The latest freshwater giants: a new Peltocephalus (Pleurodira: Podocnemididae) turtle from the Late Pleistocene of the Brazilian Amazon.

Overkill of large mammals is recognized as a key driver of Pleistocene megafaunal extinctions in the Americas and Australia. While this phenomenon primarily affected mega-mammals, its impact on large Quaternary reptiles has been debated. Freshwater turtles, due to the scarcity of giant forms in the Quaternary record, have been largely neglected in such discussions. Here we present a new giant podocnemidid turtle, Peltocephalus maturin sp. nov., from the Late Pleistocene Rio Madeira Formation in the Brazilian Amazon, that challenges this assumption. Morphological and phylogenetic analyses of the holotype, a massive partial lower jaw, reveal close affinities to extant Amazonian species and suggest an omnivorous diet. Body size regressions indicate Pe. maturin possibly reached about 180 cm in carapace length and is among the largest freshwater turtles ever found. This finding presents the latest known occurrence of giant freshwater turtles, hinting at coexistence with early human inhabitants in the Amazon.

Gonadal Morphology of Stillborn Hawksbill Sea Turtle (Eretmochelys imbricata) Hatchlings.

This study aimed to investigate sexual dimorphism in stillborn hawksbill sea turtles (Eretmochelys imbricata) through gonadal morphological characterizations. Macroscopic, light microscopy, and transmission electron analyses were performed for 30 gonad-mesonephros complexes. Female gonads were spindle-shaped and present a translucent whitish appearance with a grainy texture. Male gonads were approximately ovoid with a smooth opaque white surface. A primary sexual difference concerns different marrow structures, with females presenting organized cellularity featuring oocytes, lacunae, and blood vessels, while males presented a distinct organizational medulla pattern marked by testicular cords extending throughout the gonad length. Ultrastructurally, female's stroma presented interstitial cells and an abundant cytoplasm rich in electrodense droplets and large oval germline cells, with a conspicuous and noncentral nucleus. Males, on the other hand, presented testicular cord cells containing small amounts of heterochromatin and approximately triangular apical and basal cytoplasms with an evident nucleolus characteristic of support cells. Additionally, there were cells with a large spherical nucleus compared with the cell size and a relatively scarce cytoplasm, identified as gonocytes. These findings indicate that macroscopic, microscopic, and ultrastructural evaluations are effective and reliable techniques for the sexual identification of stillborn E. imbricata hatchlings.

Phylogenomics reconciles molecular data with the rich fossil record on the origin of living turtles.

Although a consensus exists that all living turtles fall within either Pleurodira or Cryptodira clades, estimating when these lineages split is still under debate. Most molecular studies date the split in the Triassic Period, whereas a Jurassic age is unanimous among morphological studies. Each hypothesis implies different paleobiogeographical scenarios to explain early turtle evolution. Here we explored the rich turtle fossil record with the Fossilized Birth-Death (FBD) and the traditional node dating (ND) methods using complete mitochondrial genomes (147 taxa) and a set of nuclear orthologs with over 10 million bp (25 taxa) to date the major splits in Testudines. Our results support an Early Jurassic split (191-182 Ma) for the crown Testudines with great consistency across different dating methods and datasets, with a narrow confidence interval. This result is independently supported by the oldest fossils of Testudines that postdate the Middle Jurassic (174 Ma), which were not used for calibration in this study. This age coincides with the Pangaea fragmentation and the formation of saltwater barriers such as the Atlantic Ocean and the Turgai Strait, supporting that diversification in Testudines was triggered by vicariance. Our ages of the splits in Pleurodira coincide with the geologic events of the Late Jurassic and Early Cretaceous. Conversely, the early Cryptodira radiation remained in Laurasia, and its diversification ensued as all its major lineages expanded their distribution into every continent during the Cenozoic. We provide the first detailed hypothesis of the evolution of Cryptodira in the Southern Hemisphere, in which our time estimates are correlated with each contact between landmasses derived from Gondwana and Laurasia. Although most South American Cryptodira arrived through the Great American Biotic Interchange, our results indicate that the Chelonoidis ancestor probably arrived from Africa through the chain islands of the South Atlantic during the Paleogene. Together, the presence of ancient turtle diversity and the vital role that turtles occupy in marine and terrestrial ecosystems underline South America as a chief area for conservation.

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.

No evidence for pericardial restraint in the snapping turtle (Chelydra serpentina) following pharmacologically induced bradycardia at rest or during exercise.

Most animals elevate cardiac output during exercise through a rise in heart rate (fH), whereas stroke volume (VS) remains relatively unchanged. Cardiac pacing reveals that elevating fH alone does not alter cardiac output, which is instead largely regulated by the peripheral vasculature. In terms of myocardial oxygen demand, an increase in fH is more costly than that which would incur if VS instead were to increase. We hypothesized that fH must increase because any substantial rise in VS would be constrained by the pericardium. To investigate this hypothesis, we explored the effects of pharmacologically induced bradycardia, with ivabradine treatment, on VS at rest and during exercise in the common snapping turtle (Chelydra serpentina) with intact or opened pericardium. We first showed that, in isolated myocardial preparations, ivabradine exerted a pronounced positive inotropic effect on atrial tissue but only minor effects on ventricle. Ivabradine reduced fH in vivo, such that exercise tachycardia was attenuated. Pulmonary and systemic VS rose in response to ivabradine. The rise in pulmonary VS largely compensated for the bradycardia at rest, leaving total pulmonary flow unchanged by ivabradine, although ivabradine reduced pulmonary blood flow during swimming (exercise × ivabradine interaction, P < 0.05). Although systemic VS increased, systemic blood flow was reduced by ivabradine both at rest and during exercise, despite ivabradine's potential to increase cardiac contractility. Opening the pericardium had no effect on fH, VS, or blood flows before or after ivabradine, indicating that the pericardium does not constrain VS in turtles, even during pharmacologically induced bradycardia.

Absence of atrial smooth muscle in the heart of the loggerhead sea turtle (Caretta caretta): a re-evaluation of its role in diving physiology.

Contraction of atrial smooth muscle in the hearts of semi-aquatic emydid turtles regulates ventricular filling, and it has been proposed that it could regulate stroke volume during characteristic rapid transitions in cardiac output associated with diving. For this hypothesis to be supported, atrial smooth muscle should be widely distributed in diving Testudines. To further understand the putative function and evolutionary significance of endocardial smooth muscle in Testudines, we studied the hearts of loggerhead sea turtles, Caretta caretta (n=7), using immunohistochemistry and histology. Surprisingly, we found no evidence of prominent atrial smooth muscle in C. caretta. However, smooth muscle was readily identified in the sinus venosus. Our results suggest that atrial smooth muscle does not contribute to the diving capabilities of C. caretta, indicating that the possible roles of smooth muscle in emydid turtle hearts require a re-evaluation. In sea turtles, the sinus venosus may instead contribute to regulate cardiac filling.

Morphological characterization of the digestive tube of hawksbill sea turtle (Eretmochelys imbricata) hatchlings.

Morphological studies concerning the digestive system can further information on animal diets, thus aiding in the understanding of feeding behavior. Given the scarcity of information on sea turtle digestive system morphology, the aim of the present study was to describe the digestive tube (DT) morphology of Eretmochelys imbricata hatchlings to further understand the diet of these individuals in the wild. DT samples from 10 stillborn turtles (undefined sex) were analyzed at the macro and microscopic levels. The esophagus, stomach, small intestine (SI), and large intestine (LI) are described. Histologically, the DT is formed by four tunics, the mucosa, submucosa, muscular, and adventitia or serosa. The esophagus is lined by keratinized stratified squamous epithelium, while the remainder of the DT is lined by a simple columnar epithelium. The esophagus mucosa is marked by conical, pointed papillae. The stomach comprises three regions, the cardiac, fundic, and pyloric and is covered by neutral mucous granular cells. The intestinal mucosa presents absorptive cells with microvilli, neutral and acidic goblet cells, and mucosa-associated lymphoid tissue. The SI is significantly longer than the LI (p value = 0.006841). These morphological findings are strong indications of adaptations to a carnivorous diet in this hawksbill turtle age group.

Homology and osteological correlates of pedal muscles among extant sauropsids.

Archosaurs displayed an evolutionary trend toward increasing bipedalism in their evolutionary history, that is, forelimbs tend to be reduced in contrast to the development of hindlimbs becoming major weight-bearing and locomotor appendages. The archosaurian locomotion has been extensively discussed based on their limb morphology because the latter reflects their locomotor modes very well. However, despite some attempts of reconstructing the hindlimb musculature in Archosauria, that of the most distal portion, the pes, has often been neglected. In order to rectify this trend, detailed homologies of pedal muscles among sauropsids were established based on dissections and literature reviews of adult conditions. As a result, homologies of some pedal muscles between non-avian sauropsids and avians were revised, challenging classical hypotheses. The present new hypothesis postulates that the avian m. tibialis cranialis and non-avian m. extensor digitorum longus, as well as the avian m. extensor digitorum longus and non-avian m. tibialis anterior, are homologous with each other, respectively. This is more plausible because it requires no drastical change in the attachment sites between the avian and non-avian homologues unlike the classical hypothesis. Many interosseous muscles in non-archosaurian sauropsids that have long been regarded as a part of short digital extensors or flexors are also divided into multiple distinct muscles so that they can be homologized with short pedal muscles among all sauropsids. In addition, osteological correlates of attachments are identified for most of the pedal muscles, contributing to future attempts of reconstruction of this muscle system in fossil archosaurs.

The influence of the post-pulmonary septum and submersion on the pulmonary mechanics of Trachemys scripta (Cryptodira: Emydidae).

The respiratory system of chelonians needs to function within a mostly solid carapace, with ventilation depending on movements of the flanks. When submerged, inspiration has to work against hydrostatic pressure. We examined breathing mechanics in Trachemys scripta while underwater. Additionally, as the respiratory system of T. scripta possesses a well-developed post-pulmonary septum (PPS), we investigated its role by analyzing the breathing mechanics of lungs with and without their PPS attached. Static compliance was significantly increased in submerged animals and in animals with and without their PPS, while removal of the PPS did not result in a significantly different static compliance. Dynamic compliance was significantly affected by changes in volume and frequency in every treatment, with submergence significantly decreasing dynamic compliance. The presence of the PPS significantly increased dynamic compliance. Submersion did not significantly alter work per ventilation, but caused minute work of breathing to be much greater at any frequency and ventilation level analyzed. Lungs with or without their PPS did not show significantly different work per ventilation when compared with the intact animal. Our results demonstrate that submersion results in significantly altered breathing mechanics, increasing minute work of breathing greatly. The PPS was shown to maintain a constant volume within the animal's body cavity, wherein the lungs can be ventilated more easily, highlighting the importance of this coelomic subdivision in the chelonian body cavity.

Allometric relations of respiratory variables in Amniota: Effects of phylogeny, form, and function.

Biological variables are frequently described by analyzing scaling relationships of the variable against body mass (MB). Respiratory variables are no exception and allometric relations for oxygen consumption, pulmonary ventilation, tidal volume, breathing frequency, and lung volume have been described in the literature. While the allometric relations of respiratory variables given for mammals and birds are very consistent among different studies, scaling relationships for non-avian reptiles have only been scarcely described and show considerable variation between studies. Since no comprehensive study of allometric relations of respiratory variables has been carried out comparing the different groups of non-avian reptiles, we analyzed morphological and physiological variables of the respiratory system of crocodilians, chelonians, lizards, snakes, birds, and mammals, regarding the allometric relations of each variable from a phylogenetic perspective as well as related to lung morphology. Our results indicated that few respiratory variables possess significant phylogenetic signals and that tidal volume, breathing frequency (except mammals), and air convection requirement were independent of phylogeny. Contrary to the literature, lung volume of amniotes scaled isometrically to MB, with the exception of lizards (MB0.78). Air convection requirement scaled isometrically in mammals and birds, but was more variable among non-avian reptiles, from a taxonomic perspective and in regard to different lung structures. In conclusion, respiratory variables among non-avian reptiles scaled more variably than previously expected, both according to phylogeny and to lung type, warranting future studies to explore structure-function relations of the reptilian respiratory system, especially regarding snakes and crocodilians, since these groups had very few data available for analysis.

Structural Variation of the Turtle Mitochondrial Control Region.

The present study describes the most comprehensive comparison of turtle mtD-loop regions to date. The primary structure was compared from DNA sequences accessed from GenBank from 48 species in 13 families of extant turtles, and secondary structures of the mtD-loop region were inferred from thermal stabilities, using the program Mfold, for each superfamiliy of turtles. Both primary and secondary structures were found to be highly variable across the order. The Cryptodira showed conservation in the primary structure at conserved sequence blocks (CSBs), but the Pleurodira displayed limited conservation of primary structural characters, other than the coreTAS, a binding site for the helicase TWINKLE, which was highly conserved in the Central and Right Domains across the order. No secondary structure was associated with a TAS, but an AT-rich fold (secondary structure) near the 3' terminus of the mtD-loop region was detected in all turtle superfamilies. Mapping of character states of structural features of the mtD-loop region revealed that most character states were autapomorphies and inferred a number of homoplasies. The Left Domain of turtles, containing no highly conserved structural elements, likely does not serve a functional role; therefore, the Central Domain in turtles is likely equivalent to the Left Domain of mammals. The AT-rich secondary structural element near the 3' terminus of the mtD-loop region may be conserved across turtles because of a functional role, perhaps containing the Light Strand Promotor, or perhaps interacting with the TWINKLE-coreTAS complex in the Central and Right Domains to regulate mtDNA replication and transcription.

Hepatic CT attenuation differs in three species of freshwater turtles and hepatic Hounsfield units increase with folliculogenesis in wild Blanding's turtles (Emydoidea blandingii).

Freshwater turtle species are suffering from anthropocentric-caused population declines, making preservation of professionally managed populations increasingly important. Turtles under professional care have an increased risk to develop hepatic lipidosis, potentially resulting in early death. Computed tomography can provide an antemortem screening for increased fatty liver composition. A goal of this prospective analytical cross-sectional study was to assess the hepatic attenuation measured as Hounsfield units (HU) in a wild population of a freshwater turtle species, and then compare hepatic HU to freshwater turtles under professional care. Ninety-five wild Blanding's turtles (BT; Emydoidea blandingii) as well as 10 Vietnamese Pond turtles (VPT; Mauremys annamensis) and six Northern Snake-Necked turtles (NSNT; Chelodina oblonga) under professional care underwent CT with quantification of hepatic HU. Hepatic HU were correlated to serum chemistry findings and the presence of follicles was recorded in BT. The mean (±SD) hepatic attenuation for 95 wild BT was 97.5 HU ±9.6. There were significant differences in hepatic attenuation among VPT, NSNT, and BT, with median HU values (range) of 5.39 HU (-6.45 to 61.50), 71.74 HU (59.44-94.49), and 95.43 HU (74.41-124.56), respectively. Aspartate aminotransferase (AST) values had a weak negative correlation to HU with a coefficient of -0.85 (P < .001). The hepatic attenuation was significantly higher for individuals undergoing folliculogenesis (P = .007). The decreased HU values were significantly negatively correlated with AST values. Findings supported the use of CT as an aid for guiding future management practices in freshwater turtles.

Insights into Emydid Turtle Cytogenetics: The European Pond Turtle as a Model Species.

Our knowledge of Testudines evolution is limited by the lack of modern cytogenetic data. Compared to other reptiles, there is little information even on chromosome banding, let alone molecular cytogenetic data. Here, we provide detailed information on the karyotype of the European pond turtle Emys orbicularis, a model Emydidae, employing both chromosome banding and molecular cytogenetics. We provide a high-resolution G-banded karyotype and a map of rDNA genes and telomeric sequences using fluorescence in situ hybridization. We test hypotheses of sex-determining mechanisms in Emys by comparative genomic hybridization to determine if Emys has a cryptic sex-specific region. Our results provide valuable data to guide future efforts on genome sequencing and anchoring in Emydidae and for understanding karyotype evolution in Testudines.

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.

Climatic niche evolution in turtles is characterized by phylogenetic conservatism for both aquatic and terrestrial species.

Understanding how the climatic niche of species evolved has been a topic of high interest in current theoretical and applied macroecological studies. However, little is known regarding how species traits might influence climatic niche evolution. Here, we evaluated patterns of climatic niche evolution in turtles (tortoises and freshwater turtles) and whether species habitat (terrestrial or aquatic) influences these patterns. We used phylogenetic, climatic and distribution data for 261 species to estimate their climatic niches. Then, we compared whether niche overlap between sister species was higher than between random species pairs and evaluated whether niche optima and rates varied between aquatic and terrestrial species. Sister species had higher values of niche overlap than random species pairs, suggesting phylogenetic climatic niche conservatism in turtles. The climatic niche evolution of the group followed an Ornstein-Uhlenbeck model with different optimum values for aquatic and terrestrial species, but we did not find consistent evidence of differences in their rates of climatic niche evolution. We conclude that phylogenetic climatic niche conservatism occurs among turtle species. Furthermore, terrestrial and aquatic species occupy different climatic niches but these seem to have evolved at similar evolutionary rates, reinforcing the importance of habitat in understanding species climatic niches and their evolution.

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.

Biogeographic Dating of Speciation Times Using Paleogeographically Informed Processes.

Standard models of molecular evolution cannot estimate absolute speciation times alone, and require external calibrations to do so, such as fossils. Because fossil calibration methods rely on the incomplete fossil record, a great number of nodes in the tree of life cannot be dated precisely. However, many major paleogeographical events are dated, and since biogeographic processes depend on paleogeographical conditions, biogeographic dating may be used as an alternative or complementary method to fossil dating. I demonstrate how a time-stratified biogeographic stochastic process may be used to estimate absolute divergence times by conditioning on dated paleogeographical events. Informed by the current paleogeographical literature, I construct an empirical dispersal graph using 25 areas and 26 epochs for the past 540 Ma of Earth's history. Simulations indicate biogeographic dating performs well so long as paleogeography imposes constraint on biogeographic character evolution. To gauge whether biogeographic dating may be of practical use, I analyzed the well-studied turtle clade (Testudines) to assess how well biogeographic dating fares when compared to fossil-calibrated dating estimates reported in the literature. Fossil-free biogeographic dating estimated the age of the most recent common ancestor of extant turtles to be from the Late Triassic, which is consistent with fossil-based estimates. Dating precision improves further when including a root node fossil calibration. The described model, paleogeographical dispersal graph, and analysis scripts are available for use with RevBayes.

Phylogenomic analyses of 539 highly informative loci dates a fully resolved time tree for the major clades of living turtles (Testudines).

Accurate time-calibrated phylogenies are the centerpiece of many macroevolutionary studies, and the relationship between the size and scale of molecular data sets and the density and accuracy of fossil calibrations is a key element of time tree studies. Here, we develop a target capture array specifically for living turtles, compare its efficiency to an ultraconserved element (UCE) dataset, and present a time-calibrated molecular phylogeny based on 539 nuclear loci sequenced from 26 species representing the breadth of living turtle diversity plus outgroups. Our gene array, based on three fully sequenced turtle genomes, is 2.4 times more variable across turtles than a recently published UCE data set for an identical subset of 13 species, confirming that taxon-specific arrays return more informative data per sequencing effort than UCEs. We used our genomic data to estimate the ages of living turtle clades including a mid-late Triassic origin for crown turtles and a mid-Carboniferous split of turtles from their sister group, Archosauria. By specifically excluding several of the earliest potential crown turtle fossils and limiting the age of fossil calibration points to the unambiguous crown lineage Caribemys oxfordiensis from the Late Jurassic (Oxfordian, 163.5-157.3Ma) we corroborate a relatively ancient age for living turtles. We also provide novel age estimates for five of the ten testudine families containing more than a single species, as well as several intrafamilial clades. Most of the diversity of crown turtles appears to date to the Paleogene, well after the Cretaceous-Paleogene mass extinction 66mya.