Geographic variation in incubation temperatures promoting viable offspring production in broadly co-distributed turtles.

Organisms whose early life stages are environmentally sensitive produce offspring within a relatively narrow range of suitable abiotic conditions. In reptiles, development rate and survival are often maximized if incubation temperatures remain under 31°C, though this upper bound may vary within and among species. We addressed this expectation by comparing responses to egg incubation at 30°C versus 33°C in congeneric turtle species pairs with broad syntopic geographic distributions. In the two softshell turtles (Apalone spp.), the greatest changes in development rate and phenotypic variance were observed in the northernmost population, which had a low survival rate (40%) at 33°C. The presumably suboptimal temperature (33°C) for northern populations otherwise yielded 76%-93% survival rates and fast swimming speeds in more southern populations. Still, in one species, northern hatchlings incubated at 33°C matched the elevated speeds of their southern counterparts, revealing a countergradient response. In northern populations of the two map turtles (Graptemys spp.), survival was also reduced (28%-60%) at 33°C and the development rate (relative to 30°C) increased by up to 75%. Our experiments on divergent taxa with similar nesting ecologies substantiate that the optimal thermal range for offspring production is variable. These findings encourage further work on how population- and species-level differences relate to local adaptation in widely distributed oviparous species.

Skeletal repatterning enhances the protective capacity of the shell in African hinge-back tortoises (Kinixys).

Changes in the structural association of skeletal traits are crucial to the evolution of novel forms and functions. In vertebrates, such rearrangements often occur gradually and may precede or coincide with the functional activation of skeletal traits. To illustrate this process, we examined the ontogeny of African hinge-back tortoises (Kinixys spp.). Kinixys species feature a moveable "hinge" on the dorsal shell (carapace) that enables shell closure (kinesis) when the hind limbs are withdrawn. This hinge, however, is absent in juveniles. Herein, we describe how this unusual phenotype arises via alterations in the tissue configuration and shape of the carapace. The ontogenetic repatterning of osseous and keratinous tissue coincided with shifts in morphological integration and the establishment of anterior (static) and posterior (kinetic) carapacial modules. Based on ex vivo skeletal movement and raw anatomy, we propose that Kinixys employs a "sliding hinge" shell-closing system that overcomes thoracic rigidity and enhances the protective capacity of the carapace. Universal properties of the vertebrate skeleton, such as plasticity, modularity, and secondary maturation processes, contributed to adaptive evolutionary change in Kinixys. We discuss a hypothetical model to explain the delayed emergence of skeletal traits and its relevance to the origins of novel form-to-function relationships.

Effects of the egg incubation environment on turtle carapace development.

Developing organisms are often exposed to fluctuating environments that destabilize tissue-scale processes and induce abnormal phenotypes. This might be common in species that lay eggs in the external environment and with little parental care, such as many reptiles. In turtles, morphological development has provided striking examples of abnormal phenotypic patterns, though the influence of the environment remains unclear. To this end, we compared fluctuating asymmetry, as a proxy for developmental instability, in turtle hatchlings incubated in controlled laboratory and unstable natural conditions. Wild and laboratory hatchlings featured similar proportions of supernumerary scales (scutes) on the dorsal shell (carapace). Such abnormal scutes likely elevated shape asymmetry, which was highest in natural nests. Moreover, we tested the hypothesis that hot and dry environments cause abnormal scute formation by subjecting eggs to a range of hydric and thermal laboratory incubation regimes. Shape asymmetry was similar in hatchlings incubated at five constant temperatures (26-30°C). A hot (30°C) and severely Dry substrate yielded smaller hatchlings but scutes were not overtly affected. Our study suggests that changing nest environments contribute to fluctuating asymmetry in egg-laying reptiles, while clarifying the conditions at which turtle shell development remains buffered from the external environment.

Domestication and the comparative embryology of birds.

Studies of domesticated animals have greatly contributed to our understanding of avian embryology. Foundational questions in developmental biology were motivated by Aristotle's observations of chicken embryos. By the 19th century, the chicken embryo was at the center stage of developmental biology, but how closely does this model species mirror the ample taxonomic diversity that characterizes the avian tree of life? Here, we provide a brief overview of the taxonomic breadth of comparative embryological studies in birds. We particularly focused on staging tables and papers that attempted to document the timing of developmental transformations. We show that most of the current knowledge of avian embryology is based on Galliformes (chicken and quail) and Anseriformes (duck and goose). Nonetheless, data are available for some ecologically diverse avian subclades, including Struthioniformes (e.g., ostrich, emu) and Sphenisciformes (penguins). Thus far, there has only been a handful of descriptive embryological studies in the most speciose subclade of Aves, that is, the songbirds (Passeriniformes). Furthermore, we found that temporal variances for developmental events are generally uniform across a consensus chronological sequence for birds. Based on the available data, developmental trajectories for chicken and other model species appear to be highly similar. We discuss future avenues of research in comparative avian embryology in light of the currently available wealth of data on domesticated species and beyond.

Shifts in growth, but not differentiation, foreshadow the formation of exaggerated forms under chicken domestication.

Domestication provides an outstanding opportunity for biologists to explore the underpinnings of organismal diversification. In domesticated animals, selective breeding for exaggerated traits is expected to override genetic correlations that normally modulate phenotypic variation in nature. Whether this strong directional selection affects the sequence of tightly synchronized events by which organisms arise (ontogeny) is often overlooked. To address this concern, we compared the ontogeny of the red junglefowl (RJF) (Gallus gallus) to four conspecific lineages that underwent selection for traits of economic or ornamental value to humans. Trait differentiation sequences in embryos of these chicken breeds generally resembled the representative ancestral condition in the RJF, thus revealing that early ontogeny remains highly canalized even during evolution under domestication. This key finding substantiates that the genetic cost of domestication does not necessarily compromise early ontogenetic steps that ensure the production of viable offspring. Instead, disproportionate beak and limb growth (allometry) towards the end of ontogeny better explained phenotypes linked to intense selection for industrial-scale production over the last 100 years. Illuminating the spatial and temporal specificity of development is foundational to the enhancement of chicken breeds, as well as to ongoing research on the origins of phenotypic variation in wild avian species.

Disentangling the correlated evolution of body size, life history, and ontogeny in miniaturized chelydroid turtles.

Organismal miniaturization is defined by a reduction in body size relative to a large ancestor. In vertebrate animals, miniaturization is achieved by suppressing the energetics of growth. However, this might interfere with reproductive strategies in egg-laying species with limited energy budgets for embryo growth and differentiation. In general, the extent to which miniaturization coincides with alterations in animal development remains obscure. To address the interplay among body size, life history, and ontogeny, miniaturization in chelydroid turtles was examined. The analyses corroborated that miniaturization in the Chelydroidea clade is underlain by a dampening of the ancestral growth trajectory. There were no associated shifts in the early sequence of developmental transformations, though the relative duration of organogenesis was shortened in miniaturized embryos. The size of eggs, hatchlings, and adults was positively correlated within Chelydroidea. A phylogenetically broader exploration revealed an alternative miniaturization mode wherein exceptionally large hatchlings grow minimally and thus attain diminutive adult sizes. Lastly, it is shown that miniaturized Chelydroidea turtles undergo accelerated ossification coupled with a ~10% reduction in shell bones. As in other vertebrates, the effects of miniaturization were not systemic, possibly owing to opposing functional demands and tissue geometric constraints. This underscores the integrated and hierarchical nature of developmental systems.

Transcriptomic similarities and differences between the limb bud AER and unique carapacial ridge of turtle embryos.

Evolutionary innovation may arise via major departures from an ancestral condition. Turtle shell morphogenesis depends on a unique structure known as the carapacial ridge (CR). This lateral tissue protrusion in turtle embryos exhibits similar properties as the apical ectodermal ridge (AER)-a well-known molecular signaling center involved in limb development. Still, how the CR influences shell morphogenesis is not entirely clear. The present study aimed to describe the CR transcriptome shortly before ribs were halted within its mesenchyme, as required for shell development. Analyses exposed that the mesenchymal marker VIM was one of the most highly co-expressed genes and numerous appendage formation genes were situated within the core of CR and AER co-expression networks. However, there were tissue-specific differences in the activity of these genes. For instance, WNT5A was most frequently assigned to appendage-related annotations of the CR network core, but not in the AER. Several homeobox transcription factors known to regulate limb bud patterning exhibited their highest expression levels in the AER, but were underexpressed in the CR. The results of this study corroborate that novel body plans often originate via alterations of pre-existing genetic networks. Altogether, this exploratory study enhances the groundwork for future experiments on the molecular underpinnings of turtle shell development and evolution.

An irregular hourglass pattern describes the tempo of phenotypic development in placental mammal evolution.

Organismal development is defined by progressive transformations that ultimately give rise to distinct tissues and organs. Thus, temporal shifts in ontogeny often reflect key phenotypic differences in phylogeny. Classical theory predicts that interspecific morphological divergence originates towards the end of embryonic or fetal life stages, i.e. the early conservation model. By contrast, the hourglass model predicts interspecific variation early and late in prenatal ontogeny, though with a phylogenetically similar mid-developmental period. This phylotypic period, however, remains challenging to define within large clades such as mammals. Thus, molecular and morphological tests on a mammalian hourglass have not been entirely congruent. Here, we report an hourglass-like pattern for mammalian developmental evolution. By comparing published data on the timing of 74 homologous characters across 51 placental species, we demonstrated that variation in the timing of development decreased late in embryogenesis--when organ formation is highly active. Evolutionary rates of characters related to this timeframe were lowest, coinciding with a phylotypic period that persisted well beyond the pharyngula 'stage'. The trajectory culminated with elevated variation in a handful of fetal and perinatal characters, yielding an irregular hourglass pattern. Our study invites further quantification of ontogeny across diverse amniotes and thus challenges current ideas on the universality of developmental patterns.

Excess postexercise oxygen consumption decreases with swimming duration in a labriform fish: Integrating aerobic and anaerobic metabolism across time.

Many vertebrate animals employ anaerobic pathways during high-speed exercise, even if it imposes an energetic cost during postexercise recovery, expressed as excess postexercise oxygen consumption (EPOC). In ectotherms such a fish, the initial anaerobic contribution to exercise is often substantial. Even so, fish may recover from anaerobic pathways as swimming exercise ensues and aerobic metabolism stabilizes, thus total energetic costs of exercise could depend on swimming duration and subsequent physiological recovery. To test this hypothesis, we examined EPOC in striped surfperch (Embiotoca lateralis) that swam at high speeds (3.25 L s-1 ) during randomly ordered 2-, 5-, 10-, and 20-min exercise periods. We found that EPOC was highest after the 2-min period (20.9 mg O2 kg-1 ) and lowest after the 20-min period (13.6 mg O2 kg-1 ), indicating that recovery from anaerobic pathways improved with exercise duration. Remarkably, EPOC for the 2-min period accounted for 72% of the total O2 consumption, whereas EPOC for the 20-min period only accounted for 14%. Thus, the data revealed a striking decline in the total cost of transport from 0.772 to 0.226 mg O2 ·kg-1 ·m-1 during 2- and 20-min periods, respectively. Our study is the first to combine anaerobic and aerobic swimming costs to demonstrate an effect of swimming duration on EPOC in fish. Clarifying the dynamic nature of exercise-related costs is relevant to extrapolating laboratory findings to animals in the wild.

The postembryonic transformation of the shell in emydine box turtles.

A key trend in the 210-million-year-old history of modern turtles was the evolution of shell kinesis, that is, shell movement during neck and limb retraction. Kinesis is hypothesized to enhance predator defense in small terrestrial and semiaquatic turtles and has evolved multiple times since the early Cretaceous. This complex phenotype is nonfunctional and far from fully differentiated following embryogenesis. Instead, kinesis develops slowly in juveniles, providing a unique opportunity to illustrate the postembryonic origins of an adaptive trait. To this end, we examined ventral shell (plastral) kinesis in emydine box turtles and found that hatchling plastron shape differs from that of akinetic-shelled relatives, particularly where the hinge that enables kinesis differentiates. We also demonstrated shape changes relative to plastron size in juveniles, coinciding with a shift in the carapace-plastron structural connection, rearrangement of ectodermal plates, and bone repatterning. Furthermore, because the shell grows larger relative to the head, complete concealment of the head and extremities is only achieved after relative shell proportions increase. Structural alterations that facilitate the box turtle's transformation are probably prepatterned in embryos but require function-induced changes to differentiate in juveniles. This mode of delayed trait differentiation is essential to phenotypic diversification in turtles and perhaps other tetrapods.

Transplanting gravid lizards to high elevation alters maternal and embryonic oxygen physiology, but not reproductive success or hatchling phenotype.

Increased global temperatures have opened previously inhospitable habitats, such as at higher elevations. However, the reduction of oxygen partial pressure with increase in elevation represents an important physiological constraint that may limit colonization of such habitats, even if the thermal niche is appropriate. To test the mechanisms underlying the response to ecologically relevant levels of hypoxia, we performed a translocation experiment with the common wall lizard (Podarcis muralis), a widespread European lizard amenable to establishing populations outside its natural range. We investigated the impacts of hypoxia on the oxygen physiology and reproductive output of gravid common wall lizards and the subsequent development and morphology of their offspring. Lowland females transplanted to high elevations increased their haematocrit and haemoglobin concentration within days and maintained routine metabolism compared with lizards kept at native elevations. However, transplanted lizards suffered from increased reactive oxygen metabolite production near the oviposition date, suggesting a cost of reproduction at high elevation. Transplanted females and females native to different elevations did not differ in reproductive output (clutch size, egg mass, relative clutch mass or embryonic stage at oviposition) or in post-oviposition body condition. Developing embryos reduced heart rates and prolonged incubation times at high elevations within the native range and at extreme high elevations beyond the current range, but this reduced oxygen availability did not affect metabolic rate, hatching success or hatchling size. These results suggest that this opportunistic colonizer is capable of successfully responding to novel environmental constraints in these important life-history stages.

Lizards at the Peak: Physiological Plasticity Does Not Maintain Performance in Lizards Transplanted to High Altitude.

Warming climates are facilitating the range expansion of many taxa to habitats that were formerly thermally inhospitable, including to higher latitudes and elevations. The potential for such colonization, however, varies widely among taxa. Because environmental factors may interact to affect colonization potential, an understanding of underlying physiological and behavioral mechanisms is necessary to predict how species will respond to potentially suitable habitats. For example, temperature and oxygen availability will interact to shape physiological and performance traits. Our model species, the wall lizard, Podarcis muralis, is a widely distributed ectotherm that continues to expand its range in Europe despite being limited by cold temperatures at high elevations and latitudes. To test the potential for organisms to expand to warming high-altitude environments, we conducted a transplant experiment to quantify the within-individual effects of high-altitude hypoxia on physiological and performance traits. Transplanted lizards maintained individual differences in physiological traits related to oxygen capacity and metabolism (hemoglobin concentration, hematocrit, and peak postexhaustion metabolic rate), as well as performance traits tied to fitness (sprint speed and running endurance). Although lizards altered blood biochemistry to increase oxygen-carrying capacity, their performance was reduced at high altitude. Furthermore, lizards at high altitude suffered a rapid loss of body condition over the 6-wk experiment, suggesting an energetic cost to hypoxia. Taken together, this demonstrates a limited potential for within-individual plasticity to facilitate colonization of novel high-altitude environments.

Delayed trait development and the convergent evolution of shell kinesis in turtles.

Understanding developmental processes is foundational to clarifying the mechanisms by which convergent evolution occurs. Here, we show how a key convergently evolving trait is slowly 'acquired' in growing turtles. Many functionally relevant traits emerge late in turtle ontogeny, owing to design constraints imposed by the shell. We investigated this trend by examining derived patterns of shell formation associated with the multiple (at least 8) origins of shell kinesis in small-bodied turtles. Using box turtles as a model, we demonstrate that the flexible hinge joint required for shell kinesis differentiates gradually and via extensive repatterning of shell tissue. Disproportionate changes in shell shape and size substantiate that this transformation is a delayed ontogenetic response (3-5 years post-hatching) to structural alterations that arise in embryogenesis. These findings exemplify that the translation of genotype to phenotype may reach far beyond embryonic life stages. Thus, the temporal scope for developmental origins of adaptive morphological change might be broader than generally understood. We propose that delayed trait differentiation via tissue repatterning might facilitate phenotypic diversification and innovation that otherwise would not arise due to developmental constraints.

Gene network variation and alternative paths to convergent evolution in turtles.

Diversification of the turtle's shell comprises remarkable phenotypic transformations. For instance, two divergent species convergently evolved shell-closing systems with shoulder blade (scapula) segments that enable coordinated movements with the shell. We expected these unusual structures to originate via similar changes in underlying gene networks, as skeletal segment formation is an evolutionarily conserved developmental process. We tested this hypothesis by comparing transcriptomes of scapula tissue across three stages of embryonic development in three emydid turtles from natural populations. We found that alternative strategies for skeletal segmentation were associated with interspecific differences in gene co-expression networks. Notably, mesenchyme homeobox 2 (MEOX2) and HOXA3-5 were central hubs driving the activity of 2,806 genes in a candidate network for scapula segmentation, albeit in only one species. Even so, scapula muscle overgrowth corresponded to the activity of similar myogenic networks in both species. This and other derived developmental processes were not observed in the third species, which displayed the ancestral (unsegmented) scapula condition. Differential gene expression tests against this reference lineage supported histological and network analyses. Our findings illustrate that molecular underpinnings of convergent evolution, including during the diversification of the atypical turtle "body plan," are influenced by variation in underlying developmental processes.

Is the Pelvis Sexually Dimorphic in Turtles?

Variation in the pelvis is intrinsically linked to life history evolution. This is perhaps best exemplified by sexually dimorphic pelvic variation in bipedal primates. Yet, whether this trend is applicable to other taxa is unclear. Using turtle anatomy as a model, I tested the hypothesis that the pelvis is also sexually dimorphic in egg-laying tetrapods. I sampled a natural turtle population with female-biased sexual size dimorphism (i.e., larger females). I show that the area of the egg canal (pelvic aperture) is greater in females. Morphological differences between sexes were predicted by body size, such that skeletal shape deformation of the female ilium increased proportionally with pelvic aperture area. These results suggest that sexual pelvic dimorphism might be indirectly maintained by selection for large female size, consistent with the pelvic constraint hypothesis in reptiles. However, subsampling of similarly sized individuals revealed that pelvic aperture area and shape may vary in disproportion to body size. Comparisons of pelvic ontogenetic trajectories across multiple lineages are needed to clarify the occurrence of sexual pelvic dimorphism in turtles and other egg-laying tetrapods. My findings provide impetus to further explore how sex-specific functional demands influence the architecture of the pelvic girdle. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc.

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.

Physiology at near-critical temperatures, but not critical limits, varies between two lizard species that partition the thermal environment.

The mechanisms that mediate the interaction between the thermal environment and species ranges are generally uncertain. Thermal environments may directly restrict species when environments exceed tolerance limits (i.e. the fundamental niche). However, thermal environments might also differentially affect relative performance among species prior to fundamental tolerances being met (i.e. the realized niche). We examined stress physiology (plasma glucose and corticosterone), mitochondrial performance and the muscle metabolome of congeneric lizards that naturally partition the thermal niche, Elgaria multicarinata (southern alligator lizards; SALs) and Elgaria coerulea (northern alligator lizards; NALs), in response to a thermal challenge to quantify variation in physiological performance and tolerance. Both NAL and SAL displayed physiological stress in response to high temperature, but neither showed signs of irreversible damage. NAL displayed a higher baseline mitochondrial respiration rate than SAL. Moreover, NAL substantially adjusted their physiology in response to thermal challenge, whereas SAL did not. For example, the metabolite profile of NAL shifted with changes in key energetic molecules, whereas these were unaffected in SAL. Our results indicate that near-critical high temperatures should incur greater energetic cost in NAL than SAL via an elevated metabolic rate and changes to the metabolome. Thus, SAL displace NAL in warm environments that are within NAL's fundamental thermal niche, but relatively costly. Our results suggest that subcritical thermal events can contribute to biogeographic patterns via physiological differences that alter the relative costs of living in warm or cool environments.

The turtle's shell.

Gerardo Cordero introduces the turtle shell, an evolutionary novelty.

Physiological plasticity in lizard embryos exposed to high-altitude hypoxia.

Coping with novel environments may be facilitated by plastic physiological responses that enable survival during environmentally sensitive life stages. We tested the capacity for embryos of the common wall lizard (Podarcis muralis) from low altitude to cope with low-oxygen partial pressure (hypoxia) in an alpine environment. Developing embryos subjected to hypoxic atmospheric conditions (15-16% O2 sea-level equivalent) at 2,877 m above sea level exhibited responses common to vertebrates acclimatized to or evolutionarily adapted to high altitude: suppressed metabolism, cardiac hypertrophy, and hyperventilation. These responses might have contributed to the unaltered incubation duration and hatching success relative to the ancestral, low-altitude, condition. Even so, hypoxia constrained egg energy utilization such that larger eggs produced hatchlings with relatively low mass. These findings highlight the role of physiological plasticity in maintaining fitness-relevant phenotypes in high-altitude environments, providing impetus to further explore altitudinal limits to ecological diversification in ectothermic vertebrates.

Effects of low-oxygen conditions on embryo growth in the painted turtle, Chrysemys picta.

Low-oxygen conditions (hypoxia; <21% O2 ) are considered unfavorable for growth; yet, embryos of many vertebrate taxa develop successfully in hypoxic subterranean environments. Although enhanced tolerance to hypoxia has been demonstrated in adult reptiles, such as in the painted turtle (Chrysemys picta), its effects on sensitive embryo life stages warrant attention. We tested the hypothesis that short-term hypoxia negatively affects growth during day 40 of development in C. picta, when O2 demands are highest in embryos. A brief, but severe, hypoxic event (5% O2 for 0.5 h) moderately affected embryo growth, causing a 13% reduction in mass (relative to a normoxic control). The same condition had no effect during day 27; instead, a nearly anoxic event (1% O2 for 72 h) caused a 5% mass reduction. All embryos survived the egg incubation period. Our study supports the assumption that reptilian embryos are resilient to intermittently low O2 in subterranean nests. Further work is needed to ascertain responses to suboptimal O2 levels while undergoing dynamic changes in developmental physiology.