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.

Elevation, oxygen, and the origins of viviparity.

Research focused on understanding the evolutionary factors that shape parity mode evolution among vertebrates have long focused on squamate reptiles (snakes and lizards), which contain all but one of the evolutionary transitions from oviparity to viviparity among extant amniotes. While most hypotheses have focused on the role of cool temperatures in favoring viviparity in thermoregulating snakes and lizards, there is a growing appreciation in the biogeographic literature for the importance of lower oxygen concentrations at high elevations for the evolution of parity mode. However, the physiological mechanisms underlying how hypoxia might reduce fitness, and how viviparity can alleviate this fitness decrement, has not been systematically evaluated. We qualitatively evaluated previous research on reproductive and developmental physiology, and found that (1) hypoxia can negatively affect fitness of squamate embryos, (2) oxygen availability in the circulatory system of adult lizards can be similar or greater than an egg, and (3) gravid females can possess adaptive phenotypic plasticity in response to hypoxia. These findings suggest that the impact of hypoxia on the development and physiology of oviparous and viviparous squamates would be a fruitful area of research for understanding the evolution of viviparity. To that end, we propose an integrative research program for studying hypoxia and the evolution of viviparity in squamates.

Immunochemical Detection of Modified Cytosine Species in Lampbrush Chromatin.

The lampbrush chromosomes found in the giant nucleus or germinal vesicle (GV) of amphibian oocytes provide unique opportunities for discrete closed and open chromatin structural domains to be directly observable by simple light microscopy. Moreover, the method described here for preparing spreads of lampbrush chromatin for immunostaining enables a straightforward approach to establishing the distributions of modified nucleotides within and between structurally and functionally distinctive chromatin domains.

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.

Heat tolerance of reptile embryos: Current knowledge, methodological considerations, and future directions.

Aspects of global change result in warming temperatures that threaten biodiversity across the planet. Eggs of non-avian, oviparous reptiles (henceforth "reptiles") are particularly vulnerable to warming due to a lack of parental care during incubation and limited ability to behaviorally thermoregulate. Because warming temperatures will cause increases in both mean and variance of nest temperatures, it is crucial to consider embryo responses to both chronic and acute heat stress. Although many studies have considered embryo survival across constant incubation temperatures (i.e., chronic stress) and in response to brief exposure to extreme temperatures (i.e., acute stress), there are no standard metrics or terminology for determining heat stress of embryos. This impedes comparisons across studies and species and hinders our ability to predict how species will respond to global change. In this review, we compare various methods that have been used to assess embryonic heat tolerance in reptiles and provide new terminology and metrics for quantifying embryo responses to both chronic and acute heat stress. We apply these recommendations to data from the literature to assess chronic heat tolerance in 16 squamates, 16 turtles, five crocodilians, and the tuatara and acute heat tolerance for nine squamates and one turtle. Our results indicate that there is relatively large variation in chronic and acute heat tolerance across species, and we outline directions for future research, calling for more studies that assess embryo responses to acute thermal stress, integrate embryo responses to chronic and acute temperatures in predictive models, and identify mechanisms that determine heat tolerance.

Skeletons of the Eye: An Evolutionary and Developmental Perspective.

The ocular skeleton, composed of the scleral cartilage and scleral ossicles, is present in many vertebrates. The morphology of the scleral cartilage and ossicles varies within different extant reptiles (including birds) and also varies dramatically from the morphology in extant teleosts. This incredible range of diverse morphologies is the result of millions of years of evolution. Both the position of these elements within the eye and the timing of development vary amongst different vertebrates. While the development of both the scleral cartilage and scleral ossicles is somewhat understood in reptiles and in teleosts, the functional advantage of these elements is still debated. Most reptiles have a multi-component scleral ossicle ring composed of a series of flat bone plates and a scleral cartilage cup lining the retina, some sharks have calcified cartilage plates, and some teleosts have two bones while most others only have a ring of scleral cartilage. The data presented shows that different vertebrates have adapted to similar selective pressures in different ways. However, the reason why sarcopterygians have a series of overlapping bones in the sclera remains unclear. A better understanding of the ocular skeletal diversity in Reptilia as well as a better understanding of the mechanisms of vision within different environments (i.e., air vs. water) and that used by secondarily aquatic organisms is needed. This review discusses the observed variation in morphology and development of the ocular skeleton in the context of evolution and highlights our knowledge gaps in these areas. Anat Rec, 2018. © 2018 American Association for Anatomy.

An overlooked placode: Recharacterizing the papillae in the embryonic eye of reptilia.

The papillae in the chicken embryonic eye, described as scleral papillae in the well-known Hamburger and Hamilton (1951) staging table, are one of the key anatomical features used to stage reptilian (including bird) embryos from HH30-36. These papillae are epithelial thickenings of the conjunctiva and are situated above the mesenchymal sclera. Here, we present evidence that the conjunctival papillae, which are required for the induction and patterning of the underlying scleral ossicles, require epithelial pre-patterning and have a placodal stage similar to other placode systems. We also suggest modifications to the Hamburger Hamilton staging criteria that incorporate this change in terminology (from "scleral" to "conjunctival" papillae) and provide a more detailed description of this anatomical feature that includes its placode stage. This enables a more complete and accurate description of chick embryo staging. The acknowledgment of a placode phase, which shares molecular and morphological features with other cutaneous placodes, will direct future research into the early inductive events leading to scleral ossicle formation.

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.

Insights into Germline Development and Differentiation in Molluscs and Reptiles: The Use of Molecular Markers in the Study of Non-model Animals.

When shifting research focus from model to non-model species, many differences in the working approach should be taken into account and usually methodological modifications are required because of the lack of genetics/genomics and developmental information for the vast majority of organisms. This lack of data accounts for the largely incomplete understanding of how the two components-genes and developmental programs-are intermingled in the process of evolution. A deeper level of knowledge was reached for a few model animals, making it possible to understand some of the processes that guide developmental changes during evolutionary time. However, it is often difficult to transfer the obtained information to other, even closely related, animals. In this chapter, we present and discuss some examples, such as the choice of molecular markers to be used to characterize differentiation and developmental processes. The chosen examples pertain to the study of germline in molluscs, reptiles, and other non-model animals.

Evolutionary view of pluripotency seen from early development of non-mammalian amniotes.

Early embryonic cells are capable of acquiring numerous developmental fates until they become irreversibly committed to specific lineages depending on intrinsic determinants and/or regional interactions. From fertilization to gastrulation, such pluripotent cells first increase in number and then turn to undergoing differentiation. Mechanisms regulating pluripotency in each species attract great interest in developmental biology. Also, outlining the evolutionary background of pluripotency can enhance our understanding of mammalian pluripotency and provide a broader view of early development of vertebrates. Here, we introduce integrative models of pluripotent states in amniotes (mammals, birds and reptiles) to offer a comprehensive overview of widely accepted knowledge about mammalian pluripotency and our recent findings in non-mammalian amniotes, such as chicken and gecko. In particular, we describe 1) the IL6/Stat3 signaling pathway as a positive regulator of naive pluripotency, 2) Fgf/Erk signaling as a process that prepares cells for differentiation, 3) the role of the interactions between these two signaling pathways during the transition from pluripotency to differentiation, and 4) functional diversification of two transcription factors, Class V POUs and Nanog. In the last section, we also briefly discuss possible relationships of unique cell cycle properties of early embryonic cells with signaling pathways and developmental potentials in the pluripotent cell states.

Reptile Embryology and Regeneration.

Reptiles (lizards, snakes, turtles, and crocodilians) are becoming increasingly popular as models for developmental investigations. In this review the leopard gecko, Eublepharis macularius, is presented as a reptilian model for embryonic and tissue regeneration studies. We provide details of husbandry and breeding and discuss aspects of embryonic nutrition, egg anatomy, and sex determination. We provide comprehensive protocols for transcardial perfusion, short-term anesthesia using the injectable anesthetic Alfaxan, and full-thickness cutaneous biopsy punches, used in geckos for the study of scar-free wound healing. We also provide modifications to three popular histological techniques (whole-mount histochemistry, immunohistochemistry, and double-label immunofluorescence) and provide details on bromodeoxyuridine (BrdU) labeling and immuno-detection.

Embryological development of Salvator merianae (Squamata: Teiidae).

Here we describe the embryonic development of Salvator merianae external morphologic features, as based on observation of 28 embryos across different days of incubation at 31 ± 0.5°C. Observed developmental stages were grouped and classified into the early, middle, and late periods. The early period (Stages 3-11) is distinguished by the origin of the encephalic vesicles, sensory placodes, pharyngeal arches, and degree of body flexion and rotation. The medium period (Stages 8-15) is distinguished by limb differentiation and by cranium-facial characteristics. The late period (Stages 15-18) is determined by scale patterns, pigmentation, and embryo growth.

Hox Genes in Reptile Development, Epigenetic Regulation, and Teratogenesis.

Reptiles are ancestral organisms presenting a variety of shapes, from the elongated vertebral column of the snake to the turtle dorsalized ribs or retractile neck. Body plans are specified by a conserved group of homeobox-containing genes (Hox genes), which encode transcription factors important in cell fate and vertebral architecture along the anteroposterior axis during embryonic development; thus, dysregulation of these genes may cause congenital malformations, from mild-sublethal to embryonic-lethal. The genetic pool, maternal transfer, and environmental conditions during egg incubation affect development; environmental factors such as temperature, moisture, oxygen, and pollution may alter gene expression by epigenetic mechanisms. Thus, in this review, we present information regarding Hox genes and development in reptiles, including sex determination and teratogenesis. We also present some evidence of epigenetic regulation of Hox genes and the role of the environment in epigenetic modulation of gene expression. So far, the evidence suggests that the molecular instructions encoded by Hox genes to build a snake, a lizard, or a turtle represent the interplay between genome and epigenome after years of evolution, with occasional environmentally induced molecular mistakes leading to abnormal body shapes.

A comprehensive database of thermal developmental plasticity in reptiles.

How temperature influences development has direct relevance to ascertaining the impact of climate change on natural populations. Reptiles have served as empirical models for understanding how the environment experienced by embryos can influence phenotypic variation, including sex ratio, phenology and survival. Such an understanding has important implications for basic eco-evolutionary theory and conservation efforts worldwide. While there is a burgeoning empirical literature of experimental manipulations of embryonic thermal environments, addressing widespread patterns at a comparative level has been hampered by the lack of accessible data in a format that is amendable to updates as new studies emerge. Here, we describe a database with nearly 10, 000 phenotypic estimates from 155 species of reptile, collected from 300 studies manipulating incubation temperature (published between 1974-2016). The data encompass various morphological, physiological, behavioural and performance traits along with growth rates, developmental timing, sex ratio and survival (e.g., hatching success). This resource will serve as an important data repository for addressing overarching questions about thermal plasticity of reptile embryos.

Developmental plasticity in reptiles: Critical evaluation of the evidence for genetic and maternal effects on temperature-dependent sex determination.

We critically review literature that examines variation in temperature-dependent sex determination (TSD) within species. Although variation in sex ratio among clutches of eggs from different females is common in reptiles with TSD, the underlying mechanism that causes this variation is not clear. Authors have made claims about genetic variation in TSD and maternal effects on TSD. The latter type of study usually focuses on maternally derived steroids in egg yolk. Here, we outline the types of experiments and data required to unequivocally demonstrate that variation in sex ratio among clutches (1) has a genetic basis, (2) is caused by maternally derived steroids, or (3) is influenced by both factors. To date, few studies have met these requirements.

Patterns of developmental plasticity in response to incubation temperature in reptiles.

Early life environments shape phenotypic development in important ways that can lead to long-lasting effects on phenotype and fitness. In reptiles, one aspect of the early environment that impacts development is temperature (termed 'thermal developmental plasticity'). Indeed, the thermal environment during incubation is known to influence morphological, physiological, and behavioral traits, some of which have important consequences for many ecological and evolutionary processes. Despite this, few studies have attempted to synthesize and collate data from this expansive and important body of research. Here, we systematically review research into thermal developmental plasticity across reptiles, structured around the key papers and findings that have shaped the field over the past 50 years. From these papers, we introduce a large database (the 'Reptile Development Database') consisting of 9,773 trait means across 300 studies examining thermal developmental plasticity. This dataset encompasses data on a range of phenotypes, including morphological, physiological, behavioral, and performance traits along with growth rate, incubation duration, sex ratio, and survival (e.g., hatching success) across all major reptile clades. Finally, from our literature synthesis and data exploration, we identify key research themes associated with thermal developmental plasticity, important gaps in empirical research, and demonstrate how future progress can be made through targeted empirical, meta-analytic, and comparative work.

Temperature fluctuations and maternal estrogens as critical factors for understanding temperature-dependent sex determination in nature.

Vertebrates with temperature-dependent sex determination (TSD) have justifiably received a lot of attention when it comes to the potential effects of climate change. Freshwater turtles have long been used to characterize the physiological and genetic mechanisms underlying TSD and provide a great system to investigate how changing climatic conditions will affect vertebrates with TSD. Unfortunately, most of what we know about the mechanisms underlying TSD comes from laboratory conditions that do not accurately mimic natural conditions (i.e., constant incubation temperatures and supraphysiological steroid manipulations). In this paper, we review recent advances in our understanding of how TSD operates in nature that arose from studies using more natural fluctuating incubation temperatures and natural variation in maternal estrogens within the yolk. By incorporating more natural conditions into laboratory studies, we are better able to use these studies to predict how changing climatic conditions will affect species with TSD.

Homologous Recombination between Genetically Divergent Campylobacter fetus Lineages Supports Host-Associated Speciation.

Homologous recombination is a major driver of bacterial speciation. Genetic divergence and host association are important factors influencing homologous recombination. Here, we study these factors for Campylobacter fetus, which shows a distinct intraspecific host dichotomy. Campylobacter fetus subspecies fetus (Cff) and venerealis are associated with mammals, whereas C. fetus subsp. testudinum (Cft) is associated with reptiles. Recombination between these genetically divergent C. fetus lineages is extremely rare. Previously it was impossible to show whether this barrier to recombination was determined by the differential host preferences, by the genetic divergence between both lineages or by other factors influencing recombination, such as restriction-modification, CRISPR/Cas, and transformation systems. Fortuitously, a distinct C. fetus lineage (ST69) was found, which was highly related to mammal-associated C. fetus, yet isolated from a chelonian. The whole genome sequences of two C. fetus ST69 isolates were compared with those of mammal- and reptile-associated C. fetus strains for phylogenetic and recombination analysis. In total, 5.1-5.5% of the core genome of both ST69 isolates showed signs of recombination. Of the predicted recombination regions, 80.4% were most closely related to Cft, 14.3% to Cff, and 5.6% to C. iguaniorum. Recombination from C. fetus ST69 to Cft was also detected, but to a lesser extent and only in chelonian-associated Cft strains. This study shows that despite substantial genetic divergence no absolute barrier to homologous recombination exists between two distinct C. fetus lineages when occurring in the same host type, which provides valuable insights in bacterial speciation and evolution.

How frequent and important is behavioral thermoregulation by embryonic reptiles?

The debate about behavioral thermoregulation inside reptile eggs centers on the frequency (and hence, biological significance) of the phenomenon, not about its validity. Both sides of the debate agree that large eggs in shallow nests laid in sun-exposed soil will experience clines in mean temperature and (especially) diel thermal variance; that embryos in the middle phase of development have the ability to reposition themselves, and room to do so; and that small changes in developmental temperatures can influence offspring fitness. Equally, all protagonists agree that thermal clines will be too low in some other kinds of nests, and that embryonic repositioning is impossible very early and very late in development. Based on an array of other fitness-enhancing behaviors exhibited by tetrapod embryos, and general principles for recognizing adaptation, we conclude that behavioral thermoregulation inside the egg likely is adaptive in some but not all reptile species. We identify productive directions for empirical research to resolve points of contention.

Reptile embryos are not capable of behavioral thermoregulation in the egg.

Reptile embryos have recently been observed moving within the egg in response to temperature, raising the exciting possibility that embryos might behaviorally thermoregulate analogous to adults. However, the conjecture that reptile embryos have ample opportunity and capacity to adaptively control their body temperature warrants further discussion. Using turtles as a model, we discuss the spatiotemporal constraints to movement in reptile embryos. We demonstrate that, as embryos grow, the internal egg space rapidly diminishes such that the temporal window for appreciable displacement is confined to stages that feature incomplete neuromuscular differentiation. During this time, muscles are insufficiently developed to actively and consistently control movement. These constraints are well illustrated by the Chinese softshelled turtle (Pelodiscus sinensis), the first reptile reported to behaviorally thermoregulate. Furthermore, sporadic embryo activity peaks after the temperature-sensitive period in species with temperature-dependent sex determination, thus nullifying the opportunity for embryos to exhibit control over this important phenotype. These embryonic constraints add to previously-identified environmental constraints on behavioral thermoregulation by reptile embryos. We discuss alternative hypotheses to explain previously reported patterns of behavioral thermoregulation. Based on a holistic consideration of embryonic limitations, we conclude that reptile embryos are generally unable to adaptively behaviorally thermoregulate within the egg.