The respiratory system influences flight mechanics in soaring birds.

The subpectoral diverticulum (SPD) is an extension of the respiratory system in birds that is located between the primary muscles responsible for flapping the wing1,2. Here we survey the pulmonary apparatus in 68 avian species, and show that the SPD was present in virtually all of the soaring taxa investigated but absent in non-soarers. We find that this structure evolved independently with soaring flight at least seven times, which indicates that the diverticulum might have a functional and adaptive relationship with this flight style. Using the soaring hawks Buteo jamaicensis and Buteo swainsoni as models, we show that the SPD is not integral for ventilation, that an inflated SPD can increase the moment arm of cranial parts of the pectoralis, and that pectoralis muscle fascicles are significantly shorter in soaring hawks than in non-soaring birds. This coupling of an SPD-mediated increase in pectoralis leverage with force-specialized muscle architecture produces a pneumatic system that is adapted for the isometric contractile conditions expected in soaring flight. The discovery of a mechanical role for the respiratory system in avian locomotion underscores the functional complexity and heterogeneity of this organ system, and suggests that pulmonary diverticula are likely to have other undiscovered secondary functions. These data provide a mechanistic explanation for the repeated appearance of the SPD in soaring lineages and show that the respiratory system can be co-opted to provide biomechanical solutions to the challenges of flight and thereby influence the evolution of avian volancy.

Dynamic evolutionary interplay between ontogenetic skull patterning and whole-head integration.

The arrangement and morphology of the vertebrate skull reflect functional and ecological demands, making it a highly adaptable structure. However, the fundamental developmental and macroevolutionary mechanisms leading to different vertebrate skull phenotypes remain unclear. Here we exploit the morphological diversity of squamate reptiles to assess the developmental and evolutionary patterns of skull variation and covariation in the whole head. Our geometric morphometric analysis of a complex squamate ontogenetic dataset (209 specimens, 169 embryos, 44 species), covering stages from craniofacial primordia to fully ossified bones, reveals that morphological differences between snake and lizard skulls arose gradually through changes in spatial relationships (heterotopy) followed by alterations in developmental timing or rate (heterochrony). Along with dynamic spatiotemporal changes in the integration pattern of skull bone shape and topology with surrounding brain tissues and sensory organs, we identify a relatively higher phenotypic integration of the developing snake head compared with lizards. The eye, nasal cavity and Jacobson's organ are pivotal in skull morphogenesis, highlighting the importance of sensory rearrangements in snake evolution. Furthermore, our findings demonstrate the importance of early embryonic, ontogenetic and tissue interactions in shaping craniofacial evolution and ecological diversification in squamates, with implications for the nature of cranio-cerebral relations across vertebrates.

Validating osteological correlates for the hepatic piston in the American alligator (Alligator mississippiensis).

Unlike the majority of sauropsids, which breathe primarily through costal and abdominal muscle contractions, extant crocodilians have evolved the hepatic piston pump, a unique additional ventilatory mechanism powered by the diaphragmaticus muscle. This muscle originates from the bony pelvis, wrapping around the abdominal viscera, extending cranially to the liver. The liver then attaches to the caudal margin of the lungs, resulting in a sub-fusiform morphology for the entire "pulmo-hepatic-diaphragmatic" structure. When the diaphragmaticus muscle contracts during inspiration, the liver is pulled caudally, lowering pressure in the thoracolumbar cavity, and inflating the lungs. It has been established that the hepatic piston pump requires the liver to be displaced to ventilate the lungs, but it has not been determined if the lungs are freely mobile or if the pleural tissues stretch ventrally. It has been hypothesized that the lungs are able to slide craniocaudally with the liver due to the smooth internal ceiling of the thoracolumbar cavity. We assess this through ultrasound video and demonstrate quantitatively and qualitatively that the pulmonary tissues are sliding craniocaudally across the interior thoracolumbar ceiling in actively ventilating live juvenile, sub-adult, and adult individuals (n = 7) of the American alligator (Alligator mississippiensis) during both natural and induced ventilation. The hepatic piston is a novel ventilatory mechanism with a relatively unknown evolutionary history. Questions related to when and under what conditions the hepatic piston first evolved have previously been left unanswered due to a lack fossilized evidence for its presence or absence. By functionally correlating specific characters in the axial skeleton to the hepatic piston, these osteological correlates can be applied to fossil taxa to reconstruct the evolution of the hepatic piston in extinct crocodylomorph archosaurs.

Perspectives on lung visualization: Three-dimensional anatomical modeling of computed and micro-computed tomographic data in comparative evolutionary morphology and medicine with applications for COVID-19.

The vertebrate respiratory system is challenging to study. The complex relationship between the lungs and adjacent tissues, the vast structural diversity of the respiratory system both within individuals and between taxa, its mobility (or immobility) and distensibility, and the difficulty of quantifying and visualizing functionally important internal negative spaces have all impeded descriptive, functional, and comparative research. As a result, there is a relative paucity of three-dimensional anatomical information on this organ system in all vertebrate groups (including humans) relative to other regions of the body. We present some of the challenges associated with evaluating and visualizing the vertebrate respiratory system using computed and micro-computed tomography and its subsequent digital segmentation. We discuss common mistakes to avoid when imaging deceased and live specimens and various methods for merging manual and threshold-based segmentation approaches to visualize pulmonary tissues across a broad range of vertebrate taxa, with a particular focus on sauropsids (reptiles and birds). We also address some of the recent work in comparative evolutionary morphology and medicine that have used these techniques to visualize respiratory tissues. Finally, we provide a clinical study on COVID-19 in humans in which we apply modeling methods to visualize and quantify pulmonary infection in the lungs of human patients.

Locomotor characteristics of the ground-walking chameleon Brookesia superciliaris.

Understanding the locomotor characteristics of early diverging ground-walking chameleons (members of the genera Brookesia, Rhampholeon, Palleon, and Rieppeleon) can help to explain how their unique morphology is adapted to fit their environment and mode of life. However, nearly all quantitative studies of chameleon locomotion thus far have focused on the larger "true arboreal" chameleons. We investigated kinematics and spatiotemporal gait characteristics of the Brown Leaf Chameleon (Brookesia superciliaris) on different substrates and compared them with true arboreal chameleons, nonchameleon lizards, and other small arboreal animals. Brookesia exhibits a combination of locomotor traits, some of which are traditionally arboreal, others more terrestrial, and a few that are very unusual. Like other chameleons, Brookesia moved more slowly on narrow dowels than on broad planks (simulating arboreal and terrestrial substrates, respectively), and its speed was primarily regulated by stride frequency rather than stride length. While Brookesia exhibits the traditionally arboreal trait of a high degree of humeral protraction at the beginning of stance, unlike most arboreal tetrapods, it uses smaller shoulder and hip excursions on narrower substrates, possibly reflecting its more terrestrial habits. When moving at very slow speeds, Brookesia often adopts an unusual footfall pattern, lateral-sequence lateral-couplets. Because Brookesia is a member of one of the earliest-diverging groups of chameleons, its locomotion may provide a good model for an intermediate stage in the evolution of arboreal chameleons. Thus, the transition to a fully arboreal way of life in "true arboreal" chameleons may have involved changes in spatiotemporal and kinematic characteristics as well as morphology.

Architecture of the bronchial tree in Cuvier's dwarf caiman (Paleosuchus palpebrosus).

We imaged the lungs of five Cuvier's dwarf caiman (Paleosuchus palpebrosus) via computed tomography (CT) and micro-computed tomography (µCT) and compared these data to the lungs of the American alligator (Alligator mississippiensis). These data demonstrate anatomical commonalities between the lungs of P. palpebrosus and A. mississippiensis, and a few notable differences. The structural similarities are (a) a proximally narrow, distally widened, hook-shaped primary bronchus; (b) a cervical ventral bronchus that branches of the primary bronchus and immediately makes a hairpin turn toward the apex of the lung; (c) a sequential series of dorsobronchi arising from the primary bronchus caudal to the cervical ventral bronchus; (d) intraspecifically highly variable medial sequence of secondary airways; (e) sac-like laterobronchi; and (f) grossly dead-ended caudal group bronchi in the caudal and ventral aspects of the lung. The primary differences between the two taxa are in the overall number of large bronchi (fewer in P. palpebrosus), and the number of branches that contribute to the cardiac regions. Imaging data of both a live and deceased specimen under varying states (postprandial, fasting, total lung capacity, open to atmosphere) indicate that the caudal margin and position of the lungs shift craniocaudally relative to the vertebral column. These imaging data suggest that the smooth thoracic ceiling may be correlated to visceral movement during ventilation, but this hypothesis warrants validation. These results provide the scaffolding for future comparisons between crocodilians, for generating preliminary reconstructions of the ancestral crocodilian bronchial tree, and establishing new hypotheses of bronchial homology across Archosauria.

Comparative development of limb musculature in phylogenetically and ecologically divergent lizards.

BACKGROUND: Squamate reptiles (lizards, snakes, and amphisbaenians) exhibit incredible diversity in their locomotion, behavior, morphology, and ecological breadth. Although they often are used as models of locomotor diversity, surprisingly little attention has been given to muscle development in squamate reptiles. In fact, the most detailed examination was conducted almost 80 years ago and solely focused on the proximal limb regions. Herein, we present forelimb and hindlimb muscle morphogenesis data for three lizard species with different locomotion and feeding strategies: the desert grassland whiptail lizard, the central bearded dragon, and the veiled chameleon. This study fills critical gaps in our understanding of muscle morphogenesis in squamate reptiles and presents a comparative and temporospatial analysis of muscle development. RESULTS: Our results reveal a conserved pattern of early muscle development among lizards with different adult morphologies and ecologies. The variations that exist are concentrated in distal regions, particularly the specialized autopodia of chameleons, where differentiation of muscles associated with the digits is delayed. CONCLUSIONS: The chameleon autopod provides an example of major evolutionary modifications to the skeleton with only minor disruption of the conserved order and pattern of limb muscle development. This robustness of muscle patterning facilitates the evolution of extreme yet functional phenotypes.

Environmental Thermal Stress Induces Neuronal Cell Death and Developmental Malformations in Reptiles.

Every stage of organismal life history is being challenged by global warming. Many species are already experiencing temperatures approaching their physiological limits; this is particularly true for ectothermic species, such as lizards. Embryos are markedly sensitive to thermal insult. Here, we demonstrate that temperatures currently experienced in natural nesting areas can modify gene expression levels and induce neural and craniofacial malformations in embryos of the lizard Anolis sagrei. Developmental abnormalities ranged from minor changes in facial structure to significant disruption of anterior face and forebrain. The first several days of postoviposition development are particularly sensitive to this thermal insult. These results raise new concern over the viability of ectothermic species under contemporary climate change. Herein, we propose and test a novel developmental hypothesis that describes the cellular and developmental origins of those malformations: cell death in the developing forebrain and abnormal facial induction due to disrupted Hedgehog signaling. Based on similarities in the embryonic response to thermal stress among distantly related species, we propose that this developmental hypothesis represents a common embryonic response to thermal insult among amniote embryos. Our results emphasize the importance of adopting a broad, multidisciplinary approach that includes both lab and field perspectives when trying to understand the future impacts of anthropogenic change on animal development.

Serpentovirus (Nidovirus) and Orthoreovirus Coinfection in Captive Veiled Chameleons (Chamaeleo calyptratus) with Respiratory Disease.

Serpentoviruses are an emerging group of nidoviruses known to cause respiratory disease in snakes and have been associated with disease in other non-avian reptile species (lizards and turtles). This study describes multiple episodes of respiratory disease-associated mortalities in a collection of juvenile veiled chameleons (Chamaeleo calyptratus). Histopathologic lesions included rhinitis and interstitial pneumonia with epithelial proliferation and abundant mucus. Metagenomic sequencing detected coinfection with two novel serpentoviruses and a novel orthoreovirus. Veiled chameleon serpentoviruses are most closely related to serpentoviruses identified in snakes, lizards, and turtles (approximately 40-50% nucleotide and amino acid identity of ORF1b). Veiled chameleon orthoreovirus is most closely related to reptilian orthoreoviruses identified in snakes (approximately 80-90% nucleotide and amino acid identity of the RNA-dependent RNA polymerase). A high prevalence of serpentovirus infection (>80%) was found in clinically healthy subadult and adult veiled chameleons, suggesting the potential for chronic subclinical carriers. Juvenile veiled chameleons typically exhibited a more rapid progression compared to subadults and adults, indicating a possible age association with morbidity and mortality. This is the first description of a serpentovirus infection in any chameleon species. A causal relationship between serpentovirus infection and respiratory disease in chameleons is suspected. The significance of orthoreovirus coinfection remains unknown.

Development of sexual dimorphism in two sympatric skinks with different growth rates.

Sexual size dimorphism (SSD) is widespread in animals, especially in lizards (Reptilia: Squamata), and is driven by fecundity selection, male-male competition, or other adaptive hypotheses. However, these selective pressures may vary through different life history periods; thus, it is essential to assess the relationship between growth and SSD. In this study, we tracked SSD dynamics between a "fading-tail color skink" (blue tail skink whose tail is only blue during its juvenile stage: Plestiodon elegans) and a "nonfade color" tail skink (retains a blue tail throughout life: Plestiodon quadrilineatus) under a controlled experimental environment. We fitted growth curves of morphological traits (body mass, SVL, and TL) using three growth models (Logistic, Gompertz, and von Bertalanffy). We found that both skinks have male-biased SSD as adults. Body mass has a higher goodness of fit (as represented by very high R 2 values) using the von Bertalanffy model than the other two models. In contrast, SVL and TL for both skinks had higher goodness of fit when using the Gompertz model. Two lizards displayed divergent life history tactics: P. elegans grows faster, matures earlier (at 65 weeks), and presents an allometric growth rate, whereas P. quadrilineatus grows slower, matures later (at 106 weeks), and presents an isometric growth rate. Our findings imply that species- and sex-specific trade-offs in the allocation of energy to growth and reproduction may cause the growth patterns to diverge, ultimately resulting in the dissimilar patterns of SSD.

Filling in the phylogenetic gaps: Induction, migration, and differentiation of neural crest cells in a squamate reptile, the veiled chameleon (Chamaeleo calyptratus).

Neural crest cells comprise a migratory progenitor cell population that differentiate into cell types such as neurons and glia of the peripheral nervous system, pigment cells, hormone secreting cells in glands, and skeletal and connective tissue in the head, thus making important contributions to most tissues and organs throughout the vertebrate body. The evolutionary appearance of neural crest cells is considered synonymous with the origin of vertebrates and their subsequent diversification and radiation. While the comparative biology of neural crest cells has been studied for a century and a half beginning with their discovery by Wilhelm His in 1868, most of our understanding of their development and function has come from a small number of species. Thus, critical gaps exist in our understanding of how neural crest cells mediate evolution and development. This is particularly true with respect to squamate reptiles (lizards, snakes, amphisbaenians), which account for approximately one-third of all living tetrapods. Here, we present veiled chameleons (Chamaeleo calyptratus) as a model system for studying neural crest cell development in squamates. Chameleons exhibit various morphological specializations associated with an arboreal lifestyle that may have been facilitated through neural crest cells acting as a conduit for evolutionary change.

The transcriptome of the veiled chameleon (Chamaeleo calyptratus): A resource for studying the evolution and development of vertebrates.

PURPOSE: The veiled chameleon (Chamaeleo calyptratus) is an emerging model system for studying functional morphology and evolutionary developmental biology (evo-devo). Chameleons possess body plans that are highly adapted to an arboreal life style, featuring laterally compressed bodies, split hands/ft for grasping, a projectile tongue, turreted independently moving eyes, and a prehensile tail. Despite being one of the most phenotypically divergent clades of tetrapods, genomic resources for chameleons are severely lacking. METHODS: To address this lack of resources, we used RNAseq to generate 288 million raw Illumina sequence reads from four adult tissues (male and female eyes and gonads) and whole embryos at three distinct developmental stages. We used these data to assemble a largely complete de novo transcriptome consisting of only 82 952 transcripts. In addition, a majority of assembled transcripts (67%) were successfully annotated. RESULTS: We then demonstrated the utility of these data in the context of studying visual system evolution by examining the content of veiled chameleon opsin genes to show that chameleons possess all five ancestral tetrapod opsins. CONCLUSION: We present this de novo, annotated, multi-tissue transcriptome assembly for the Veiled Chameleon, Chamaeleo calyptratus, as a resource to address a range of evolutionary and developmental questions. The associated raw reads and final annotated transcriptome assembly are freely available for use on NCBI and Figshare, respectively.

Lifting the Veil on Reptile Embryology: The Veiled Chameleon (Chamaeleo calyptratus) as a Model System to Study Reptilian Development.

Living amniotes comprise three major phylogenetic lineages: mammals, birds, and non-avian reptiles. Mouse and avian embryos continue to be the primary species used in experimental settings to further our knowledge and understanding of the genetics and embryology of amniotes. In comparison, non-avian reptiles, which constitute up to 40% of all living amniotes, have played a comparatively minor role. Studies of non-avian reptiles are, however, paramount for providing insights into the evolutionary changes that occurred in the transition from reptilian-like amniote ancestors to derived mammalian and avian species. Here, we introduce the Veiled Chameleon, a squamate reptile, as a new experimental model for examining fundamental questions in development, evolution, and disease.

Comparative musculoskeletal anatomy of chameleon limbs, with implications for the evolution of arboreal locomotion in lizards and for teratology.

Chameleon species have recently been adopted as models for evo-devo and macroevolutionary processes. However, most anatomical and developmental studies of chameleons focus on the skeleton, and information about their soft tissues is scarce. Here, we provide a detailed morphological description based on contrast enhanced micro-CT scans and dissections of the adult phenotype of all the forelimb and hindlimb muscles of the Veiled Chameleon (Chamaeleo calyptratus) and compare these muscles with those of other chameleons and lizards. We found the appendicular muscle anatomy of chameleons to be surprisingly conservative considering the remarkable structural and functional modifications of the limb skeleton, particularly the distal limb regions. For instance, the zygodactyl autopodia of chameleons are unique among tetrapods, and the carpals and tarsals are highly modified in shape and number. However, most of the muscles usually present in the manus and pes of other lizards are present in the same configuration in chameleons. The most obvious muscular features related to the peculiar opposable autopodia of chameleons are: (1) presence of broad, V-shaped plantar and palmar aponeuroses, and absence of intermetacarpales and intermetatarsales, between the digits separated by the cleft in each autopod; (2) oblique orientation of the superficial short flexors originating from these aponeuroses, which may allow these muscles to act as powerful adductors of the "super-digits"; and (3) well-developed abductor digiti minimi muscles and abductor pollicis/hallucis brevis muscles, which may act as powerful abductors of the "super-digits."

Dinosaurs, Chameleons, Humans, and Evo-Devo Path: Linking Étienne Geoffroy's Teratology, Waddington's Homeorhesis, Alberch's Logic of "Monsters," and Goldschmidt Hopeful "Monsters".

Since the rise of evo-devo (evolutionary developmental biology) in the 1980s, few authors have attempted to combine the increasing knowledge obtained from the study of model organisms and human medicine with data from comparative anatomy and evolutionary biology in order to investigate the links between development, pathology, and macroevolution. Fortunately, this situation is slowly changing, with a renewed interest in evolutionary developmental pathology (evo-devo-path) in the past decades, as evidenced by the idea to publish this special, and very timely, issue on "Developmental Evolution in Biomedical Research." As all of us have recently been involved, independently, in works related in some way or another with evolution and developmental anomalies, we decided to join our different perspectives and backgrounds in the present contribution for this special issue. Specifically, we provide a brief historical account on the study of the links between evolution, development, and pathologies, followed by a review of the recent work done by each of us, and then by a general discussion on the broader developmental and macroevolutionary implications of our studies and works recently done by other authors. Our primary aims are to highlight the strength of studying developmental anomalies within an evolutionary framework to understand morphological diversity and disease by connecting the recent work done by us and others with the research done and broader ideas proposed by authors such as Étienne Geoffroy Saint-Hilaire, Waddington, Goldschmidt, Gould, and Per Alberch, among many others to pave the way for further and much needed work regarding abnormal development and macroevolution.

Hand/foot splitting and the 're-evolution' of mesopodial skeletal elements during the evolution and radiation of chameleons.

BACKGROUND: One of the most distinctive traits found within Chamaeleonidae is their split/cleft autopodia and the simplified and divergent morphology of the mesopodial skeleton. These anatomical characteristics have facilitated the adaptive radiation of chameleons to arboreal niches. To better understand the homology of chameleon carpal and tarsal elements, the process of syndactyly, cleft formation, and how modification of the mesopodial skeleton has played a role in the evolution and diversification of chameleons, we have studied the Veiled Chameleon (Chamaeleo calyptratus). We analysed limb patterning and morphogenesis through in situ hybridization, in vitro whole embryo culture and pharmacological perturbation, scoring for apoptosis, clefting, and skeletogenesis. Furthermore, we framed our data within a phylogenetic context by performing comparative skeletal analyses in 8 of the 12 currently recognized genera of extant chameleons. RESULTS: Our study uncovered a previously underappreciated degree of mesopodial skeletal diversity in chameleons. Phylogenetically derived chameleons exhibit a 'typical' outgroup complement of mesopodial elements (with the exception of centralia), with twice the number of currently recognized carpal and tarsal elements considered for this clade. In contrast to avians and rodents, mesenchymal clefting in chameleons commences in spite of the maintenance of a robust apical ectodermal ridge (AER). Furthermore, Bmp signaling appears to be important for cleft initiation but not for maintenance of apoptosis. Interdigital cell death therefore may be an ancestral characteristic of the autopodium, however syndactyly is an evolutionary novelty. In addition, we find that the pisiform segments from the ulnare and that chameleons lack an astragalus-calcaneum complex typical of amniotes and have evolved an ankle architecture convergent with amphibians in phylogenetically higher chameleons. CONCLUSION: Our data underscores the importance of comparative and phylogenetic approaches when studying development. Body size may have played a role in the characteristic mesopodial skeletal architecture of chameleons by constraining deployment of the skeletogenic program in the smaller and earliest diverged and basal taxa. Our study challenges the 're-evolution' of osteological features by showing that 're-evolving' a 'lost' feature de novo (contrary to Dollo's Law) may instead be due to so called 'missing structures' being present but underdeveloped and/or fused to other adjacent elements (cryptic features) whose independence may be re-established under changes in adaptive selective pressure.

Captive Care, Raising, and Breeding of the Veiled Chameleon (Chamaeleo calyptratus).

Squamate reptiles comprise approximately one-third of all living amniotes. In most of these species, it is difficult to study gastrulation and neurulation because the embryos are at a late stage of development at the time of oviposition. This is not the case, however, in veiled chameleons (Chamaeleo calyptratus), which are increasingly being used as a model organism to study these and other developmental and evolutionary phenomena. Originating from the Arabian Peninsula, veiled chameleons are arboreal specialists that possess extensive morphological specializations for climbing. They naturally inhabit semitropical habitats, but they also have an almost 30-yr history of being bred in captivity. Veiled chameleons breed readily and do not require a period of cooling to induce the reproductive cycle, and females can produce ∼45-90 eggs multiple times per year. Thus, compared with other reptiles, relatively few animals are needed to maintain a productive breeding colony. Herein, we present the conditions, equipment, and techniques required for proper husbandry and breeding of veiled chameleons within a laboratory environment.

The Veiled Chameleon (Chamaeleo calyptratus Duméril and Duméril 1851): A Model for Studying Reptile Body Plan Development and Evolution.

Vertebrate model organisms have facilitated the discovery and exploration of morphogenetic events and developmental pathways that underpin normal and pathological embryological events. In contrast to amniotes such as Mus musculus (Mammalia) and Gallus gallus (Aves), our understanding of early patterning and developmental events in reptiles (particularly nonavians) remains weak. Squamate reptiles (lizards, snakes, and amphisbaenians) comprise approximately one-third of all living amniotes. But studies of early squamate development have been limited because, in most members of this lineage, embryo development at the time of oviposition is very advanced (limb bud stages and older). In many cases, squamates give birth to fully developed offspring. However, in the veiled chameleon (Chamaeleo calyptratus), embryos have progressed only to a primitive pregastrula stage at the time of oviposition. Furthermore, the body plan of the veiled chameleon is highly specialized for climbing in an arboreal environment. It possesses an entire suite of skeletal and soft anatomical modifications, including cranioskeletal ornamentation, lingual anatomy and biomechanics for projection, autopodial clefting for grasping, adaptations for rapid integumental color changes, a prehensile tail with a lack of caudal autotomy, the loss of the tympanum in the middle ear, and the acquisition of turreted eyes. Thus, C. calyptratus is an important model organism for studying the role of ecological niche specialization, as well as genetic and morphological evolution within an adaptive framework. More importantly, this species is easily bred in captivity, with only a small colony (<10 individuals) needed to obtain hundreds of embryos every year.

Bi-modal strategy of gastrulation in reptiles.

BACKGROUND: Amniote gastrulation is often described with respect to human, mouse and chick development by the presence of the primitive streak, a posterior-to-anterior midline morphological cell ingression feature that has come to define Amniote gastrulation. How this midline, ingression-based strategy of gastrulation evolved from the ancestral blastopore, a circumferential involution event in Anamniotes, is unknown. However, within the Amniote clade there exists a more diverse range of gastrulation strategies than just the primitive streak. Investigating gastrulation in a wider range of Amniotes provides a way to understand evolutionary transition from blastopore to the primitive streak. RESULTS: We analysed early to late gastrulation stages of Chamaeleo calyptratus, showing their unique morphology through confocal imaging of F-actin and laminin-stained embryos to visualise cell morphology and assess basal lamina integrity. We analysed the expression pattern of core mesodermal markers Brachyury and Fgf8 and complimented this analysis with that of the turtle, Trachemys scripta. CONCLUSIONS: Our analysis suggests that reptile gastrulation is bi-modal; primary internalization occurs anteriorly by means of an incomplete blastopore-like opening, while posteriorly the cells undergo ingression in the Brachyury-expressing blastoporal plate. This strategy stands mid-way between Anamniotes and Avians/Mammals, suggesting that blastoporal plate is a precursor of the avian primitive streak. Developmental Dynamics 244:1144-1157, 2015. © 2015 Wiley Periodicals, Inc.