Snake embryos are often tightly coiled, but the biological basis for this developmental trait remains a puzzle. To understand how and why they coil, we first investigated directions of coiling within and across 39 snake and other limbless squamate species. In early developmental stages, coiling orientation appears to be nearly fixed right-handed across multiple species. We present evidence that accelerated somitogenesis outpaces gastrointestinal extension at these stages, causing the axial column and somitic series to coil around the delayed visceral tissues. To allow this, the body axis and the gut are physically separate, where the latter forms a pillar-like structure from the stomach to the cloaca. The initial dextral directional bias in these early embryos is likely passively determined by the left-sided yolk mass. Coiling becomes looser and more inconsistent later in development when the embryos acquire mobility through maturation of axial musculoskeletal tissues and with a sufficiently extended gut. These consilient lines of observations reveal, in embryonic coiling, a series of responses of the growing tissues to spatial limitations during snake embryogenesis. As these parameters conflict at some developmental stages (e.g., the offset growth curves between the gut and the body axis), snakes apparently solve some of the many challenges to patterning their startlingly elongate bodies by coiling, which takes advantage of what could otherwise be discordant features of embryonic growth.

Digit loss has occurred independently multiple times in the order Caudata. Within the family Amphiumidae, significant digit reduction has taken place in the three species: Amphiuma tridactylum (Three-toed Amphiuma), A. means (Two-toed Amphiuma), and A. pholeter (One-toed Amphiuma). Species within this genus present a unique system to study the evolution and development of limbs due to their elongated body, reduced limb length, and digit loss along an apparent taxonomic morphocline. Individuals within the genus Amphiuma lack limb regeneration after amputation, thus the mesopodial variation results from alterations in the developmental processes. Significant mesopodial variation was present in each species with several novel arrangements, but predominately shared mesopodial patterns to other species with the same digit number. Our results showed more variation in the mesopodial pattern of the forelimbs than the hind limbs in all three species, and more variation in A. tridactylum and A. means compared to A. pholeter. Limbs of A. pholeter had fewer elements present, which we hypothesize as a consequence of the species being in the process of complete limb loss. Lastly, specimens of A. pholeter were consistently found with two digits present bound in hard syndactyly, contrary to the previously assumed one digit.

The structure of sarcomeres imposes limits to the capacity of striated muscle to change length and produce force, with z-disc and myosin filament interactions constraining shortening. Conversely, supercontracting muscles, hitherto only known among vertebrates in the tongue retractor muscle (m. hyoglossus) of chameleons, have perforated z-discs that allow myosin filaments to extend through them into adjacent sarcomeres, permitting continued shortening and force development. Additional hyolingual muscles in chameleons undergo extreme length changes during feeding as well and may benefit from supercontractile properties. We compared length-tension relationship data and transmission electron microscopy images from four chameleon muscles to test for the presence of additional supercontracting muscle. We document the second known example of a supercontracting muscle among vertebrates (the m. sternohyoideus superficialis) and show that the m. sternohyoideus profundus exhibits functional convergence with supercontracting muscles by increasing the range of muscle lengths over which it can exert force through the exploitation of sarcomere length non-uniformity across its muscle fibres. Additionally, we show that chameleon supercontracting muscles may share common contractile and structural properties due to a common origin from occipital somites. These results provide important insights into the developmental and evolutionary patterns associated with supercontracting muscle and extreme muscle elongation.

Background: Pulmonary development in tetrapods is a complex process, especially within squamates, where single-chambered, transitional, and multi-chambered lungs can be found in adult animals. While the embryological development of the respiratory system of lizards and snakes was studied in a number of species between the 1830s and 1940s, the subject has only received sporadic attention since then. With the advancement of imaging technology, non-invasive methods can be used to explore the degree of respiratory system development in embryos of different ages. Results: Micro-computed tomography (micro-CT) was used to reconstruct three-dimensional extrapulmonary airways and pulmonary structures and to analyze lung development in five species of lizards (two species of Teiidae, one Anguidae, one Iguanidae, and one Tropiduridae) and one species of snake (Lamprophiidae). Results indicate that pulmonary parenchyma development was undetectable in the earliest embryonic stages, likely due to technical or developmental limitations. In later stages, structures such as faveolar parenchyma and intrapulmonary subdivisions were clearly observable, resembling the morphology seen in adult animals. Conclusions: These findings provide valuable insights into the viability of micro-CT scans to investigate embryonic respiratory systems, as well as into the evolution and development of the respiratory system in Squamata.

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. An investigation of the subpectoral diverticulum—an inflatable air sac structure between the major flight muscles—in 68 avian species reveals that the respiratory system has a role in the mechanics of flight in soaring birds.

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.

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.

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.

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.

Innovations in three-dimensional (3D) imaging and segmentation have facilitated unprecedented levels of anatomical investigation into the detailed structures of the respiratory system that are often difficult to study in situ. Recent hypotheses of homology between crocodilians and birds have facilitated quantitative comparative analyses of bronchial trees and in situ models reveal new complexities in the relationship between the respiratory and skeletal systems. Here we quantitatively compare the bronchial trees of two crocodilians, the American alligator (Alligator mississippiensis) and Cuvier's dwarf caiman (Paleosuchus palpebrosus) with select birds, including the ostrich (Struthio camelus), the African gray parrot (Psittacus erithacus), and the red-tailed hawk (Buteo jamaicensis). Notably, the relative distances from the carina to the secondary bronchi measured are conserved, indicating a possible ancestral or constrained trait. With respect to interspecific avian comparisons, we found grossly observable variation within a single taxon in air sac morphology (e.g., P. erithacus), as well as substantial differences between the individual taxa via segmented surface models - particularly in the expansions of the interclavicular sacs, the extent of the diverticula, and the size of the abdominal sacs. Furthermore, we found that specific sac contribution to postcranial pneumatization varies substantially across our dataset. Individual specimens imaged for this study also revealed multiple pathologies, including scoliosis, foreign objects inside the animals, and broken bones, which have been incorporated into the anatomical models for clinical surgical atlases that are under development. While these data are preliminary, they provide a framework for larger scale comparisons and hypotheses of the ancestral archosaurian pulmonary system.

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.

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.

Since its inception in the late 1970s/early 1980s, Evolutionary Developmental Biology (Evo-Devo) has produced much insight into the evolution of organismal design. Despite the obvious roots connecting Evo-Devo studies to pathology, malformations have been ignored as possible adaptive phenotypes within a different ecological or phylogenetic context and may provide insight into studies of macroevolution. While early opponents of the Modern Synthesis such as Richard Goldschmidt rooted for “hopeful monsters,” this idea was poorly supported and until recently has gone without many examples. Evo-Devo Path [1, 2] builds upon the theoretical framework of early biologists like Pere Alberch while also bridging our current understanding of genomics and development with the hierarchical organization of pathologies established by Isidore Gregory Saint-Hilaire and morphological disparity, thus further challenging the Modern Synthesis and interrupting the boundary between disease and novelty and serving as a bridge between the fields of biomedical research and natural history. Mutations in the regulation and expression of genes within our genome recur as categorizable malformations through the perturbation of key signaling pathways. With the advent of Evo-Devo and genomic advancements over the last few decades, we are able to revisit the micro-evolutionary-centered Modern Synthesis and the idea of “hopeful monsters” to find that unlike the contentious aura which surrounded Goldschmidt’s work, we may now be in a position to re-examine his theoretical framework along with the tenets of the modern synthesis.

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.

The discovery of unidirectional airflow patterns in the lungs of numerous sauropsids has renewed interest in the evolution, anatomy, and structural diversity of the respiratory structures in archosaurs. In order to understand the origin and evolution of the crocodilian lung, it is first necessary to map out the bronchial architecture across the crocodilian phylogenetic tree. To this end, we describe the anatomy of the respiratory system of Cuvier's dwarf caiman ( Paleosuchus palpebrosus ) based upon micro‐computed tomography (uCT) data of the respiratory system in situ (n=4). The lungs were inflated artificially via a syringe, and imaged at total lung capacity. Two P. palpebrosus were artificially deflated via a syringe to an approximate functional residual capacity, and re‐imaged to visualize changes in the bronchial tree and lung position at the end of a hypothetical natural exhalation. For each of the scans, the lung surface and bronchial tree were segmented into surface models in the scientific visualization program Avizo 7.1. Specific measures of the bronchial tree were acquired in the DICOM viewer OsiriX MD for intraspecific comparisons: (1) the distance from the carina to the first three large secondary bronchi; (2) the area of the primary bronchus at the first three large secondary bronchi; and, (3) the area of the ostium of the first three large secondary bronchi. These data were then compared to the same quantitative measures previously acquired from Alligator mississippiensis (n=10), and Crocodylus niloticus (n=3), allowing for interspecific comparisons across Crocodylia. As expected, the shape of the cartilaginous regions of the bronchial tree were unaffected by changes in the inspiratory volume. Overall, the basic bauplan of the bronchial tree of P. palpebrosus is more similar to that of A. mississippiensis with fewer large secondary airways (both medial and dorsobronchi), and fewer small secondary saccular bronchi in the caudal and ventral regions of the lung. Additionally, P. palpebrosus demonstrates a decreased density of the respiratory parenchyma throughout the lung, similar to the hatchling A. mississippiensis and in contrast to the specimens of C. niloticus ; however, it is possible that this is due to factors unrelated to anatomy or phylogeny, and could be associated with something else entirely (e.g., environmental conditions). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Abstract 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.