2018 em diferentes locais de Punjab. Platyceps rhodorachis foi identificada com base na morfologia e confirmada por meio de sequências do gene COI. As sequências de DNA obtidas mostraram identificação de espécies confiável e exata. Sequências de DNA de Platyceps rhodorachis recém-produzidas foram submetidas ao GenBank e os números de acesso foram obtidos (MK936174.1, MK941839.1 e MT790210.1). Árvore N-J baseada em sequências COI de Platyceps rhodorachis claramente separadas como out-group com outros membros da família Colubridae com base na distância-p. A variação genética intraespecífica varia de 12% a 18%. As sequências de DNA de Platyceps rhodorachis kashmirensis, Platyceps rhodorachis ladacensis, Platyceps ventromaculatus, Platyceps ventromaculatus bengalensis e Platyceps ventromaculatus indusai não estão disponíveis no NCBI para validar suas posições taxonômicas. Em nossas recomendações, uma identificação de base molecular em grande escala da herpetofauna do Paquistão é necessária para relatar mais novas ou subespécies do país.

Live birth in a 47-million-year-old snake.

Viviparity is a widespread reproductive trait in snakes, although fossil evidence bearing on its evolution is extremely sparse. Here, we report an exceptional specimen of the minute booid snake Messelophis variatus recovered in the paleolake of the Messel Formation (early-middle Eocene, Germany). This gravid female contains at least two embryos located in the posterior third of the trunk region. The morphology, size, and degree of ossification of the cranial and postcranial remains indicate they correspond with late embryos. This specimen documents the first occurrence of viviparity in a fossil snake and extends the temporal distribution of this reproductive strategy in booid snakes by over 47 Ma. The evolution of viviparity in squamates has traditionally been associated with cold climates, but its presence at the dawn of the evolution of booids during early Palaeogene thermal peaks indicates that viviparity may have evolved under different selective pressures in this clade.

Synchrotron tomography of a stem lizard elucidates early squamate anatomy.

Squamates (lizards and snakes) include more than 10,000 living species, descended from an ancestor that diverged more than 240 million years ago from that of their closest living relative, Sphenodon. However, a deficiency of fossil evidence1-7, combined with serious conflicts between molecular and morphological accounts of squamate phylogeny8-13 (but see ref. 14), has caused uncertainty about the origins and evolutionary assembly of squamate anatomy. Here we report the near-complete skeleton of a stem squamate, Bellairsia gracilis, from the Middle Jurassic epoch of Scotland, documented using high-resolution synchrotron phase-contrast tomography. Bellairsia shares numerous features of the crown group, including traits related to cranial kinesis (an important functional feature of many extant squamates) and those of the braincase and shoulder girdle. Alongside these derived traits, Bellairsia also retains inferred ancestral features including a pterygoid-vomer contact and the presence of both cervical and dorsal intercentra. Phylogenetic analyses return strong support for Bellairsia as a stem squamate, suggesting that several features that it shares with extant gekkotans are plesiomorphies, consistent with the molecular phylogenetic hypothesis that gekkotans are early-diverging squamates. We also provide confident support of stem squamate affinities for the enigmatic Oculudentavis. Our findings indicate that squamate-like functional features of the suspensorium, braincase and shoulder girdle preceded the origin of their palatal and vertebral traits and indicate the presence of advanced stem squamates as persistent components of terrestrial assemblages up to at least the middle of the Cretaceous period.

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.

The relationship between hard and soft tissue structures of the eye in extant lizards.

The sizes of the eye structures, such as the lens diameter and the axial length, are important factors for the visual performance and are considered to be related to the mode of life. Although the size of these soft structures cannot be directly observed in fossil taxa, such information may be obtained from measuring size and morphology of the bony scleral ossicle ring, which is present in the eyes of extant saurospids, excluding crocodiles and snakes, and is variously preserved in fossil taxa. However, there have been only a few studies investigating the relationships between the size, the scleral ossicle ring, and soft structures of the eye. We investigated such relationships among the eye structures in extant Squamata, to establish the basis for inferring the size of the soft structures in the eye in fossil squamates. Three-dimensional morphological data on the eye and head region of 59 lizard species covering most major clades were collected using micro-computed tomography scanners. Strong correlations were found between the internal and external diameters of the scleral ossicle ring and soft structures. The tight correlations found here will allow reliable estimations of the sizes of soft structures and inferences on the visual performance and mode of life in fossil squamates, based on the diameters of their preserved scleral ossicle rings. Furthermore, the comparison of the allometric relationships between structures in squamates eyes with those in avian eyes suggest the possibility that the similarities of these structures closely reflect the mechanism of accommodation.

Coordinating tiny limbs and long bodies: Geometric mechanics of lizard terrestrial swimming.

Although typically possessing four limbs and short bodies, lizards have evolved diverse morphologies, including elongate trunks with tiny limbs. Such forms are hypothesized to aid locomotion in cluttered/fossorial environments but propulsion mechanisms (e.g., the use of body and/or limbs to interact with substrates) and potential body/limb coordination remain unstudied. Here, we use biological experiments, a geometric theory of locomotion, and robophysical models to investigate body-limb coordination in diverse lizards. Locomotor field studies in short-limbed, elongate lizards (Brachymeles and Lerista) and laboratory studies of fully limbed lizards (Uma scoparia and Sceloporus olivaceus) and a snake (Chionactis occipitalis) reveal that body-wave dynamics can be described by a combination of standing and traveling waves; the ratio of the amplitudes of these components is inversely related to the degree of limb reduction and body elongation. The geometric theory (which replaces laborious calculation with diagrams) helps explain our observations, predicting that the advantage of traveling-wave body undulations (compared with a standing wave) emerges when the dominant thrust-generation mechanism arises from the body rather than the limbs and reveals that such soil-dwelling lizards propel via "terrestrial swimming" like sand-swimming lizards and snakes. We test our hypothesis by inducing the use of traveling waves in stereotyped lizards via modulating the ground-penetration resistance. Study of a limbed/undulatory robophysical model demonstrates that a traveling wave is beneficial when propulsion is generated by body-environment interaction. Our models could be valuable in understanding functional constraints on the evolutionary processes of elongation and limb reduction as well as advancing robot designs.

Cranial osteology of Hypoptophis (Aparallactinae: Atractaspididae: Caenophidia), with a discussion on the evolution of its fossorial adaptations.

Fossoriality evolved early in snakes, and has left its signature on the cranial morphology of many extinct Mesozoic and early Caenozoic forms. Knowledge of the cranial osteology of extant snakes is indispensable for associating the crania of extinct lineages with a particular mode of life; this applies to fossorial taxa as well. In the present work, we provide a detailed description of the cranium of Hypoptophis wilsonii, a member of the subfamily Aparallactinae, using micro-computed tomography (CT). This is also the first thorough micro-CT-based description of any snake assigned to this African subfamily of predominantly mildly venomous, fossorial, and elusive snakes. The cranium of Hypoptophis is adapted for a fossorial lifestyle, with increased consolidation of skull bones. Aparallactines show a tendency toward reduction of maxillary length by bringing the rear fangs forward. This development attains its pinnacle in the sister subfamily Atractaspidinae, in which the rear fang has become the "front fang" by a loss of the part of the maxilla lying ahead of the fang. These dentitional changes likely reflect adaptation to subdue prey in snug burrows. An endocast of the inner ear of Hypoptophis shows that this genus has the inner ear typical of fossorial snakes, with a large, globular sacculus. A phylogenetic analysis based on morphology recovers Hypoptophis as a sister taxon to Aparallactus. We also discuss the implications of our observations on the burrowing origin hypothesis of snakes.

Sharp and fully loaded: 3D tissue reconstruction reveals how snake fangs stay deadly during fang replacement.

Snake venom is produced, transported and delivered by the sophisticated venom delivery system (VDS). When snakes bite, the venom travels from the venom gland through the venom duct into needle-like fangs that inject it into their prey. To counteract breakages, fangs are continuously replaced throughout life. Currently, the anatomy of the connection between the duct and the fang has not been described, and the mechanism by which the duct is reconnected to the replacement fang has not been identified. We examined the VDS in 3D in representative species from two families and one subfamily (Elapidae, Viperidae, Atractaspidinae) using contrast-enhanced microCT (diceCT), followed by dissection and histology. We observed that the venom duct bifurcates immediately anterior to the fangs so that both the original and replacement fangs are separately connected and functional in delivering venom. When a fang is absent, the canal leading to the empty position is temporarily closed. We found that elapid snakes have a crescent-shaped venom reservoir where venom likely pools before it enters the fang. These findings form the final piece of the puzzle of VDS anatomy in front-fanged venomous snakes. Additionally, they provide further evidence for independent evolution of the VDS in these three snake taxa.