Demographic history and genomic signatures of selection in a widespread vertebrate ectotherm.

Environmental conditions vary greatly across large geographic ranges, and yet certain species inhabit entire continents. In such species, genomic sequencing can inform our understanding of colonization history and the impact of selection on the genome as populations experience diverse local environments. As ectothermic vertebrates are among the most vulnerable to environmental change, it is critical to understand the contributions of local adaptation to population survival. Widespread ectotherms offer an opportunity to explore how species can successfully inhabit such differing environments and how future climatic shifts will impact species' survival. In this study, we investigated the widespread painted turtle (Chrysemys picta) to assess population genomic structure, demographic history, and genomic signatures of selection in the western extent of the range. We found support for a substantial role of serial founder effects in shaping population genomic structure: demographic analysis and runs of homozygosity were consistent with bottlenecks of increasing severity from eastern to western populations during and following the Last Glacial Maximum, and edge populations were more strongly diverged and had less genetic diversity than those from the centre of the range. We also detected outlier loci, but allelic patterns in many loci could be explained by either genetic surfing or selection. While range expansion complicates the identification of loci under selection, we provide candidates for future study of local adaptation in a long-lived, widespread ectotherm that faces an uncertain future as the global climate continues to rapidly change.

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.

Age Predicts Risky Investment Better Than Residual Reproductive Value.

AbstractLife-history theory predicts that investment in reproduction should increase as future reproductive potential (i.e., residual reproductive value [RRV]) decreases. Researchers have thus intuitively used age as a proxy for RRV and assume that RRV decreases with age when interpreting age-specific investment. Yet age is an imperfect proxy for RRV and may even be a poor correlate in some systems. We used a 31-year study of the nesting ecology of painted turtles (Chrysemys picta) to assess how age and RRV compare in explaining variation in a risky investment behavior. We predicted that RRV would be a better predictor of risky investment than age because RRV accounts for variation in future reproductive potential across life. We found that RRV was high in early life, slowly decreased until midlife, and then steadily decreased to terminal reproduction. However, age predicted risky behavior better than RRV. This finding suggests that stronger correlates of age (e.g., size) may be more responsible for this behavior in turtles. This study highlights that researchers should not assume that age-specific investment is driven by RRV and that future work should quantify RRV to more directly test this key element of life-history theory.

Predicting the effects of climate change on incubation in reptiles: methodological advances and new directions.

The unprecedented advancement of global climate change is affecting thermal conditions across spatial and temporal scales. Reptiles with temperature-dependent sex determination (TSD) are uniquely vulnerable to even fine-scale variation in incubation conditions and are a model system for investigating the impacts of shifting temperatures on key physiological and life-history traits. The ways in which current and predicted future climatic conditions translate from macro- to ultra-fine scale temperature traces in subterranean nests is insufficiently understood. Reliably predicting the ways in which fine-scale, daily and seasonally fluctuating nest temperatures influence embryonic development and offspring phenotypes is a goal that remains constrained by many of the same logistical challenges that have persisted throughout more than four decades of research on TSD. However, recent advances in microclimate and developmental modeling should allow us to move farther away from relatively coarse metrics with limited predictive capacity and towards a fully mechanistic model of TSD that can predict incubation conditions and phenotypic outcomes for a variety of reptile species across space and time and for any climate scenario.

Becoming creatures of habit: Among- and within-individual variation in nesting behaviour shift with age.

The quantification of repeatability has enabled behavioural and evolutionary ecologists to assess the heritable potential of traits. For behavioural traits that vary across life, age-related variation should be accounted for to prevent biasing the microevolutionary estimate of interest. Moreover, to gain a mechanistic understanding of ontogenetic variation in behaviour, among- and within-individual variance should be quantified across life. We leveraged a 30-year study of painted turtles (Chrysemys picta) to assess how age contributes to variation in the repeatability of nesting behaviours. We found that four components of nesting behaviour were repeatable and that accounting for age increased the repeatability estimate for maternal choice of canopy cover over nests. We detected canalization (diminished within-individual variance with age) of canopy cover choice in a reduced data set despite no shift in repeatability. Additionally, random regression analysis revealed that females became more divergent from each other in their choice of canopy cover with age. Thus, properly modelling age-related variance should more precisely estimate heritable potential, and assessing among- and within-individual variance components in addition to repeatability will offer a more mechanistic understanding of behavioural variation across age.

Development-specific transcriptomic profiling suggests new mechanisms for anoxic survival in the ventricle of overwintering turtles.

Oxygen deprivation swiftly damages tissues in most animals, yet some species show remarkable abilities to tolerate little or even no oxygen. Painted turtles exhibit a development-dependent tolerance that allows adults to survive anoxia approximately four times longer than hatchlings: adults survive ∼170 days and hatchlings survive ∼40 days at 3°C. We hypothesized that this difference is related to development-dependent differences in ventricular gene expression. Using a comparative ontogenetic approach, we examined whole transcriptomic changes before, during and 5 days after a 20-day bout of anoxic submergence at 3°C. Ontogeny accounted for more gene expression differences than treatment (anoxia or recovery): 1175 versus 237 genes, respectively. Of the 237 differences, 93 could confer protection against anoxia and reperfusion injury, 68 could be injurious and 20 may be constitutively protective. Most striking during anoxia was the main expression pattern of all 76 annotated ribosomal protein (R-protein) mRNAs, which decreased in anoxia-tolerant adults, but increased in anoxia-sensitive hatchlings, suggesting adult-specific regulation of translational suppression. These genes, along with 60 others that decreased their levels in adults and either increased or remained unchanged in hatchlings, implicate antagonistic pleiotropy as a mechanism to resolve the long-standing question about why hatchling painted turtles overwinter in terrestrial nests, rather than emerge and overwinter in water during their first year. In summary, developmental differences in the transcriptome of the turtle ventricle revealed potentially protective mechanisms that contribute to extraordinary adult-specific anoxia tolerance, and provide a unique perspective on differences between the anoxia-induced molecular responses of anoxia-tolerant and anoxia-sensitive phenotypes within a species.

Do Covariances Between Maternal Behavior and Embryonic Physiology Drive Sex-Ratio Evolution Under Environmental Sex Determination?

Fisherian sex-ratio theory predicts sexual species should have a balanced primary sex ratio. However, organisms with environmental sex determination (ESD) are particularly vulnerable to experiencing skewed sex ratios when environmental conditions vary. Theoretical work has modeled sex-ratio dynamics for animals with ESD with regard to 2 traits predicted to be responsive to sex-ratio selection: 1) maternal oviposition behavior and 2) sensitivity of embryonic sex determination to environmental conditions, and much research has since focused on how these traits influence offspring sex ratios. However, relatively few studies have provided estimates of univariate quantitative genetic parameters for these 2 traits, and the existence of phenotypic or genetic covariances among these traits has not been assessed. Here, we leverage studies on 3 species of reptiles (2 turtle species and a lizard) with temperature-dependent sex determination (TSD) to assess phenotypic covariances between measures of maternal oviposition behavior and thermal sensitivity of the sex-determining pathway. These studies quantified maternal behaviors that relate to nest temperature and sex ratio of offspring incubated under controlled conditions. A positive covariance between these traits would enhance the efficiency of sex-ratio selection when primary sex ratio is unbalanced. However, we detected no such covariance between measures of these categories of traits in the 3 study species. These results suggest that maternal oviposition behavior and thermal sensitivity of sex determination in embryos might evolve independently. Such information is critical to understand how animals with TSD will respond to rapidly changing environments that induce sex-ratio selection.

Decades of field data reveal that turtles senesce in the wild.

Lifespan and aging rates vary considerably across taxa; thus, understanding the factors that lead to this variation is a primary goal in biology and has ramifications for understanding constraints and flexibility in human aging. Theory predicts that senescence-declining reproduction and increasing mortality with advancing age-evolves when selection against harmful mutations is weaker at old ages relative to young ages or when selection favors pleiotropic alleles with beneficial effects early in life despite late-life costs. However, in many long-lived ectotherms, selection is expected to remain strong at old ages because reproductive output typically increases with age, which may lead to the evolution of slow or even negligible senescence. We show that, contrary to current thinking, both reproduction and survival decline with adult age in the painted turtle, Chrysemys picta, based on data spanning >20 y from a wild population. Older females, despite relatively high reproductive output, produced eggs with reduced hatching success. Additionally, age-specific mark-recapture analyses revealed increasing mortality with advancing adult age. These findings of reproductive and mortality senescence challenge the contention that chelonians do not age and more generally provide evidence of reduced fitness at old ages in nonmammalian species that exhibit long chronological lifespans.

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.

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.

Sex-specific growth, shape, and their impacts on the life history of a long-lived vertebrate.

BACKGROUND: Individual growth rates both comprise and determine life-history phenotypes. Despite decades of interest in understanding the relationship between individual growth and life history, chelonian longevity has limited our ability to robustly estimate individual growth curves that span the life of both sexes. QUESTIONS: (1) Do patterns of growth in size and shape differ between the sexes of the painted turtle, Chrysemys picta? (2) Does individual variation in size and shape affect female reproductive effort? METHODS: Using 30 years of field data on shell morphology of a single population of painted turtles, we used principal components analysis to summarize multivariate size and shape. We assessed the ability of three non-linear growth models - the logistic, Gompertz, and von Bertalanffy - to predict size-at-age and used model comparison to justify sex-specific model fits. We correlated age-specific size and shape of females with their reproductive efforts. RESULTS: Model comparison supported separate fits of the von Bertalanffy growth function for each sex; non-overlapping confidence intervals imply differences in sex-specific asymptotic size, but not growth rate. Higher-order axes of variation in shell morphology described significant sexual dimorphism in shell shape related to the sphericity and curviness of the shell. Shell sphericity of females covaried with clutch size, mean egg mass, and total clutch mass. Irrespective of shell morphology, we found evidence of an egg number versus egg mass trade-off. Yet, females who matured at a larger size produced greater reproductive efforts.

Rapid molecular evolution across amniotes of the IIS/TOR network.

The insulin/insulin-like signaling and target of rapamycin (IIS/TOR) network regulates lifespan and reproduction, as well as metabolic diseases, cancer, and aging. Despite its vital role in health, comparative analyses of IIS/TOR have been limited to invertebrates and mammals. We conducted an extensive evolutionary analysis of the IIS/TOR network across 66 amniotes with 18 newly generated transcriptomes from nonavian reptiles and additional available genomes/transcriptomes. We uncovered rapid and extensive molecular evolution between reptiles (including birds) and mammals: (i) the IIS/TOR network, including the critical nodes insulin receptor substrate (IRS) and phosphatidylinositol 3-kinase (PI3K), exhibit divergent evolutionary rates between reptiles and mammals; (ii) compared with a proxy for the rest of the genome, genes of the IIS/TOR extracellular network exhibit exceptionally fast evolutionary rates; and (iii) signatures of positive selection and coevolution of the extracellular network suggest reptile- and mammal-specific interactions between members of the network. In reptiles, positively selected sites cluster on the binding surfaces of insulin-like growth factor 1 (IGF1), IGF1 receptor (IGF1R), and insulin receptor (INSR); whereas in mammals, positively selected sites clustered on the IGF2 binding surface, suggesting that these hormone-receptor binding affinities are targets of positive selection. Further, contrary to reports that IGF2R binds IGF2 only in marsupial and placental mammals, we found positively selected sites clustered on the hormone binding surface of reptile IGF2R that suggest that IGF2R binds to IGF hormones in diverse taxa and may have evolved in reptiles. These data suggest that key IIS/TOR paralogs have sub- or neofunctionalized between mammals and reptiles and that this network may underlie fundamental life history and physiological differences between these amniote sister clades.

Phenotypic plasticity of nest timing in a post-glacial landscape: how do reptiles adapt to seasonal time constraints?

Life histories evolve in response to constraints on the time available for growth and development. Nesting date and its plasticity in response to spring temperature may therefore be important components of fitness in oviparous ectotherms near their northern range limit, as reproducing early provides more time for embryos to complete development before winter. We used data collected over several decades to compare air temperature and nest date plasticity in populations of painted turtles and snapping turtles from a relatively warm environment (southeastern Michigan) near the southern extent of the last glacial maximum to a relatively cool environment (central Ontario) near the northern extent of post-glacial recolonization. For painted turtles, population-level differences in reaction norm elevation for two phenological traits were consistent with adaptation to time constraints, but no differences in reaction norm slopes were observed. For snapping turtle populations, the difference in reaction norm elevation for a single phenological trait was in the opposite direction of what was expected under adaptation to time constraints, and no difference in reaction norm slope was observed. Finally, among-individual variation in individual plasticity for nesting date was detected only in the northern population of snapping turtles, suggesting that reaction norms are less canalized in this northern population. Overall, we observed evidence of phenological adaptation, and possibly maladaptation, to time constraints in long-lived reptiles. Where present, (mal)adaptation occurred by virtue of differences in reaction norm elevation, not reaction norm slope. Glacial history, generation time, and genetic constraint may all play an important role in the evolution of phenological timing and its plasticity in long-lived reptiles.

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.

Reptile Embryos Lack the Opportunity to Thermoregulate by Moving within the Egg.

Historically, egg-bound reptile embryos were thought to passively thermoconform to the nest environment. However, recent observations of thermal taxis by embryos of multiple reptile species have led to the widely discussed hypothesis that embryos behaviorally thermoregulate. Because temperature affects development, such thermoregulation could allow embryos to control their fate far more than historically assumed. We assessed the opportunity for embryos to behaviorally thermoregulate in nature by examining thermal gradients within natural nests and eggs of the common snapping turtle (Chelydra serpentina; which displays embryonic thermal taxis) and by simulating thermal gradients within nests across a range of nest depths, egg sizes, and soil types. We observed little spatial thermal variation within nests, and thermal gradients were poorly transferred to eggs. Furthermore, thermal gradients sufficiently large and constant for behavioral thermoregulation were not predicted to occur in our simulations. Gradients of biologically relevant magnitude have limited global occurrence and reverse direction twice daily when they do exist, which is substantially faster than embryos can shift position within the egg. Our results imply that reptile embryos will rarely, if ever, have the opportunity to behaviorally thermoregulate by moving within the egg. We suggest that embryonic thermal taxis instead represents a play behavior, which may be adaptive or selectively neutral, and results from the mechanisms for behavioral thermoregulation in free-living stages coming online prior to hatching.

The anti-predator role of within-nest emergence synchrony in sea turtle hatchlings.

Group formation is a common behaviour among prey species. In egg-laying animals, despite the various factors that promote intra-clutch variation leading to asynchronous hatching and emergence from nests, synchronous hatching and emergence occurs in many taxa. This synchrony may be adaptive by reducing predation risk, but few data are available in any natural system, even for iconic examples of the anti-predator function of group formation. Here, we show for the first time that increased group size (number of hatchlings emerging together from a nest) reduces green turtle (Chelonia mydas) hatchling predation. This effect was only observed earlier in the night when predation pressure was greatest, indicated by the greatest predator abundance and a small proportion of predators preoccupied with consuming captured prey. Further analysis revealed that the effect of time of day was due to the number of hatchlings already killed in an evening; this, along with the apparent lack of other anti-predatory mechanisms for grouping, suggests that synchronous emergence from a nest appears to swamp predators, resulting in an attack abatement effect. Using a system with relatively pristine conditions for turtle hatchlings and their predators provides a more realistic environmental context within which intra-nest synchronous emergence has evolved.

Temperature-Dependent Sex Determination under Rapid Anthropogenic Environmental Change: Evolution at a Turtle's Pace?

Organisms become adapted to their environment by evolving through natural selection, a process that generally transpires over many generations. Currently, anthropogenically driven environmental changes are occurring orders of magnitude faster than they did prior to human influence, which could potentially outpace the ability of some organisms to adapt. Here, we focus on traits associated with temperature-dependent sex determination (TSD), a classic polyphenism, in a model turtle species to address the evolutionary potential of species with TSD to respond to rapid climate change. We show, first, that sex-ratio outcomes in species with TSD are sensitive to climatic variation. We then identify the evolutionary potential, in terms of heritability, of TSD and quantify the evolutionary potential of 3 key traits involved in TSD: pivotal temperature, maternal nest-site choice, and nesting phenology. We find that these traits display different patterns of adaptive potential: pivotal temperature exhibits moderate heritable variation, whereas nest-site choice and nesting phenology, with considerable phenotypic plasticity, have only modest evolutionary potential to alter sex ratios. Therefore, the most likely response of species with TSD to anthropogenically induced climate change may be a combination of microevolution in thermal sensitivity of the sex-determining pathway and of plasticity in maternal nesting behavior.

Swimming against the tide: resilience of a riverine turtle to recurrent extreme environmental events.

Extreme environmental events (EEEs) are likely to exert deleterious effects on populations. From 1996 to 2012 we studied the nesting dynamics of a riverine population of painted turtles (Chrysemys picta) that experienced seven years with significantly definable spring floods. We used capture-mark-recapture methods to estimate the relationships between more than 5 m and more than 6 m flood events and population parameters. Contrary to expectations, flooding was not associated with annual differences in survival, recruitment or annual population growth rates of the adult female segment of the population. These findings suggest that female C. picta exhibit resiliency to key EEE, which are expected to increase in frequency under climate change.

Exogenous application of estradiol to eggs unexpectedly induces male development in two turtle species with temperature-dependent sex determination.

Steroid hormones affect sex determination in a variety of vertebrates. The feminizing effects of exposure to estradiol and the masculinizing effects of aromatase inhibition during development are well established in a broad range of vertebrate taxa, but paradoxical findings are occasionally reported. Four independent experiments were conducted on two turtle species with temperature-dependent sex determination (Chrysemys picta and Chelydra serpentina) to quantify the effects of egg incubation temperature, estradiol, and an aromatase inhibitor on offspring sex ratios. As expected, the warmer incubation temperatures induced female development and the cooler temperatures produced primarily males. However, application of an aromatase inhibitor had no effect on offspring sex ratios, and exogenous applications of estradiol to eggs produced male offspring across all incubation temperatures. These unexpected results were remarkably consistent across all four experiments and both study species. Elevated concentrations of estradiol could interact with androgen receptors or inhibit aromatase expression, which might result in relatively high testosterone concentrations that lead to testis development. These findings add to a short list of studies that report paradoxical effects of steroid hormones, which addresses the need for a more comprehensive understanding of the role of sex steroids in sexual development.

Increased nest temperature during winter does not affect residual yolk metabolism of hatchling painted turtles (Chrysemys picta).

Rising global temperatures have a wide range of effects at organismal, population, and ecosystem levels. Increased winter temperatures are expected to alter the energetics of species that are dormant during this time. Hatchling painted turtles (Chrysemys picta) spend their first ∼8 months in shallow nests on land, where they putatively rely on residual yolk reserves to fuel energetic demands during this period of inactivity before they emerge in the spring. We performed a laboratory experiment to characterize changes in residual yolk quantity in hatchling C. picta and experimentally tested the effect of temperature on residual yolk, hatchling size, and survival over the winter brumation period. We manipulated winter nest temperature by simulating two natural thermal regimes ("low" vs "high" treatments) and one regime that approximates warmer temperatures expected by 2100 ("future" treatment). Because high temperature increases metabolism, we predicted that the future temperature treatment would decrease the amount of residual yolk remaining by the end of winter and reduce hatchling mass and survival. Residual yolk over winter did not differ from that before winter, and the temperature had no effect on the quantity of residual yolk or hatchling survival by the following spring. However, hatchlings that experienced future temperatures lost more mass over winter than those from the other treatments. These results correspond with previous work indicating that residual yolk does not fuel the energetic needs of hatchlings during winter. The effect of future warming temperatures on body mass may have negative consequences during energetically demanding activities during spring emergence and dispersal.