Capillary cell-type specialization in the alveolus.

In the mammalian lung, an apparently homogenous mesh of capillary vessels surrounds each alveolus, forming the vast respiratory surface across which oxygen transfers to the blood1. Here we use single-cell analysis to elucidate the cell types, development, renewal and evolution of the alveolar capillary endothelium. We show that alveolar capillaries are mosaics; similar to the epithelium that lines the alveolus, the alveolar endothelium is made up of two intermingled cell types, with complex 'Swiss-cheese'-like morphologies and distinct functions. The first cell type, which we term the 'aerocyte', is specialized for gas exchange and the trafficking of leukocytes, and is unique to the lung. The other cell type, termed gCap ('general' capillary), is specialized to regulate vasomotor tone, and functions as a stem/progenitor cell in capillary homeostasis and repair. The two cell types develop from bipotent progenitors, mature gradually and are affected differently in disease and during ageing. This cell-type specialization is conserved between mouse and human lungs but is not found in alligator or turtle lungs, suggesting it arose during the evolution of the mammalian lung. The discovery of cell type specialization in alveolar capillaries transforms our understanding of the structure, function, regulation and maintenance of the air-blood barrier and gas exchange in health, disease and evolution.

A claustrum in reptiles and its role in slow-wave sleep.

The mammalian claustrum, owing to its widespread connectivity with other forebrain structures, has been hypothesized to mediate functions that range from decision-making to consciousness1. Here we report that a homologue of the claustrum, identified by single-cell transcriptomics and viral tracing of connectivity, also exists in a reptile-the Australian bearded dragon Pogona vitticeps. In Pogona, the claustrum underlies the generation of sharp waves during slow-wave sleep. The sharp waves, together with superimposed high-frequency ripples2, propagate to the entire neighbouring pallial dorsal ventricular ridge (DVR). Unilateral or bilateral lesions of the claustrum suppress the production of sharp-wave ripples during slow-wave sleep in a unilateral or bilateral manner, respectively, but do not affect the regular and rapidly alternating sleep rhythm that is characteristic of sleep in this species3. The claustrum is thus not involved in the generation of the sleep rhythm itself. Tract tracing revealed that the reptilian claustrum projects widely to a variety of forebrain areas, including the cortex, and that it receives converging inputs from, among others, areas of the mid- and hindbrain that are known to be involved in wake-sleep control in mammals4-6. Periodically modulating the concentration of serotonin in the claustrum, for example, caused a matching modulation of sharp-wave production there and in the neighbouring DVR. Using transcriptomic approaches, we also identified a claustrum in the turtle Trachemys scripta, a distant reptilian relative of lizards. The claustrum is therefore an ancient structure that was probably already present in the brain of the common vertebrate ancestor of reptiles and mammals. It may have an important role in the control of brain states owing to the ascending input it receives from the mid- and hindbrain, its widespread projections to the forebrain and its role in sharp-wave generation during slow-wave sleep.

A Triassic stem turtle with an edentulous beak.

The early evolution of turtles continues to be a contentious issue in vertebrate palaeontology. Recent reports have suggested that they are diapsids1-6, but the position of turtles within Diapsida is controversial7-12 and the sequence of acquisition of turtle synapomorphies remains unclear1-3. Here we describe a Triassic turtle from China that has a mixture of derived characters and plesiomorphic features. To our knowledge, it represents the earliest known stem turtle with an edentulous beak and a rigid puboischiadic plate. The discovery of this new form reveals a complex early history of turtles.

Macro- and micro-anatomical investigation of the oropharyngeal roof of landform greek tortoise (Testudo graeca graeca) and semi-aquatic red-eared slider turtle (Trachemys scripta elegans).

The present investigation examined the oropharyngeal roof of two turtles having different feeding behaviors: the landform Greek tortoise (Testudo graeca graeca) primarily herbivores and the semi-aquatic red-eared slider turtle (Trachemys scripta elegans) lives in freshwater that opportunistic omnivorous grossly and by scanning and light microscopes. Grossly, the Greek tortoise had a V-shaped roof consisting of the upper rhamphotheca, peri-palatine region, upper alveolar ridge, peripheral palatine ridge, median palatine ridge, vomer, choanae, caudal palatine part, and pharynx. At the same time, the red-eared slider had a semilunar roof consisting of upper rhamphotheca, two peripheral palatine ridges, core of palatine ridges, upper alveolar band, vomer, choanae, caudal palatine part, and pharynx. SEM revealed that the red-eared slider roof appeared more straightforward. The upper rhamphotheca is sharp, with a median premaxillary notch in the red-eared slider that gives a powerful bite for cutting to compensate absence of the teeth. Additionally, the red-eared slider's upper alveolar band is interrupted by a single upper alveolar ridge that appears spiky, pointed, and longer as it needs powerful chewing of prey and there are two types of teeth-like projections at its peri-palatine area for food-crushing and chewing. The Greek tortoise palatine region had numerous ridges and folds to provide roughness for food processing. Greek tortoises had small-sized choanae with two choanal folds to minimize choanal openings when eating dusty grasses. Histologically, Greek tortoise palate was rostrally thicker and more keratinized than caudally, and the caudal palatine region was characterized by a single pair of circumvallate-like papilla with multiple mucous openings and secretions, while red-eared slider palate was slightly keratinized at the peri-choanal region, and the rest of the palate was non-keratinized with few mucous openings. The current investigation found various structural oropharyngeal roof adaptations to feeding behavior in the omnivore red-eared slide compared to the herbivorous Greek turtle.

Morphological comparison of the larynx and trachea of Chelonia mydas (Linnaeus, 1758), Caiman yacare (Daudin, 1802) and Caiman latirostris (Daudin, 1802).

The larynx is in the lower respiratory tract and has the function of protecting the airways, controlling, and modulating breathing, assisting the circulatory system, and vocalizing. This study aims to describe the anatomy and histology of the skeleton of the larynx and trachea of the species Chelonia mydas, Caiman yacare and Caiman latirostris. The study was conducted at the Federal University of Espírito Santo (UFES), using nine specimens of Ch. mydas, 20 of Ca. yacare and four of Ca. latirostris. Samples of the larynx and trachea were collected, fixed, and sent for dissection of the structures and subsequent macroscopic analysis. For histology, samples were processed by the routine paraffin embedding method and stained with hematoxylin-eosin and Verhoeff. For the three species, two arytenoid cartilages, a cricoid cartilage, a hyoid apparatus composed of a base and two horns were found. In Ch. mydas, two structures called thyroid wings were observed, not found in crocodilians. The trachea of crocodilians presented incomplete tracheal rings and musculature, while the trachea of Ch. mydas presented complete tracheal rings. Histologically, the entire cartilaginous skeleton of the larynx of the three species, as well as the tracheal rings, are constituted by hyaline cartilage.

Histological features and Gαolf expression patterns in the nasal cavity of sea turtles.

Sea turtles use olfaction to detect volatile and water-soluble substances. The nasal cavity of green turtles (Chelonia mydas) comprises morphologically defined the anterodorsal, anteroventral, and posterodorsal diverticula, as well as a single posteroventral fossa. Here, we detailed the histological features of the nasal cavity of a mature female green turtle. The posterodorsal diverticulum contained spongy-like venous sinuses and a wave-shaped sensory epithelium that favored ventilation. Secretory structures that were significant in sensory and non-sensory epithelia were probably involved in protection against seawater. These findings suggested that green turtles efficiently intake airborne substances and dissolve water-soluble substances in mucous, while suppressing the effects of salts. In addition, positive staining of Gαs/olf that couples with olfactory, but not vomeronasal, receptors was predominant in all three types of sensory epithelium in the nasal cavity. Both of airborne and water-soluble odorants seemed to be detected in cells expressing Gαolf and olfactory receptors.

Quaternary megafauna extinctions altered body size distribution in tortoises.

The late Quaternary is characterized by the extinction of many terrestrial megafauna, which included tortoises (Family: Testudinidae). However, limited information is available on how extinction shaped the phenotype of surviving taxa. Here, based on a global dataset of straight carapace length, we investigate the temporal variation, spatial distribution and evolution of tortoise body size over the past 23 million years, thereby capturing the effects of Quaternary extinctions in this clade. We found a significant change in body size distribution characterized by a reduction of both mean body size and maximum body size of extant tortoises relative to fossil taxa. This reduction of body size occurred earlier in mainland (Early Pleistocene 2.588-0.781 Ma) than in island tortoises (Late Pleistocene/Holocene 0.126-0 Ma). Despite contrasting body size patterns between fossil and extant taxa on a spatial scale, tortoise body size showed limited variation over time until this decline. Body size is a fundamental functional trait determining many aspects of species ecologies, with large tortoises playing key roles as ecosystem engineers. As such, the transition from larger sized to smaller sized classes indicated by our findings likely resulted in the homogenization of tortoises' ecological functions and diminished the role of tortoises in structuring the vegetation community.

EXTRAVASATION OF CONTRAST MEDIA AFTER SUBCARAPACIAL VESSEL INJECTION IN THREE CHELONIAN SPECIES.

The subcarapacial vessel is a popular site for venipuncture and intravenous medication administration in chelonians. Reports of adverse effects when using this site have increased, prompting evaluation of its safety. This study aimed to evaluate the anatomy of the subcarapacial vessel in 25 individual chelonians (2 box turtles, 3 red-eared sliders, and 20 red-footed tortoises) using computed tomography (CT). Individuals were sedated and administered contrast in the subcarapacial vessel. The vessel was visualized in 50% of the box turtles and red-footed tortoises, and 100% of the red-eared sliders. All species had contrast extravasation in the subarachnoid space, with red-footed tortoises having the largest percentage (70% compared to 50% and 33% of box turtles and red-eared sliders, respectively). Extravasation of contrast in the trachea or bronchi (70%) and lungs (80%) was seen in the red-footed tortoises only. Higher prevalence of contrast extravasation in the red-footed tortoises is likely because of anatomical differences, including a more cranially extending lung field and domed-shaped carapace compared to the other species. These findings highlight the risk associated with using the subcarapacial vessel for intravenous medication administration in certain species of chelonian.

Turning turtle: scaling relationships and self-righting ability in Chelydra serpentina.

Testudines are susceptible to inversion and self-righting using their necks, limbs or both, to generate enough mechanical force to flip over. We investigated how shell morphology, neck length and self-righting biomechanics scale with body mass during ontogeny in Chelydra serpentina, which uses neck-powered self-righting. We found that younger turtles flipped over twice as fast as older individuals. A simple geometric model predicted the relationships of shell shape and self-righting time with body mass. Conversely, neck force, power output and kinetic energy increase with body mass at rates greater than predicted. These findings were correlated with relatively longer necks in younger turtles than would be predicted by geometric similarity. Therefore, younger turtles self-right with lower biomechanical costs than predicted by simple scaling theory. Considering younger turtles are more prone to inverting and their shells offer less protection, faster and less costly self-righting would be advantageous in overcoming the detriments of inversion.

A Middle Triassic stem-turtle and the evolution of the turtle body plan.

The origin and early evolution of turtles have long been major contentious issues in vertebrate zoology. This is due to conflicting character evidence from molecules and morphology and a lack of transitional fossils from the critical time interval. The ∼220-million-year-old stem-turtle Odontochelys from China has a partly formed shell and many turtle-like features in its postcranial skeleton. Unlike the 214-million-year-old Proganochelys from Germany and Thailand, it retains marginal teeth and lacks a carapace. Odontochelys is separated by a large temporal gap from the ∼260-million-year-old Eunotosaurus from South Africa, which has been hypothesized as the earliest stem-turtle. Here we report a new reptile, Pappochelys, that is structurally and chronologically intermediate between Eunotosaurus and Odontochelys and dates from the Middle Triassic period (∼240 million years ago). The three taxa share anteroposteriorly broad trunk ribs that are T-shaped in cross-section and bear sculpturing, elongate dorsal vertebrae, and modified limb girdles. Pappochelys closely resembles Odontochelys in various features of the limb girdles. Unlike Odontochelys, it has a cuirass of robust paired gastralia in place of a plastron. Pappochelys provides new evidence that the plastron partly formed through serial fusion of gastralia. Its skull has small upper and ventrally open lower temporal fenestrae, supporting the hypothesis of diapsid affinities of turtles.

Evolutionary origin of the turtle skull.

Transitional fossils informing the origin of turtles are among the most sought-after discoveries in palaeontology. Despite strong genomic evidence indicating that turtles evolved from within the diapsid radiation (which includes all other living reptiles), evidence of the inferred transformation between an ancestral turtle with an open, diapsid skull to the closed, anapsid condition of modern turtles remains elusive. Here we use high-resolution computed tomography and a novel character/taxon matrix to study the skull of Eunotosaurus africanus, a 260-million-year-old fossil reptile from the Karoo Basin of South Africa, whose distinctive postcranial skeleton shares many unique features with the shelled body plan of turtles. Scepticism regarding the status of Eunotosaurus as the earliest stem turtle arises from the possibility that these shell-related features are the products of evolutionary convergence. Our phylogenetic analyses indicate strong cranial support for Eunotosaurus as a critical transitional form in turtle evolution, thus fortifying a 40-million-year extension to the turtle stem and moving the ecological context of its origin back onto land. Furthermore, we find unexpected evidence that Eunotosaurus is a diapsid reptile in the process of becoming secondarily anapsid. This is important because categorizing the skull based on the number of openings in the complex of dermal bone covering the adductor chamber has long held sway in amniote systematics, and still represents a common organizational scheme for teaching the evolutionary history of the group. These discoveries allow us to articulate a detailed and testable hypothesis of fenestral closure along the turtle stem. Our results suggest that Eunotosaurus represents a crucially important link in a chain that will eventually lead to consilience in reptile systematics, paving the way for synthetic studies of amniote evolution and development.

CLINICAL OPHTHALMIC PARAMETERS OF THE TEXAS TORTOISE (GOPHERUS BERLANDIERI).

Ophthalmic studies of the Texas tortoise (Gopherus berlandieri) established normal ophthalmic parameters for select diagnostic tests in captive tortoises and assessment of differences among individuals of differing size and health status. Sixty-one tortoises of varying weight, shell size, Mycoplasma seroprevalence, and herpesvirus exposure were included. Complete ophthalmic examinations, including neuro-ophthalmic reflexes, phenol red thread test, rebound tonometry, fluorescein staining, palpebral fissure length measurement, slit lamp biomicroscopy, indirect fundoscopy, and ocular ultrasound measurements of axial globe length, anterior chamber depth, lens thickness, and vitreous length, were recorded. All tortoises had negative dazzle and pupillary light reflexes, inconsistent menace responses, and positive palpebral reflexes. Mean ± SD tear production and intraocular pressure (IOP) were 14.2 ± 5.6 mm/15 sec and 13.8 ± 2.4 mm Hg in healthy tortoises, respectively. Mycoplasma-seropositive tortoises (with or without herpesvirus exposure) had significantly increased tear production (20.2 ± 8.1 and 19.9 ± 8.9 mm/15 sec, respectively) compared with healthy seronegative tortoises (14.2 ± 5.6 mm/15 sec; P = 0.02). As body size decreased, so too did palpebral fissure length and ocular ultrasound measurements, while IOP increased. Overall, palpebral fissure length appeared relatively small, and tear production relatively increased compared with other chelonian species, likely on the basis of the relatively arid native habitat. Further work is recommended to establish baseline values in related species, as well as comparison in aquatic versus terrestrial chelonians. The authors further suggest that the finding of relatively increased tear production in tortoises may indicate the need to rule out mycoplasmosis as a cause of upper respiratory tract disease.

Melanosome evolution indicates a key physiological shift within feathered dinosaurs.

Inference of colour patterning in extinct dinosaurs has been based on the relationship between the morphology of melanin-containing organelles (melanosomes) and colour in extant bird feathers. When this relationship evolved relative to the origin of feathers and other novel integumentary structures, such as hair and filamentous body covering in extinct archosaurs, has not been evaluated. Here we sample melanosomes from the integument of 181 extant amniote taxa and 13 lizard, turtle, dinosaur and pterosaur fossils from the Upper-Jurassic and Lower-Cretaceous of China. We find that in the lineage leading to birds, the observed increase in the diversity of melanosome morphologies appears abruptly, near the origin of pinnate feathers in maniraptoran dinosaurs. Similarly, mammals show an increased diversity of melanosome form compared to all ectothermic amniotes. In these two clades, mammals and maniraptoran dinosaurs including birds, melanosome form and colour are linked and colour reconstruction may be possible. By contrast, melanosomes in lizard, turtle and crocodilian skin, as well as the archosaurian filamentous body coverings (dinosaur 'protofeathers' and pterosaur 'pycnofibres'), show a limited diversity of form that is uncorrelated with colour in extant taxa. These patterns may be explained by convergent changes in the key melanocortin system of mammals and birds, which is known to affect pleiotropically both melanin-based colouration and energetic processes such as metabolic rate in vertebrates, and may therefore support a significant physiological shift in maniraptoran dinosaurs.

Osteohistological correlates of muscular attachment in terrestrial and freshwater Testudines.

Sharpey's fibers are considered the anatomical structures integrated to the muscles. Since these fibers leave marks at the microscopic level, their presence and distribution are used as evidence of muscle attachment in extinct and extant forms. In recent years, studies have been focusing on muscle-bone and tendon-bone interaction mostly on mammals. The main objective of this work is to contribute to the morphological and histological knowledge of muscle attachment in other amniotes, such as reptiles, and their variation related to different locomotor habits. In this way, a study was performed on terrestrial and aquatic turtles. The musculature related to the movement of the humerus, and pectoral girdle in Chelonoidis chilensis, Phrynops hilarii and Hydromedusa tectifera was analyzed. Dissections were performed mapping the origins and insertions of each muscle and undecalcified thin sections were performed in specific muscular attachment sites. We found some differences which were not previously reported, related to the insertion of the m. pectoralis, the m. coracobrachialis magnus and the origin of the m. tractor radii. The osteohistology revealed the presence of Sharpey's fibers in the cortex of all the bone elements analyzed. Patterns were established in relation to the orientation and density of Sharpey's fibers, which were used for the categorization of each muscle attachment site. The comparative micro-anatomical study of these areas did not reveal any important differences between terrestrial and freshwater turtles in muscles involved with the rotation, abduction and adduction of the humerus. In this way, the preliminary results suggest an absence of correlation between the distribution and density of Sharpey's fibers between different habitat forms, at least in the bones and species analyzed.

Comparative analysis of the shape and size of the middle ear cavity of turtles reveals no correlation with habitat ecology.

The middle ear of turtles differs from other reptiles in being separated into two distinct compartments. Several ideas have been proposed as to why the middle ear is compartmentalized in turtles, most suggesting a relationship with underwater hearing. Extant turtle species span fully marine to strictly terrestrial habitats, and ecomorphological hypotheses of turtle hearing predict that this should correlate with variation in the structure of the middle ear due to differences in the fluid properties of water and air. We investigate the shape and size of the air-filled middle ear cavity of 56 extant turtles using 3D data and phylogenetic comparative analysis to test for correlations between habitat preferences and the shape and size of the middle ear cavity. Only weak correlations are found between middle ear cavity size and ecology, with aquatic taxa having proportionally smaller cavity volumes. The middle ear cavity of turtles exhibits high shape diversity among species, but we found no relationship between this shape variation and ecology. Surprisingly, the estimated acoustic transformer ratio, a key functional parameter of impedance-matching ears in vertebrates, also shows no relation to habitat preferences (aquatic/terrestrial) in turtles. We suggest that middle ear cavity shape may be controlled by factors unrelated to hearing, such as the spatial demands of surrounding cranial structures. A review of the fossil record suggests that the modern turtle ear evolved during the Early to Middle Jurassic in stem turtles broadly adapted to freshwater and terrestrial settings. This, combined with our finding that evolutionary transitions between habitats caused only weak evolutionary changes in middle ear structure, suggests that tympanic hearing in turtles evolved as a compromise between subaerial and underwater hearing.

Auditory brainstem responses in the red-eared slider Trachemys scripta elegans (Testudoformes: Emydidae) reveal sexually dimorphic hearing sensitivity.

Hearing sensitivity is of general interest from the perspective of understanding the functionality and evolution of vertebrate auditory systems. Sexual dimorphism of auditory systems has been reported in several species of vertebrates, but little is known about this phenomenon in turtles. Some morphological characteristics, such as middle ear and tympanic membrane that influence the hearing sensitivity of animals can result in hearing sexual dimorphism. To examine whether sexual dimorphism in hearing sensitivity occurs in turtles and to compare hearing characteristics with respect to the shape of the tympanic membrane, we measured the hearing sensitivity and tympanum diameter in both sexes of Trachemys scripta elegans. The results showed that, with the exception of 0.9 kHz, auditory brainstem response thresholds were significantly lower in females than in males for frequencies in the 0.2-1.1 kHz range, indicating that the hearing of females shows greater sensitivity. No significant differences were detected in the tympanum diameter of both sexes. These results showed that sexually dimorphic hearing sensitivity has evolved in turtles; however, this difference does not appear to be related to differences in the size of the tympanic membrane. The possible origin and function of the sexual differences in auditory characteristic are discussed.

Radiographic anatomy and barium sulfate contrast study of the gastrointestinal tract of eastern box turtles (Terrapene carolina carolina).

Gastrointestinal disorders are an important cause of morbidity in box turtles (Terrapene carolina Carolina), however published information is currently lacking on the normal radiographic anatomy, transit, and emptying times of the gastrointestinal tract. A total of 15 healthy box turtles were recruited for this prospective, anatomic, reference interval study. Three-view radiographic series (vertical beam dorsoventral, horizontal beam latero-lateral, and horizontal beam rostrocaudal views) were acquired prior to contrast administration, and following contrast administration at 0, 20, 40, 60, and 90 min, 2, 4, 8, 12, and 24 h post administration, and every 24 h thereafter until all contrast was eliminated (15 mL/kg barium sulfate diluted to 30% weight per volume was administered via orogastric gavage). Vertical beam dorsoventral and horizontal beam latero-lateral views were of excellent quality to identify gastrointestinal structures. The horizontal beam rostrocaudal view immediately postcontrast administration provided gastric and pyloric identification but had lesser diagnostic use at later time points due to anatomical superimposition. The gastrointestinal tract was composed of a tubular stomach, a pyloric sphincter near midline, a duodenum with a cranial flexure in the right cranial coelomic cavity, small intestines within the right coelom, a small cecal bulb, and a transverse and descending colon. Contrast media entered the large intestine by 24 h in all turtles, and a pyloro-colic indentation was noted at the proximal descending colon. The large intestinal emptying was highly variable due to the interindividual variability of contrast sequestration within the cecal bulb. Findings from the current study serve as a reference on the gastrointestinal anatomy, transit, and emptying times in healthy eastern box turtles; and introduce a novel, horizontal beam, rostrocaudal view for gastrointestinal contrast studies in chelonians.

MEASURING FAT CONTENT USING COMPUTED TOMOGRAPHY TO ESTABLISH A BODY CONDITION INDEX IN FREE-RANGING BLANDING'S TURTLES (EMYDOIDEA BLANDINGII) IN ILLINOIS.

Health assessment of free-ranging populations requires an integrated approach, often incorporating a method to measure mass as a representation of the animals' ability to utilize environmental resources. In chelonians, direct measurements of mass have historically served as a corollary for body condition. However, this method may not accurately represent the true fat volume (FV) and may be skewed by the presence of eggs, shell size, or muscle mass. The objective of this study was to use computed tomography (CT) to develop a model for determining body condition index (BCI) in free-ranging Blanding's turtles (Emydoidea blandingii). Mass, shell measurements, and FV were measured by CT in 65 free-ranging Blanding's turtles from Lake and DuPage counties in Illinois. Twenty-one different models were built for BCI using both FV and fat percentage (FP) as dependent variables. The best fit model for FP included the relationship between mass and carapace length with nearly 60% model support. The model for FV demonstrated a similar relationship but had only 18% support. Linear models with BCI as the dependent variable showed that juveniles had a higher FP than adults and females with more eggs had a lower FP. FP can be calculated in the field with nearly 60% accuracy compared to CT-assessed FP as a component of a physical exam and population health survey to assess the effectiveness of conservation efforts for the endangered Blanding's turtle.