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.

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.

Mother knows best: nest-site choice homogenizes embryo thermal environments among populations in a widespread ectotherm.

Species with large geographical ranges provide an excellent model for studying how different populations respond to dissimilar local conditions, particularly with respect to variation in climate. Maternal effects, such as nest-site choice greatly affect offspring phenotypes and survival. Thus, maternal behaviour has the potential to mitigate the effects of divergent climatic conditions across a species' range. We delineated natural nesting areas of six populations of painted turtles (Chrysemys picta) that span a broad latitudinal range and quantified spatial and temporal variation in nest characteristics. To quantify microhabitats available for females to choose, we also identified sites within the nesting area of each location that were representative of available thermal microhabitats. Across the range, females nested non-randomly and targeted microhabitats that generally had less canopy cover and thus higher nest temperatures. Nest microhabitats differed among locations but did not predictably vary with latitude or historic mean air temperature during embryonic development. In conjunction with other studies of these populations, our results suggest that nest-site choice is homogenizing nest environments, which buffers embryos from thermally induced selection and could slow embryonic evolution. Thus, although effective at a macroclimatic scale, nest-site choice is unlikely to compensate for novel stressors that rapidly increase local temperatures. This article is part of the theme issue 'The evolutionary ecology of nests: a cross-taxon approach'.

On the origin of patterns of temperature-dependent sex determination.

Species with temperature-dependent sex determination (TSD) exhibit significant variation in the relationship between incubation temperatures and the sex ratios they produce, making this an ideal system for comparing processes producing variation above and below the species level. Furthermore, a deeper mechanistic understanding of TSD macro- and microevolution may help reveal the currently unknown adaptive significance of this variation or of TSD as a whole. Here, we probe these topics by examining the evolutionary dynamics of this sex-determining mechanism in turtles. Our ancestral state reconstructions of discrete patterns of TSD suggest that producing females at cool incubation temperatures is derived and potentially adaptive. However, the ecological irrelevance of these cool temperatures and a strong genetic correlation across the sex-ratio reaction norm in Chelydra serpentina both contradict this interpretation. We further find the phenotypic consequence of this genetic correlation in C. serpentina reflected across all turtle species, suggesting that a single genetic architecture underlies both intra- and interspecific variation in TSD in this clade. This correlated architecture can explain the macroevolutionary origin of discrete TSD patterns without assigning cool-temperature female production an adaptive value. However, this architecture may also constrain adaptive microevolutionary responses to ongoing climate 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.

Diverse aging rates in ectothermic tetrapods provide insights for the evolution of aging and longevity.

Comparative studies of mortality in the wild are necessary to understand the evolution of aging; yet, ectothermic tetrapods are underrepresented in this comparative landscape, despite their suitability for testing evolutionary hypotheses. We present a study of aging rates and longevity across wild tetrapod ectotherms, using data from 107 populations (77 species) of nonavian reptiles and amphibians. We test hypotheses of how thermoregulatory mode, environmental temperature, protective phenotypes, and pace of life history contribute to demographic aging. Controlling for phylogeny and body size, ectotherms display a higher diversity of aging rates compared with endotherms and include phylogenetically widespread evidence of negligible aging. Protective phenotypes and life-history strategies further explain macroevolutionary patterns of aging. Analyzing ectothermic tetrapods in a comparative context enhances our understanding of the evolution of aging.

ROSIE, a database of reptilian offspring sex ratios and sex-determining mechanisms, beginning with Testudines.

In contrast to genotypic sex determination (GSD), temperature-dependent sex determination (TSD) in amniotic vertebrates eludes intuitive connections to Fisherian sex-ratio theory. Attempts to draw such connections have driven over 50 years of research on the evolution of sex-determining mechanisms (SDM), perhaps most prominently among species in the order Testudines. Despite regular advancements in our understanding of this topic, no efforts have been published compiling the entirety of data on the relationships between incubation temperature and offspring sex in any taxonomic group. Here, we present the Reptilian Offspring Sex and Incubation Environment (ROSIE) database, a comprehensive set of over 7,000 individual measurements of offspring sex ratios in the order Testudines as well as SDM classifications for 149 species. As the name suggests, we plan to expand the taxonomic coverage of ROSIE to include all non-avian reptiles and will regularly release updates to maintain its comprehensive nature. This resource will enable crucial future research probing the ecology and evolution of SDM, including the presumed sensitivity of TSD to rapid environmental change.

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.

Insights from Population Genomics to Enhance and Sustain Biological Control of Insect Pests.

Biological control-the use of organisms (e.g., nematodes, arthropods, bacteria, fungi, viruses) for the suppression of insect pest species-is a well-established, ecologically sound and economically profitable tactic for crop protection. This approach has served as a sustainable solution for many insect pest problems for over a century in North America. However, all pest management tactics have associated risks. Specifically, the ecological non-target effects of biological control have been examined in numerous systems. In contrast, the need to understand the short- and long-term evolutionary consequences of human-mediated manipulation of biological control organisms for importation, augmentation and conservation biological control has only recently been acknowledged. Particularly, population genomics presents exceptional opportunities to study adaptive evolution and invasiveness of pests and biological control organisms. Population genomics also provides insights into (1) long-term biological consequences of releases, (2) the ecological success and sustainability of this pest management tactic and (3) non-target effects on native species, populations and ecosystems. Recent advances in genomic sequencing technology and model-based statistical methods to analyze population-scale genomic data provide a much needed impetus for biological control programs to benefit by incorporating a consideration of evolutionary consequences. Here, we review current technology and methods in population genomics and their applications to biological control and include basic guidelines for biological control researchers for implementing genomic technology and statistical modeling.

Joint estimation of growth and survival from mark-recapture data to improve estimates of senescence in wild populations.

Understanding age-dependent patterns of survival is fundamental to predicting population dynamics, understanding selective pressures, and estimating rates of senescence. However, quantifying age-specific survival in wild populations poses significant logistical and statistical challenges. Recent work has helped to alleviate these constraints by demonstrating that age-specific survival can be estimated using mark-recapture data even when age is unknown for all or some individuals. However, previous approaches do not incorporate auxiliary information that can improve age estimates of individuals. We introduce a survival estimator that combines a von Bertalanffy growth model, age-specific hazard functions, and a Cormack-Jolly-Seber mark-recapture model into a single hierarchical framework. This approach allows us to obtain information about age and its uncertainty based on size and growth for individuals of unknown age when estimating age-specific survival. Using both simulated and real-world data for two painted turtle (Chrysemys picta) populations, we demonstrate that this additional information substantially reduces the bias of age-specific hazard rates, which allows for the testing of hypotheses related to aging. Estimating patterns of senescence is just one practical application of jointly estimating survival and growth; other applications include obtaining better estimates of the timing of recruitment and improved understanding of life-history trade-offs between growth and survival.

Sex and Incubation Temperature Independently Affect Embryonic Development and Offspring Size in a Turtle with Temperature-Dependent Sex Determination.

Developmental environments can have lasting effects on an individual's phenotype. In many reptiles, for example, egg incubation temperature permanently determines offspring sex (temperature-dependent sex determination, TSD) and also influences a suite of morphological, physiological, and behavioral traits. Thus, the contributions of sex and incubation temperature to phenotypic variation are difficult to identify because these factors are confounded under TSD. We used chemical manipulations to experimentally decouple gonadal sex and incubation temperature in a turtle with TSD (Chrysemys picta) to examine their relative and interactive effects on variation in incubation duration and offspring size. We show that warm incubation temperature accelerates development as expected and that exogenous estradiol treatment to eggs further shortens incubation duration across all incubation temperatures. Moreover, estradiol unexpectedly induced male development, resulting in male offspring hatching sooner than female offspring. Variation in offspring size was also influenced by incubation temperature and gonadal sex, but interactions between these two variables were relatively small or nonsignificant. The fitness consequences of these effects are unknown, but we provide preliminary results from our attempts at examining the long-term and sex-specific effects of incubation temperature. Manipulative experimental approaches, combined with longer-term experiments that track individuals through reproduction, will provide novel insights into the adaptive significance of developmental plasticity in long-lived organisms.

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.

Geographic variation in thermal sensitivity of early life traits in a widespread reptile.

Taxa with large geographic distributions generally encompass diverse macroclimatic conditions, potentially requiring local adaptation and/or phenotypic plasticity to match their phenotypes to differing environments. These eco-evolutionary processes are of particular interest in organisms with traits that are directly affected by temperature, such as embryonic development in oviparous ectotherms. Here we examine the spatial distribution of fitness-related early life phenotypes across the range of a widespread vertebrate, the painted turtle (Chrysemys picta). We quantified embryonic and hatchling traits from seven locations (in Idaho, Minnesota, Oregon, Illinois, Nebraska, Kansas, and New Mexico) after incubating eggs under constant conditions across a series of environmentally relevant temperatures. Thermal reaction norms for incubation duration and hatchling mass varied among locations under this common-garden experiment, indicating genetic differentiation or pre-ovulatory maternal effects. However, latitude, a commonly used proxy for geographic variation, was not a strong predictor of these geographic differences. Our findings suggest that this macroclimatic proxy may be an unreliable surrogate for microclimatic conditions experienced locally in nests. Instead, complex interactions between abiotic and biotic factors likely drive among-population phenotypic variation in this system. Understanding spatial variation in key life-history traits provides an important perspective on adaptation to contemporary and future climatic conditions.

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.

Environmentally induced phenotypic plasticity explains hatching synchrony in the freshwater turtle Chrysemys picta.

Environmentally cued hatching allows embryos to alter the time of hatching in relation to environment through phenotypic plasticity. Spatially variable temperatures within shallow nests of many freshwater turtles cause asynchronous development of embryos within clutches, yet neonates still hatch synchronously either by hatching early or via metabolic compensation. Metabolic compensation and changes in circadian rhythms presumably enable embryos to adjust their developmental rates to catch up to more advanced embryos within the nest. Hatchlings of the North American freshwater turtle Chrysemys picta usually overwinter within the nest and emerge the following spring, but still hatch synchronously via hatching early. Here, we used rates of oxygen consumption and heart rate profiles to investigate the metabolic rates of clutches of C. picta developing in conditions that result in asynchronous development to determine if compensatory changes in metabolism occur during incubation. Embryos hatched synchronously and displayed circadian rhythms throughout incubation, but exhibited no evidence of metabolic compensation. Phenotypic traits of hatchlings, including body size and righting performance, were also not affected by asynchronous development. We conclude that less developed embryos of C. picta hatch synchronously with their clutch-mates by hatching early, which does not appear to inflict a fitness cost to individuals. The ultimate mechanism for synchronous hatching in C. picta could be for hatchlings to ensure an optimal overwintering position within the center of the nest. Consequently, immediate fitness costs will not hinder hatchling survival. The geographic location, as well as environmental and genetic factors unique to populations, can all influence hatching behavior in turtles through phenotypic plasticity. Hence, synchronous hatching is an adaptive bet-hedging strategy in turtles, but the mechanisms to achieve it are diverse.

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.