In vitro culture of reptile PGCS to preserve endangered species.

Primordial germ cells (PGCs), are the source of gametes in vertebrates. There are similarities in the development of PGCs of reptiles with avian and mammalian species PGCs development. PGCs culture has been performed for avian and mammalian species but there is no report for reptilian PGCs culture. In vitro culture of PGCs is needed to produce transgenic animals, preservation of endangered animals and for studies on cell behaviour and research on fertility. Reptiles are traded as exotic pets and a source of food and they are valuable for their skin and they are useful as model for medical research. Transgenic reptile has been suggested to be useful for pet industry and medical research. In this research different aspects of PGCs development was compared in three main classes of vertebrates including mammalian, avian and reptilian species. It is proposed that a discussion on similarities between reptilian PGCs development with avian and mammalian species helps to find clues for studies of reptilian PGCs development details and finding an efficient protocol for in vitro culture of reptilian PG.

Ontogenetic variation in the skull of Stenopterygius quadriscissus with an emphasis on prenatal development.

The availability of a large sample size from a range of ontogenetic stages makes Stenopterygius quadriscissus a good model to study ontogenetic variation in a fossil sauropsid. We qualitatively examined pre- and postnatal ontogenetic changes in the cranium of S. quadriscissus. The prenatal ossification sequence is similar to other diapsids, exhibiting delayed chondrocranial ossification compared to the dermatocranium. In the dermatocranium, the circumorbital area is more ossified earlier in development relative to other elements, especially those of the skull roof where ossification is comparatively weaker across prenatal stages. Perinatally all cranial elements are ossified, and many scarf and step joints are already closed. We propose four prenatal and three postnatal stages in S. quadriscissus on the basis of relative ossification, size and qualitative cranial characters pertaining to the jugal, parietal, frontal, pterygoid and surangular. These will provide a basis for determining ontogenetic stages in other ichthyosaurs. Moreover, our postnatal observations aid in refining ontogenetic characters for phylogenetic studies. Lastly, we observed that the antimeric sutures of the midline of the skull roof are open perinatally and that fusion of the midline only appears in the adult stage. We hypothesize that the loose connection of the midline functions as a fontanelle, limiting potential damage during birth.

Sex chromosome evolution among amniotes: is the origin of sex chromosomes non-random?

Sex chromosomes are a great example of a convergent evolution at the genomic level, having evolved dozens of times just within amniotes. An intriguing question is whether this repeated evolution was random, or whether some ancestral syntenic blocks have significantly higher chance to be co-opted for the role of sex chromosomes owing to their gene content related to gonad development. Here, we summarize current knowledge on the evolutionary history of sex determination and sex chromosomes in amniotes and evaluate the hypothesis of non-random emergence of sex chromosomes. The current data on the origin of sex chromosomes in amniotes suggest that their evolution is indeed non-random. However, this non-random pattern is not very strong, and many syntenic blocks representing putatively independently evolved sex chromosomes are unique. Still, repeatedly co-opted chromosomes are an excellent model system, as independent co-option of the same genomic region for the role of sex chromosome offers a great opportunity for testing evolutionary scenarios on the sex chromosome evolution under the explicit control for the genomic background and gene identity. Future studies should use these systems more to explore the convergent/divergent evolution of sex chromosomes. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part II)'.

The thermal ecology and physiology of reptiles and amphibians: A user's guide.

Research on the thermal ecology and physiology of free-living organisms is accelerating as scientists and managers recognize the urgency of the global biodiversity crisis brought on by climate change. As ectotherms, temperature fundamentally affects most aspects of the lives of amphibians and reptiles, making them excellent models for studying how animals are impacted by changing temperatures. As research on this group of organisms accelerates, it is essential to maintain consistent and optimal methodology so that results can be compared across groups and over time. This review addresses the utility of reptiles and amphibians as model organisms for thermal studies by reviewing the best practices for research on their thermal ecology and physiology, and by highlighting key studies that have advanced the field with new and improved methods. We end by presenting several areas where reptiles and amphibians show great promise for further advancing our understanding of how temperature relations between organisms and their environments are impacted by global climate change.

MeDAS: a Metazoan Developmental Alternative Splicing database.

Alternative splicing is widespread throughout eukaryotic genomes and greatly increases transcriptomic diversity. Many alternative isoforms have functional roles in developmental processes and are precisely temporally regulated. To facilitate the study of alternative splicing in a developmental context, we created MeDAS, a Metazoan Developmental Alternative Splicing database. MeDAS is an added-value resource that re-analyses publicly archived RNA-seq libraries to provide quantitative data on alternative splicing events as they vary across the time course of development. It has broad temporal and taxonomic scope and is intended to assist the user in identifying trends in alternative splicing throughout development. To create MeDAS, we re-analysed a curated set of 2232 Illumina polyA+ RNA-seq libraries that chart detailed time courses of embryonic and post-natal development across 18 species with a taxonomic range spanning the major metazoan lineages from Caenorhabditis elegans to human. MeDAS is freely available at https://das.chenlulab.com both as raw data tables and as an interactive browser allowing searches by species, tissue, or genomic feature (gene, transcript or exon ID and sequence). Results will provide details on alternative splicing events identified for the queried feature and can be visualised at the gene-, transcript- and exon-level as time courses of expression and inclusion levels, respectively.

Development, structure, and protein composition of reptilian claws and hypotheses of their evolution.

Here, we review the development, morphology, genes, and proteins of claws in reptiles. Claws likely form owing to the inductive influence of phalangeal mesenchyme on the apical epidermis of developing digits, resulting in hyperproliferation and intense protein synthesis in the dorsal epidermis, which forms the unguis. The tip of claws results from prevalent cell proliferation and distal movement along most of the ungueal epidermis in comparison to the ventral surface forming the subunguis. Asymmetrical growth between the unguis and subunguis forces beta-cells from the unguis to rotate into the apical part of the subunguis, sharpening the claw tip. Further sharpening occurs by scratching and mechanical wearing. Ungueal keratinocytes elongate, form an intricate perimeter and cementing junctions, and remain united impeding desquamation. In contrast, thin keratinocytes in the subunguis form a smooth perimeter, accumulate less corneous beta proteins (CBPs) and cysteine-poor intermediate filament (IF)-keratins, and desquamate. In addition to prevalent glycine-cysteine-tyrosine rich CBPs, special cysteine-rich IF-keratins are also synthesized in the claw, generating numerous SS bonds that harden the thick and compact corneous material. Desquamation and mechanical wear at the tip ensure that the unguis curvature remains approximately stable over time. Reptilian claws are likely very ancient in evolution, although the unguis differentiated like the outer scale surface of scales, while the subunguis might have derived from the inner scale surface. The few hair-like IF-keratins synthesized in reptilian claws indicate that ancestors of sauropsids and mammals shared cysteine-rich IF-keratins. However, the number of these keratins remained low in reptiles, while new types of CBPs function to strengthen claws.

Determinate growth is predominant and likely ancestral in squamate reptiles.

Body growth is typically thought to be indeterminate in ectothermic vertebrates. Indeed, until recently, this growth pattern was considered to be ubiquitous in ectotherms. Our recent observations of a complete growth plate cartilage (GPC) resorption, a reliable indicator of arrested skeletal growth, in many species of lizards clearly reject the ubiquity of indeterminate growth in reptiles and raise the question about the ancestral state of the growth pattern. Using X-ray micro-computed tomography (µCT), here we examined GPCs of long bones in three basally branching clades of squamate reptiles, namely in Gekkota, Scincoidea and Lacertoidea. A complete loss of GPC, indicating skeletal growth arrest, was the predominant finding. Using a dataset of 164 species representing all major clades of lizards and the tuataras, we traced the evolution of determinate growth on the phylogenetic tree of Lepidosauria. The reconstruction of character states suggests that determinate growth is ancestral for the squamate reptiles (Squamata) and remains common in the majority of lizard lineages, while extended (potentially indeterminate) adult growth evolved several times within squamates. Although traditionally associated with endotherms, determinate growth is coupled with ectothermy in this lineage. These findings combined with existing literature suggest that determinate growth predominates in both extant and extinct amniotes.

Ecological corridors for the amphibians and reptiles in the Natura 2000 sites of Romania.

Landscape heterogeneity and fragmentation are key challenges for biodiversity conservation. As Earth's landscape is increasingly dominated by anthropogenic land use, it is clear that broad-scale systems of nature reserves connected by corridors are needed to enable the dispersal of flora and fauna. The European Union currently supports a continent-wide network of protected areas, the Natura 2000 program, but this program lacks the necessary connectivity component. To examine whether a comprehensive network could be built in order to protect amphibians and reptiles, two taxonomic groups sensitive to environmental changes due to their physiological constrains and low dispersal capacity, we used species' distribution maps, the sites of community interest (SCIs) in Romania, and landscape resistance rasters. Except Vipera ursinii rakosiensis, all amphibians and reptiles had corridors mapped that, when assembled, provided linkages for up to 27 species. Natura 2000 species were not good candidates for umbrella species as these linkages covered only 17% of the corridors for all species. Important Areas for Connectivity were identified in the Carpathian Mountains and along the Danube River, further confirming these regions as hot spots for biodiversity in Europe, where successful linkages are most likely. In the end, while such corridors may not be created just for amphibians and reptiles, they can easily be incorporated into more complex linkages with corridors for more charismatic species, therefore enhancing the corridors' value in terms of quality and structure.

Bone histology of varanopids (Synapsida) from Richards Spur, Oklahoma, sheds light on growth patterns and lifestyle in early terrestrial colonizers.

Varanopids were a group of small to medium-sized synapsids whose fossil record spans the Carboniferous through middle Permian. Although their phylogenetic relationships have received some interest in recent years, little is known about other aspects of their palaeobiology, including their skeletal growth, allometry and habitat preference. Here, we describe varanopid long bone histology based on a sample of well-preserved femora from the lower Permian Richards Spur fissure fill locality, Comanche County, Oklahoma, USA. The sample includes five femora from at least two varanopid taxa-Mycterosaurus and the large varanodontine Varanops brevirostris-and four additional mycterosaurine femora not diagnosed to genus. Prior work on femoral bone compactness provided a baseline to make lifestyle inferences and evaluate whether varanopids were ancestrally terrestrial. Moreover, the large availability of specimens spanning different sizes made possible an assessment of size-related ontogenetic histovariability. All specimens revealed moderately dense cortical bone tissues composed of sparsely vascularized parallel-fibred and lamellar bone with radially arranged rows of longitudinal canals (mostly simple), and many preserved regularly spaced growth marks (annuli and lines of arrested growth) as in modern varanids. We show that bone histology has the potential to explain how ballast was shed and the skeleton lightened for terrestrial mobility in ancestral synapsids and their basal amniote kin, as well as how adjustments in postnatal growth influenced the evolution of larger body sizes in the terrestrial frontier. This article is part of the theme issue 'Vertebrate palaeophysiology'.

Developmental mechanisms driving complex tooth shape in reptiles.

BACKGROUND: In mammals, odontogenesis is regulated by transient signaling centers known as enamel knots (EKs), which drive the dental epithelium shaping. However, the developmental mechanisms contributing to formation of complex tooth shape in reptiles are not fully understood. Here, we aim to elucidate whether signaling organizers similar to EKs appear during reptilian odontogenesis and how enamel ridges are formed. RESULTS: Morphological structures resembling the mammalian EK were found during reptile odontogenesis. Similar to mammalian primary EKs, they exhibit the presence of apoptotic cells and no proliferating cells. Moreover, expression of mammalian EK-specific molecules (SHH, FGF4, and ST14) and GLI2-negative cells were found in reptilian EK-like areas. 3D analysis of the nucleus shape revealed distinct rearrangement of the cells associated with enamel groove formation. This process was associated with ultrastructural changes and lipid droplet accumulation in the cells directly above the forming ridge, accompanied by alteration of membranous molecule expression (Na/K-ATPase) and cytoskeletal rearrangement (F-actin). CONCLUSIONS: The final complex shape of reptilian teeth is orchestrated by a combination of changes in cell signaling, cell shape, and cell rearrangement. All these factors contribute to asymmetry in the inner enamel epithelium development, enamel deposition, ultimately leading to the formation of characteristic enamel ridges.

Adaptive responses of the embryos of birds and reptiles to spatial and temporal variations in nest temperatures.

Natural nests of egg-laying birds and reptiles exhibit substantial thermal variation, at a range of spatial and temporal scales. Rates and trajectories of embryonic development are highly sensitive to temperature, favouring an ability of embryos to respond adaptively (i.e. match their developmental biology to local thermal regimes). Spatially, thermal variation can be significant within a single nest (top to bottom), among adjacent nests (as a function of shading, nest depth etc.), across populations that inhabit areas with different weather conditions, and across species that differ in climates occupied and/or nest characteristics. Thermal regimes also vary temporally, in ways that generate differences among nests within a single population (e.g. due to seasonal timing of laying), among populations and across species. Anthropogenic activities (e.g. habitat clearing, climate change) add to this spatial and temporal diversity in thermal regimes. We review published literature on embryonic adaptations to spatio-temporal heterogeneity in nest temperatures. Although relatively few taxa have been studied in detail, and proximate mechanisms remain unclear, our review identifies many cases in which natural selection appears to have fine-tuned embryogenesis to match local thermal regimes. Developmental rates have been reported to differ between uppermost versus lower eggs within a single nest, between eggs laid early versus late in the season, and between populations from cooler versus warmer climates. We identify gaps in our understanding of thermal adaptations of early (embryonic) phases of the life history, and suggest fruitful opportunities for future research.

Effects of Chlorella vulgaris polysaccharides accumulation on growth characteristics of Trachemys scripta elegans.

The present study investigated the effects of the accumulated polysaccharides in Chlorella vulgaris microalgae on the growth characteristics of Trachemys scripta elegans. Sodium alginate was used to prepare immobilized C. vulgaris, and the antioxidant effects of the accumulated polysaccharides in it were determined using Caenorhabditis elegans as a model. We determined the specific growth rates of T. s. elegans (10 in each group) and their levels of non-specific immune-related indexes (including alkaline phosphatase; total superoxide dismutase; catalase; malondialdehyde). Under optimal culturing conditions, the accumulated polysaccharide content in C. vulgaris reached 32.7% (dry weight). Polysaccharides from C. vulgaris significantly improved the hydrogen peroxide-induced oxidative stress resistance and resulted in the enhancement of stress resistance-related antioxidant enzymes, including total superoxide dismutase and catalase (p < 0.05). The accumulated polysaccharides in C. vulgaris were heteropolysaccharides comprising rhamnose, ribose, arabinose, xylose, 2-deoxy-D-glucose, mannose, glucose, galactose, and glucosamine with a molar ratio of 0.26: 0.62: 0.21: 0.10: 0.08: 0.18: 1.00: 0.42: 0.17. Compared with the control group with common feeds, suspended and immobilized C. vulgaris with higher accumulated polysaccharide levels had a positive effect on the specific growth rate of the T. s. elegans (p < 0.05). Further, the suspended and immobilized C. vulgaris with higher accumulated polysaccharide levels significantly increased serum alkaline phosphatase, total superoxide dismutase and catalase activity (p < 0.05) and decreased serum malondialdehyde levels of T. s. elegans (p < 0.05).

Elastic instability during branchial ectoderm development causes folding of the Chlamydosaurus erectile frill.

We study the morphogenesis and evolutionary origin of the spectacular erectile ruff of the frilled dragon (Chlamydosaurus kingii). Our comparative developmental analyses of multiple species suggest that the ancestor of Episquamata reptiles developed a neck fold from the hyoid branchial arch by preventing it to fully fuse with posterior arches. We also show that the Chlamydosaurus embryonic neck fold dramatically enlarges and its anterior surface wrinkles, establishing three convex ridges on each lobe of the frill. We suggest that this robust folding pattern is not due to localised increased growth at the positions of the ridges, but emerges from an elastic instability during homogeneous growth of the frill skin frustrated by its attachment to adjacent tissues. Our physical analog experiments and 3D computational simulations, using realistic embryonic tissue growth, thickness and stiffness values, recapitulate the transition from two to three ridges observed during embryonic development of the dragon's frill.

Prenatal development in pterosaurs and its implications for their postnatal locomotory ability.

Recent fossil finds in China and Argentina have provided startling new insights into the reproductive biology and embryology of pterosaurs, Mesozoic flying reptiles. Nineteen embryos distributed among four species representing three distinct clades have been described and all are assumed to be at, or near, term. We show here how the application of four contrasting quantitative approaches allows a more precise identification of the developmental status of embryos revealing, for the first time to our knowledge, the presence of middle and late developmental stages as well as individuals that were at term. We also identify a predicted relationship between egg size and shape and the developmental stage of embryos contained within. Small elongate eggs contain embryos at an earlier stage of development than larger rounder eggs which contain more fully developed embryos. Changes in egg shape and size probably reflect the uptake of water, consistent with a pliable shell reported for several pterosaurs. Early ossification of the vertebral column, limb girdles and principal limb bones involved some heterochronic shifts in appearance times, most notably of manus digit IV, and facilitated full development of the flight apparatus prior to hatching. This is consistent with a super-precocial flight ability and, while not excluding the possibility of parental care in pterosaurs, suggests that it was not an absolute requirement.

Extreme thermal fluctuations from climate change unexpectedly accelerate demographic collapse of vertebrates with temperature-dependent sex determination.

Global climate is warming rapidly, threatening vertebrates with temperature-dependent sex determination (TSD) by disrupting sex ratios and other traits. Less understood are the effects of increased thermal fluctuations predicted to accompany climate change. Greater fluctuations could accelerate feminization of species that produce females under warmer conditions (further endangering TSD animals), or counter it (reducing extinction risk). Here we use novel experiments exposing eggs of Painted Turtles (Chrysemys picta) to replicated profiles recorded in field nests plus mathematically-modified profiles of similar shape but wider oscillations, and develop a new mathematical model for analysis. We show that broadening fluctuations around naturally male-producing (cooler) profiles feminizes developing embryos, whereas embryos from warmer profiles remain female or die. This occurs presumably because wider oscillations around cooler profiles expose embryos to very low temperatures that inhibit development, and to feminizing temperatures where most embryogenesis accrues. Likewise, embryos incubated under broader fluctuations around warmer profiles experience mostly feminizing temperatures, some dangerously high (which increase mortality), and fewer colder values that are insufficient to induce male development. Therefore, as thermal fluctuations escalate with global warming, the feminization of TSD turtle populations could accelerate, facilitating extinction by demographic collapse. Aggressive global CO2 mitigation scenarios (RCP2.6) could prevent these risks, while intermediate actions (RCP4.5 and RCP6.0 scenarios) yield moderate feminization, highlighting the peril that insufficient reductions of greenhouse gas emissions pose for TSD taxa. If our findings are generalizable, TSD squamates, tuatara, and crocodilians that produce males at warmer temperatures could suffer accelerated masculinization, underscoring the broad taxonomic threats of climate change.

Disparate Growth Strategies within Aetosauria: Novel Histologic Data from the Aetosaur Coahomasuchus chathamensis.

Aetosaurs comprise a clade of quadrupedal, armored, omnivores to herbivores that lived across much of the supercontinent of Pangea during the Late Triassic. Their relative abundance in many units, and the rarity of other Triassic herbivores, points to them as key components of Late Triassic ecosystems. Debate persists about whether they were growing more or less slowly when compared to extant crocodylians, and bone histology is sparsely sampled within the group. We undertook a histological examination of Coahomasuchus chathamensis to address its ontogenetic trajectory and characterize its histology. We sampled a paramedian osteoderm from the holotype specimen, as well as five osteoderms (two paramedian, one lateral, and two of uncertain position) and two incomplete limb bones (radius and fibula), from referred specimens discovered at the type locality. Using these we estimated specimen ages with lines of arrested growth (LAGs) to determine that the study individuals reached from 2 to 7 years old. All of the sampled elements contained woven-fibered bone with extensive vasculature within the internal cortex. In some specimens, more poorly vascularized, parallel-fibered bone is evident externally. The holotype of C. chathamensis represents a juvenile individual, and raises the possibility that the holotype of C. kahleorum is a juvenile as well, complicating aetosaur systematics and diversity. When compared to aetosaurs of similar size, it is clear that C. chathamensis was growing comparatively rapidly, about 3 times the rate of similarly sized specimens of Aetosauroides scagliai from Argentina. This discovery reveals the presence of disparate growth strategies within Aetosauria. Anat Rec, 302:1504-1515, 2019. © 2018 American Association for Anatomy.

Dental ontogeny in extinct synapsids reveals a complex evolutionary history of the mammalian tooth attachment system.

The mammalian dentition is uniquely characterized by a combination of precise occlusion, permanent adult teeth and a unique tooth attachment system. Unlike the ankylosed teeth in most reptiles, mammal teeth are supported by a ligamentous tissue that suspends each tooth in its socket, providing flexible and compliant tooth attachment that prolongs the life of each tooth and maintains occlusal relationships. Here we investigate dental ontogeny through histological examination of a wide range of extinct synapsid lineages to assess whether the ligamentous tooth attachment system is unique to mammals and to determine how it evolved. This study shows for the first time that the ligamentous tooth attachment system is not unique to crown mammals within Synapsida, having arisen in several non-mammalian therapsid clades as a result of neoteny and progenesis in dental ontogeny. Mammalian tooth attachment is here re-interpreted as a paedomorphic condition relative to the ancestral synapsid form of tooth attachment.

Introduction to the special issue-Developmental plasticity in reptiles: Physiological mechanisms and ecological consequences.

Scientific interest in developmental plasticity spans many disciplines, and research on reptiles has provided many insights into this field. We highlight these contributions, review the field's history, and introduce the special issue on this topic .

Developmental temperatures and phenotypic plasticity in reptiles: a systematic review and meta-analysis.

Early environments can profoundly influence an organism in ways that persist over its life. In reptiles, early thermal environments (nest temperatures) can impact offspring phenotype and survival in important ways, yet we still lack an understanding of whether general trends exist and the magnitude of impact. Understanding these patterns is important in predicting how climate change will affect reptile populations and the role of phenotypic plasticity in buffering populations. We compiled data from 175 reptile studies to examine, and quantify, the effect of incubation temperature on phenotype and survival. Using meta-analytic approaches (standardized mean difference between incubation treatments, Hedges' g), we show that across all trait types examined there is, on average, a moderate to large magnitude of effect of incubation temperatures (absolute effect: |g| = 0.75). Unsurprisingly, this influence was extremely large for incubation duration, as predicted, with warmer temperatures decreasing incubation time overall (g = -8.42). Other trait types, including behaviour, physiology, morphology, performance, and survival experienced reduced, but still mostly moderate to large effects, with particularly strong effects on survival. Moreover, the impact of incubation temperature persisted at least one-year post-hatching, suggesting that these effects have the potential to impact fitness in the long term. The magnitude of effect increased as the change in temperature increased (e.g. 6°C versus 2°C) in almost all cases, and tended to decrease when temperatures of the treatments fluctuated around a mean temperature compared to when they were constant. The effect also depended on the mid-temperature of the comparison, but not in consistent ways, with some traits experiencing the greatest effects at extreme temperatures, while others did not. The highly heterogeneous nature of the effects we observe, along with a large amount of unexplained variability, indicates that the shape of reaction norms between phenotype and temperature, along with ecological and/or experimental factors, are important when considering general patterns. Our analyses provide new insights into the effects of incubation environments on reptile phenotype and survival and allow general, albeit coarse, predictions for taxa experiencing warming nest temperatures under climatic change.

The old and new faces of morphology: the legacy of D'Arcy Thompson's 'theory of transformations' and 'laws of growth'.

In 1917, the publication of On Growth and Form by D'Arcy Wentworth Thompson challenged both mathematicians and naturalists to think about biological shapes and diversity as more than a confusion of chaotic forms generated at random, but rather as geometric shapes that could be described by principles of physics and mathematics. Thompson's work was based on the ideas of Galileo and Goethe on morphology and of Russell on functionalism, but he was first to postulate that physical forces and internal growth parameters regulate biological forms and could be revealed via geometric transformations in morphological space. Such precise mathematical structure suggested a unifying generative process, as reflected in the title of the book. To Thompson it was growth that could explain the generation of any particular biological form, and changes in ontogeny, rather than natural selection, could then explain the diversity of biological shapes. Whereas adaptationism, widely accepted in evolutionary biology, gives primacy to extrinsic factors in producing morphological variation, Thompson's 'laws of growth' provide intrinsic directives and constraints for the generation of individual shapes, helping to explain the 'profusion of forms, colours, and other modifications' observed in the living world.