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Leaving the water to bask (usually in the sun) is a common behavior for many freshwater turtles, with some species also engaging in “nocturnal basking.” Ectoparasite removal is an obvious hypothesis to explain nocturnal basking and has also been proposed as a key driver of diurnal basking. However, the efficacy of basking, day or night, to remove leeches has not been experimentally tested. Therefore, we examined the number of leeches that were removed from Krefft's river turtles (Emydura macquarii krefftii) after experimentally making turtles bask at a range of times of day, durations, and temperatures. Turtles had high initial leech loads, with a mean of 32.1 leeches per turtle. Diurnal basking under a heat lamp for 3 hr at ~28°C significantly reduced numbers of leeches relative to controls. In diurnal trials, 90.9% of turtles lost leeches (mean loss of 7.1 leeches per turtle), whereas basking for 30 min under the same conditions was not effective (no turtles lost leeches, and all turtles were still visibly wet). Similarly, “nocturnal basking” at ~23°C for 3 hr was not effective at removing leeches. Only 18% of turtles lost leeches (one turtle lost one leech and another lost four leeches). Diurnal basking outdoors under direct sunlight for 20 min (mean temp = 34.5°C) resulted in a small reduction in leeches, with 50% of turtles losing leeches and an average loss of 0.7 leeches per turtle. These results indicate basking can remove leeches if temperatures are high or basking durations are long. However, it was only effective at unusually long basking durations in this system. Our data showed even the 20‐min period was longer than 70.1% of natural diurnal basking events, many of which took place at cooler temperatures. Therefore, leech removal does not appear to be the purpose of the majority of basking events., Aerial basking has long been hypothesized to reduce ectoparasites in turtles. We experimentally tested if basking reduced leech loads in an Australian freshwater turtle. While basking can reduce leech loads, the basking duration and heat intensity greatly influence the results. Thus, we have determined that mean basking times in nature were not sufficient to effectively reduce leech loads, making leech removal unlikely to be the primary mechanism driving basking behavior.

Anthropogenic land use changes have broad impacts on biological diversity, often resulting in shifts in community composition. While many studies have documented negative impacts on occurrence and abundance of species, less attention has been given to native species that potentially benefit from anthropogenic land use changes. For many species reaching high densities in human‐dominated landscapes, it is unclear whether these environments represent higher quality habitat than more natural environments. We examined the influence of landscape ecological integrity on relative abundance and body condition of two native generalist freshwater turtle species that are prevalent in anthropogenic systems, the painted turtle (Chrysemys picta) and red‐eared slider (Trachemys scripta elegans). Relative abundance was negatively associated with ecological integrity for both species, but the relationship was not strongly supported for painted turtles. Body condition was positively associated with ecological integrity for painted turtles, with no strong association for red‐eared sliders. Our study suggests that both species benefitted at the population level from reduced ecological integrity, but individual‐level habitat quality was reduced for painted turtles. The differing responses between these two habitat generalists could partially explain why red‐eared sliders have become a widespread exotic invasive species, while painted turtles have not., Identifying the differences in the relationship between landscape integrity and body condition for painted turtles and red‐eared sliders may porovide insight on how red‐eared sliders have become a widespread invasive species, while painted turtles have not. We collected and collated turtle capture and morphometric data to create model‐estimated relationships between mean 2.5 km landscape condition value (LCV) and standardized body condition index (BCI) scores for (A) painted turtles (Chrysemys picta; n = 625) sampled at 46 wetlands across 10 counties in West Virginia and (B) red‐eared sliders (Trachemys scripta elegans; n = 715) sampled at 42 wetlands across five counties in Texas.

A frequent response of organisms to climate change is altering the timing of reproduction, and advancement of reproductive timing has been a common reaction to warming temperatures in temperate regions. We tested whether this pattern applied to two common North American turtle species over the past three decades in Nebraska, USA. The timing of nesting (either first date or average date) of the Common Snapping Turtle (Chelydra serpentina) was negatively correlated with mean December maximum temperatures of the preceding year and mean May minimum and maximum temperatures in the nesting year and positively correlated with precipitation in July of the previous year. Increased temperatures during the late winter and spring likely permit earlier emergence from hibernation, increased metabolic rates and feeding opportunities, and accelerated vitellogenesis, ovulation, and egg shelling, all of which could drive earlier nesting. However, for the Painted Turtle (Chrysemys picta), the timing of nesting was positively correlated with mean minimum temperatures in September, October, December of the previous year, February of the nesting year, and April precipitation. These results suggest warmer fall, and winter temperature may impose an increased metabolic cost to painted turtles that impedes fall vitellogenesis, and April rains may slow the completion of vitellogenesis through decreased basking opportunities. For both species, nest deposition was highly correlated with body size, and larger females nested earlier in the season. Although average annual ambient temperatures have increased over the last four decades of our overall fieldwork at our study site, spring temperatures have not yet increased, and hence, nesting phenology has not advanced at our site for Chelydra. While Chrysemys exhibited a weak trend toward later nesting, this response was likely due to increased recruitment of smaller females into the population due to nest protection and predator control (Procyon lotor) in the early 2000s. Should climate change result in an increase in spring temperatures, nesting phenology would presumably respond accordingly, conditional on body size variation within these populations., A frequent response of organisms to climate change is altering the timing of reproduction. Over the past three decades, the timing of nesting of the Common Snapping Turtle (Chelydra serpentina) was negatively correlated with mean December maximum temperatures of the preceding year and mean May minimum and maximum temperatures in the nesting year and positively correlated with precipitation in July of the previous year. For the Painted Turtle (Chrysemys picta), the timing of nesting was positively correlated with mean minimum temperatures in September, October, December of the previous year, February of the nesting year, and April precipitation.

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.

The western painted turtle (Chrysemys picta bellii) can survive extended periods of anoxia via a series of mechanisms that serve to reduce its energetic needs. Central to these mechanisms is the response of mitochondria, which depolarize in response to anoxia in turtle pyramidal neurons due to an influx of K+. It is currently unknown how mitochondrial matrix pH is affected by this response and we hypothesized that matrix pH acidifies during anoxia due to increased K+/H+ exchanger activity. Inhibition of K+/H+ exchange via quinine led to a collapse of mitochondrial membrane potential (Ψm) during oxygenated conditions in turtle cortical neurons, as indicated by rhodamine-123 fluorescence, and this occurred twice as quickly during anoxia which indicates an elevation in K+ conductance. Mitochondrial matrix pH acidified during anoxia, as indicated by SNARF-1 fluorescence imaged via confocal microscopy, and further acidification occurred during anoxia when the F1Fo-ATPase was inhibited with oligomycin-A, indicating that ΔpH collapse is prevented during anoxic conditions. Collectively, these results indicate that the mitochondrial proton electrochemical gradient is actively preserved during anoxia to prevent a collapse of Ψm and ΔpH.

Landscape-scale alterations that accompany urbanization may negatively affect the population structure of wildlife species such as freshwater turtles. Changes to nesting sites and higher mortality rates due to vehicular collisions and increased predator populations may particularly affect immature turtles and mature female turtles. We hypothesized that the proportions of adult female and immature turtles in a population will negatively correlate with landscape urbanization. As a collaborative effort of the Ecological Research as Education Network (EREN), we sampled freshwater turtle populations in 11 states across the central and eastern United States. Contrary to expectations, we found a significant positive relationship between proportions of mature female painted turtles (Chrysemys picta) and urbanization. We did not detect a relationship between urbanization and proportions of immature turtles. Urbanization may alter the thermal environment of nesting sites such that more females are produced as urbanization increases. Our approach of creating a collaborative network of scientists and students at undergraduate institutions proved valuable in terms of testing our hypothesis over a large spatial scale while also allowing students to gain hands-on experience in conservation science.

Low-oxygen conditions (hypoxia

Bycatch, the incidental capture of non-target organisms, occurs in most commercial fisheries. Although immediate bycatch mortality is frequently documented in fisheries, detrimental sub-lethal effects and potential post-release mortality remain largely unknown despite the potential population-level consequences. Turtles are captured as bycatch and their populations are vulnerable to slight increases in adult mortality. In eastern Ontario, turtles are frequently captured as bycatch in a small-scale freshwater commercial fyke-net fishery and, currently, the fate of discarded turtles is unknown. We wished to determine the effect of fyke-net capture on post-release survival and behaviour in eastern musk turtles Sternotherus odoratus and painted turtles Chrysemys picta. We used biologgers equipped with tri-axial acceleration, depth and temperature sensors to document locomotor activity, vertical distribution, and temperature use of entrapped (exposed to forced submergence for 4 h) and control turtles upon release. Overall dynamic body acceleration was used as a measure of post-release activity for the first hour, first 6 h, and first 48 h. Post-release mortality was not detected. Turtles subjected to entrapment exhibited lower activity during the first 6 h following release, and their vertical distribution and temperature use differed in the first 2 h following release, but these effects disappeared after 48 h, suggesting turtles have the ability to recover. Quantifying the post-release mortality and sub-lethal effects of entrapment is important for estimating the population effects associated with bycatch.

Retinal progenitors in the circumferential marginal zone (CMZ) and Muller glia-derived progenitors have been well described for the eyes of fish, amphibians, and birds. However, there is no information regarding a CMZ and the nature of retinal glia in species phylogenetically bridging amphibians and birds. The purpose of this study was to examine the retinal glia and investigate whether a CMZ is present in the eyes of reptilian species. We used immunohistochemical analyses to study retinal glia, neurons that could influence CMZ progenitors, the retinal margin, and the nonpigmented epithelium of ciliary body of garter snakes, queen snakes, anole lizards, snapping turtles, and painted turtles. We compare our observations on reptile eyes to the CMZ and glia of fish, amphibians, and birds. In all species, Sox9, Pax6, and the glucocorticoid receptor are expressed by Muller glia and cells at the retinal margin. However, proliferating cells were found only in the CMZ of turtles and not in the eyes of anoles and snakes. Similar to eyes of chickens, the retinal margin in turtles contains accumulations of GLP1/glucagonergic neurites. We find that filamentous proteins, vimentin and GFAP, are expressed by Muller glia, but have different patterns of subcellular localization in the different species of reptiles. We provide evidence that the reptile retina may contain nonastrocytic inner retinal glial cells, similar to those described in the avian retina. We conclude that the retinal glia, glucagonergic neurons, and CMZ of turtles appear to be most similar to those of fish, amphibians, and birds.

Key points Anoxia induces hyper-excitability and cell death in mammalian brain but in the western painted turtle (Chrysemys picta bellii) enhanced GABA transmission prevents injury. The mechanism responsible for increased GABA transmission is unknown; however, reactive oxygen species (ROS) generated by mitochondria may play a role because this is an oxygen-sensitive process. In this study, we show that inhibition of mitochondrial ROS production is sufficient to initiate a redox-sensitive GABA signalling cascade that suppresses pyramidal neuron action potential frequency. These results further our understanding of the turtle's unique strategy for reducing ATP consumption during anoxia and highlights a natural mechanism in which to explore therapies to protect mammalian brain from low-oxygen insults (e.g. cerebral stroke). Abstract Anoxia induces hyper-excitability and cell death in mammalian brain but in the anoxia-tolerant western painted turtle (Chrysemys picta bellii) neuronal electrical activity is suppressed (i.e. spike arrest), adenosine triphosphate (ATP) consumption is reduced, and cell death does not occur. Electrical suppression is primarily the result of enhanced γ-aminobutyric acid (GABA) transmission; however, the underlying mechanism responsible for initiating oxygen-sensitive GABAergic spike arrest is unknown. In turtle cortical pyramidal neurons there are three types of GABAA receptor-mediated currents: spontaneous inhibitory postsynaptic currents (IPSCs), giant IPSCs and tonic currents. The aim of this study was to assess the effects of reactive oxygen species (ROS) scavenging on these three currents since ROS levels naturally decrease with anoxia and may serve as a redox signal to initiate spike arrest. We found that anoxia, pharmacological ROS scavenging, or inhibition of mitochondrial ROS generation enhanced all three types of GABA currents, with tonic currents comprising ∼50% of the total current. Application of hydrogen peroxide inhibited all three GABA currents, demonstrating a reversible redox-sensitive signalling mechanism. We conclude that anoxia-mediated decreases in mitochondrial ROS production are sufficient to initiate a redox-sensitive inhibitory GABA signalling cascade that suppresses electrical activity when oxygen is limited. This unique strategy for reducing neuronal ATP consumption during anoxia represents a natural mechanism in which to explore therapies to protect mammalian brain from low-oxygen insults.

During anoxia, overall protein synthesis is almost undetectable in the brain of the western painted turtle. The aim of this investigation was to address the question of whether there are alterations to specific proteins by comparing the normoxic and anoxic brain proteomes. Reductions in creatine kinase, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase reflected the reduced production of adenosine triphosphate (ATP) during anoxia while the reduction in transitional endoplasmic reticulum ATPase reflected the conservation of ATP or possibly a decrease in intracellular Ca(2+). In terms of neural protection programed cell death 6 interacting protein (PDCD6IP; a protein associated with apoptosis), dihydropyrimidinase-like protein, t-complex protein, and guanine nucleotide protein G(o) subunit alpha (Go alpha; proteins associated with neural degradation and impaired cognitive function) also declined. A decline in actin, gelsolin, and PDCD6IP, together with an increase in tubulin, also provided evidence for the induction of a neurological repair response. Although these proteomic alterations show some similarities with the crucian carp (another anoxia-tolerant species), there are species-specific responses, which supports the theory of no single strategy for anoxia tolerance. These findings also suggest the anoxic turtle brain could be an etiological model for investigating mammalian hypoxic damage and clinical neurological disorders.

Climate change may subject animals to increasingly stressful environmental conditions, which could have negative physiological consequences if stress levels are elevated for long periods. We conducted a manipulative experiment to determine the effects of a novel climate on stress levels and immune function in a model reptile species, the painted turtle. We collected turtles from four populations across the species' geographic range and housed them in a common-garden in one population's local climate. We measured levels of the stress hormone corticosterone and tested two aspects of innate immune function, bactericidal capacity and natural antibody agglutination, at the time of capture (baseline) and three additional time points over 1 year. The four populations did not differ in corticosterone levels over the course of 1 year, and corticosterone levels were also similar at each sampling period except that post-hibernation corticosterone levels were significantly lower than the previous three time points. Furthermore, we found no evidence that elevated corticosterone depressed immune function in the painted turtle. Our study suggests that turtles exposed to novel climatic conditions did not display a detectable stress response, nor did the novel climate depress immune function in the transplanted populations. Therefore, in terms of innate immune function, turtles may be relatively resilient to at least small changes in climatic conditions.

Summary 1. Phenotypic variation is strongly impacted by environmental conditions experienced during development. Substantial laboratory research has shown that reptiles with flexible-shelled eggs are particularly sensitive to hydric conditions, yet research on nests in the wild is sparse. 2. In this 2-year field experiment, we explore the influence of hydric conditions during incubation on phenotypic traits of hatchling painted turtles (Chrysemys picta). Using a split-clutch design, we created two artificial nests adjacent to each maternally selected nest site. Half the eggs incubated in a nest that received regular supplemental watering, while the control nest was exposed to natural precipitation only. 3. Our results suggest that the influence of the hydric environment on developing reptilian embryos is context dependent. Supplemental water applied to nests in a drier than normal season elicited the expected biotic responses, based on laboratory experiments. However, when the soil surrounding C. picta eggs was already highly moist, the additional water from supplemental application effectively stunted embryonic development. 4. Our experiment confirms that hydric conditions of the soil during incubation in the wild can substantially influence phenotypic variation of reptiles with flexible-shelled eggs. Additionally, our experiment highlights the importance of complex interactions in the field that are often unexplored in laboratory experiments, reiterating the importance of validating laboratory work with field experiments.

Summary Offspring phenotypic variation can be substantially influenced by non-genetic factors such as maternal effects, which ultimately can influence organismal fitness. For oviparous organisms that lack parental care, oviposition-site choice and egg size are maternal effects that can greatly affect offspring traits. Yet, few studies examine the consequences of these traits in the wild. We manipulated the contents of natural painted turtle nests such that offspring spent two life stages (incubation and hibernation) in either maternally selected nest sites or randomly selected nest sites and quantified treatment differences in environmental parameters and offspring phenotypes. Additionally, we tracked the fates of individual eggs and hatchlings, which allowed us to quantify the strength and form of selection acting on egg size during incubation and, for the first time, body size during hibernation. Maternally selected nest sites were warmer and produced offspring that were longer and hatched earlier than their siblings emerging from cooler, randomly selected nests. Treatments did not affect any measured traits during hibernation. We detected no selection on egg size during the incubation stage, but significant linear selection favouring larger hatchlings during hibernation. Our results suggest that nest-site choice allows mothers to partially control the environment of their incubating eggs, but is less effective at controlling hatchling environments during hibernation. Additionally, we provide novel support for the ‘bigger-is-better’ hypothesis in turtles by showing a positive relationship between size and survival during the hibernation stage.

SUMMARY Evolutionary developmental biology requires integration of knowledge from multiple levels of organization. Information at the genome level is quickly outpacing crucial descriptive information on developmental processes at the cell, tissue, and organ levels. Unprecedented progress in genomics has enabled the rise of so-called non-traditional reference species. However, a substantial, though overlooked, amount of phenotypic information has long been available for species such as the Painted Turtle (Chrysemys picta), which recently became the first turtle to feature a fully sequenced and annotated nuclear genome. In this commentary, I highlight research on the comparative embryology of Chrysemys during the twentieth century and briefly discuss future directions for research on this re-emerging species for evo-devo.

Bycatch of non-target species in commercial fishing nets can have adverse impacts on their populations. Freshwater turtle populations are particularly susceptible to increases in adult mortality, and freshwater turtles are among the most threatened vertebrates. As a case study, the population-level impacts of bycatch mortality on freshwater turtles were evaluated in Lake Opinicon, Ontario, Canada, a lake that supports a small-scale commercial fishery. Using population viability analyses, the impacts of bycatch on common snapping turtles (Chelydra serpentina), eastern musk turtles (Sternotherus odoratus), northern map turtles (Graptemys geographica), and painted turtles (Chrysemys picta) were evaluated. In all four species, even low levels of additional annual female mortality as a result of bycatch were sufficient either to reduce population size or to cause extirpation of the local population within 500 years. Bycatch reduction programmes, such as seasonal closures and implementation of bycatch reduction devices, can help alleviate the risk of extirpation. Changes to fishing season length could help reduce the number of snapping turtles and musk turtles captured. Installation of simple bycatch reduction devices can exclude between 95% and 100% of snapping turtles and between 0% and 97% of the other three species, depending on the width of the exclusion device. If combined, these two bycatch reduction methods would help prevent adult female mortality and help maintain turtle populations in Lake Opinicon. Although these findings are specific to the study area, the same principles apply to other areas where similar simple bycatch reduction strategies can be employed to prevent the extirpation of other freshwater turtle species. Considering the consequences of bycatch and of bycatch reduction programmes on populations provides managers with important information to support development of risk-averse conservation strategies. Copyright © 2014 John Wiley & Sons, Ltd.

Normal developmental staging tables often undergo expansion and enhancement in response to advancing research paradigms and technologies. The Painted Turtle, Chrysemys picta, has long been a preferred reference taxon for comparative embryology and recently became the first turtle species to feature a sequenced genome. However, modern descriptive studies on embryogenesis are lacking and an earlier developmental staging table has been ignored. To address these problems, we re-evaluated descriptions of developmental stages by studying embryos under standardized laboratory conditions. We created an enhanced normal developmental staging table that clarifies and validates previous descriptions of developmental processes in this species. Moreover, we emphasized description of turtle-specific developmental characters such as the carapacial ridge. We demonstrated that embryo growth rate, length of incubation period, and timing to developmental stages are predictable under controlled environmental conditions. The appearance of characters associated with eye, limb, and shell anatomy was congruent with observations made in other turtle species. To reduce experimental bias, we recommend the use of our enhanced staging table when describing embryogenesis in the Painted Turtle.

Increases in extreme environmental events are predicted to be major results of ongoing global climate change and may impact the persistence of species. We examined the effects of heat and cold waves during embryonic development of painted turtles (Chrysemys picta) in natural nests on the occurrence of abnormal shell morphologies in hatchlings. We found that nests exposed to extreme hot temperatures for >60 h produced more hatchlings with abnormalities than nests exposed to extreme hot temperatures for shorter periods, regardless of whether or not nesting females displayed abnormal morphologies. We observed no effect of extreme cold nest temperatures on the occurrence of hatchlings with abnormalities. Moreover, the frequency of nesting females with abnormal shell morphologies was approximately 2-fold lower than that of their offspring, suggesting that such abnormalities are negatively correlated with survival and fitness. Female turtles could potentially buffer their offspring from extreme heat by altering aspects of nesting behavior, such as choosing shadier nesting sites. We addressed this hypothesis by examining the effects of shade cover on extreme nest temperatures and the occurrence of hatchling abnormalities. While shade cover was negatively correlated with the occurrence of extreme hot nest temperatures, it was not significantly correlated with abnormalities. Therefore, female choice of shade cover does not appear to be a viable target for selection to reduce hatchling abnormalities. Our results suggest that increases in the frequency and intensity of heat waves associated with climate change might perturb developmental programs and thereby reduce the fitness of entire cohorts of turtles.

Maternal ability to match nest characteristics with environmental conditions can influence offspring survival and quality, and may provide a mechanism by which animals can keep pace with climate change. In species with temperature-dependent sex determination that construct subterranean nests, the depth of the nest may affect incubation temperatures, and thus offspring sex ratio. Maternal adjustment of nest depth may be a mechanism by which climate change-induced sex ratio skews could be prevented in globally imperiled taxa such as turtles. We experimentally manipulated nest depth within a biologically relevant range in nests of the model turtle species Chrysemys picta. We then quantified the effects of nest depth on incubation regime, offspring sex ratio and offspring performance. We found no effect of nest depth on six parameters of incubation regime, nor on resultant offspring survival, size or sex ratio. However, deeper nests produced hatchlings that weighed less, and were faster at righting themselves and swimming, than hatchlings from shallower nests. We suggest that cues used by females in adjusting nest depth are unreliable as predictors of future incubation conditions, and the adjustment in nest depth required to affect sex ratio in this species may be too great to keep pace with climate change. Therefore, maternal adjustment of nest depth seems unlikely to compensate for climate change-induced sex ratio skews in small-bodied, freshwater turtles.

Selection is expected to maintain primary sex ratios at an evolutionary equilibrium. In organisms with temperature-dependent sex determination (TSD), targets of sex-ratio selection include the thermal sensitivity of the sex-determining pathway (hereafter, sex determination threshold) and nest-site choice. However, offspring sex may be canalized for nests located in thermally extreme environments; thus, genetic variance for the sex determination threshold is not expressed and is invisible to direct selection. The concept of 'effective heritability' accounts for this dependence and provides a more realistic prediction of the expected evolutionary response to selection in the wild. Past estimates of effective heritability of the sex determination threshold, which were derived from laboratory data, suggested that the potential for the sex determination threshold to evolve in the wild was extremely low. We re-evaluated original estimates of this parameter by analysing field-collected measures of nest temperatures, vegetation cover and clutch sex ratios from nests in a population of painted turtles (Chrysemys picta). We coupled these data with measurements of broad-sense heritability of the sex determination threshold in C. picta, using an experiment that splits clutches of eggs between a constant temperature (i.e. typical laboratory incubation) and a daily fluctuating temperature (i.e. similar to natural nests) with the same mean. We found that (i) the effective heritability of the sex determination threshold appears to have been historically underestimated and the effective heritability of nest-site choice has been overestimated and (ii) significant family-by-incubation treatment interaction exists for sex for C. picta between constant- and fluctuating-temperature regimes. Our results suggest that the thermal sensitivity of the sex-determining pathway may play a larger, more complex role in the microevolution of TSD than traditionally thought.

Conditions experienced early in life can influence phenotypes in ecologically important ways, as exemplified by organisms with environmental sex determination. For organisms with temperature-dependent sex determination (TSD), variation in nest temperatures induces phenotypic variation that could impact population growth rates. In environments that vary over space and time, how does this variation influence key demographic parameters (cohort sex ratio and hatchling recruitment) in early life stages of populations exhibiting TSD? We leverage a 17-year data set on a population of painted turtles, Chrysemys picta, to investigate how spatial variation in nest vegetation cover and temporal variation in climate influence early life-history demography. We found that spatial variation in nest cover strongly influenced nest temperature and sex ratio, but was not correlated with clutch size, nest predation, total nest failure, or hatching success. Temporal variation in climate influenced percentage of total nest failure and cohort sex ratio, but not depredation rate, mean clutch size, or mean hatching success. Total hatchling recruitment in a year was influenced primarily by temporal variation in climate-independent factors, number of nests constructed, and depredation rate. Recruitment of female hatchlings was determined by stochastic variation in nest depredation and annual climate and also by the total nest production. Overall population demography depends more strongly on annual variation in climate and predation than it does on the intricacies of nest-specific biology. Finally, we demonstrate that recruitment of female hatchlings translates into recruitment of breeding females into the population, thus linking climate (and other) effects on early life stages to adult demographics.

Successful overwintering under ice by an air-breathing vertebrate requires either effective aquatic respiration if dissolved O(2) is available or the capacity for prolonged anaerobic metabolism if O(2) supplies are limiting. Frogs can remain aerobic for many weeks when submerged at low temperature, even at water PO(2) as low as 30 mmHg, but are unable to survive even 1 week in anoxic water. Fuel reserves of hibernating frogs limit aerobic submergence, whereas acidosis may limit anoxic submergence. Freshwater turtles can also satisfy all or most of their O(2) needs in well-aerated water at low temperature by aquatic respiration, but certain species, in particular painted and snapping turtles, can also survive for up to 4-5 months without O(2). Key adaptations of the painted turtles, and presumably snapping turtles, include metabolic depression and the exploitation of the shell and other bones to buffer lactic acid. As in frogs, glycogen and glucose are the only fuel sources during anoxia, and stores do not seem to be limiting in the painted turtle. Significant differences in anoxia tolerance exist among chelonian species that can be attributed, at least in part, to the magnitude of metabolic depression, the effectiveness of lactic acid buffering, and the size of glycogen stores.

Summary 1. Synchrony in the timing of births is thought to have evolved as a general predator avoidance strategy. In turtles, synchronous hatching may facilitate group emergence from the nest, which in turn, may limit predation by diluting an individual’s risk of predation or by simply swamping predators upon emergence. However, synchronizing hatching should not be easily achieved in natural nests because of thermal gradients affecting developmental rates. 2. We evaluated pipping synchrony in the painted turtle (Chrysemys picta), where the drive to hatch synchronously may be reduced, because in many populations, hatching and emergence are dissociated through hatchlings overwintering within a nest. 3. We also assessed developmental mechanisms through which synchrony occurs and explored potential trade-offs between pipping synchrony and individual fitness by determining potential short and long-term neuromuscular developmental costs to hatching prematurely. These data were also used to develop a theoretical model to determine how differential embryonic maturation rates affect hatching synchrony. 4. Underdeveloped embryos pipped much earlier than expected and at similar times to their more advanced sibs. In addition, a trade-off between hatching synchronously and neuromuscular development (motility) was evident several days after hatching and up to 9 months later, after the overwintering period. 5. Synchronous hatching is an ancestral trait in turtles, but its relevance today is not solely for predator avoidance in all species. 6. Abiotic factors during incubation (e.g. temperature regime and moisture) have long-term effects on reptiles during ontogeny but it is also clear that incubation behaviour is major factor for the persistence of these developmental costs.

We used landscape ecology concepts to test the importance of upland–wetland linkages on the distribution of two common wetland species, the northern watersnake Nerodia sipedon sipedon and midland painted turtle Chrysemys picta marginata, and two rare wetland species, the copper-bellied watersnake Nerodia erythrogaster neglecta and Blanding’s turtle Emydoidea blandingii. We tested if connectivity (proximity to other wetlands), connectivity quality (wetland distance to roads and forest area within 30, 125, 250, 500 and 1000 m of the wetland), and patch size (wetland size and shoreline length) affected the distribution of these four species. Our results show that both common species were more likely to occur in larger, less isolated wetlands, but their distribution were not influenced by proximity to roads or the amount of adjacent forest area surrounding the wetland. Both rare species were more likely to occur in wetlands that were farther away from roads and that had more surrounding forest. Proximity to other wetlands was not a significant predictor of either rare species’ distribution. Our results suggest that management practices should focus on protecting wetland complexes and maintaining upland–wetland linkages by improving landscape connectivity, increasing forest area surrounding wetlands and reducing road effects.

Hypoxic mammalian neurons undergo excitotoxic cell death, whereas painted turtle neurons survive prolonged anoxia without apparent injury. Anoxic survival is possibly mediated by a decrease in N-methyl-d-aspartate receptor (NMDAR) activity and maintenance of cellular calcium concentrations ([Ca(2+)](c)) within a narrow range during anoxia. In mammalian ischaemic models, activation of mitochondrial ATP-sensitive K(+) (mK(ATP)) channels partially uncouples mitochondria resulting in a moderate increase in [Ca(2+)](c) and neuroprotection. The aim of this study was to determine the role of mK(ATP) channels in anoxic turtle NMDAR regulation and if mitochondrial uncoupling and [Ca(2+)](c) changes underlie this regulation. In isolated mitochondria, the K(ATP) channel activators diazoxide and levcromakalim increased mitochondrial respiration and decreased ATP production rates, indicating mitochondria were 'mildly' uncoupled by 10-20%. These changes were blocked by the mK(ATP) antagonist 5-hydroxydecanoic acid (5HD). During anoxia, [Ca(2+)](c) increased 9.3 +/- 0.3% and NMDAR currents decreased 48.9 +/- 4.1%. These changes were abolished by K(ATP) channel blockade with 5HD or glibenclamide, Ca(2+)(c) chelation with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or by activation of the mitochondrial Ca(2+) uniporter with spermine. Similar to anoxia, diazoxide or levcromakalim increased [Ca(2+)](c) 8.9 +/- 0.7% and 3.8 +/- 0.3%, while decreasing normoxic whole-cell NMDAR currents by 41.1 +/- 6.7% and 55.4 +/- 10.2%, respectively. These changes were also blocked by 5HD or glibenclamide, BAPTA, or spermine. Blockade of mitochondrial Ca(2+)-uptake decreased normoxic NMDAR currents 47.0 +/- 3.1% and this change was blocked by BAPTA but not by 5HD. Taken together, these data suggest mK(ATP) channel activation in the anoxic turtle cortex uncouples mitochondria and reduces mitochondrial Ca(2+) uptake via the uniporter, subsequently increasing [Ca(2+)](c) and decreasing NMDAR activity.

The painted turtle, Chrysemys picta, and the snapping turtle, Chelydra serpentina, occur in aquatic habitats north of Lake Superior in boreal forest and cold prairie areas across Canada, and have by far the most northern range of any modern North American chelonian taxa. These two species are also the dominant turtles of the North American Pleistocene glacial ages and were among the first species to invade formerly glaciated areas at the end of the Wisconsinan. These Pleistocene occurrences are documented here. Several remarkable behavioral, physiological and reproductive adaptations that may act together to allow these species to survive in such northern areas are discussed.

Classic egg size theory predicts that, in a given environment, there is a level of maternal investment per offspring that will maximize maternal fitness. However, positive correlations among egg size and female body size are observed within populations in diverse animal taxa. A popular explanation for this phenomenon is that, in some populations, morphological constraints on egg size, such as ovipositor size (insects) or pelvic aperture width (lizards and turtles), limit egg size. Egg size may therefore increase with female body size due to body size-specific constraints on investment per offspring, coupled with selection towards an optimal egg size. We use 17 years of data from a population of painted turtles Chrysemys picta to evaluate this hypothesis. In accordance with our predictions, we find that (1) morphological constraints on egg size are apparent only in relatively small females, similarly (2) egg mass exhibits a strong asymptotic relationship with female body size, suggesting egg mass is optimized only at large body sizes, (3) clutch size, not egg mass, varies with female condition, and (4) clutch size varies more than egg mass across years. Contrary to our predictions, we observe that (5) the egg mass-clutch size tradeoff is not less pronounced at large body sizes. Our data do not fully support the traditional hypothesis, and recent models suggest that this hypothesis is indeed overly simplistic. When the selective environment of a female's offspring is influenced by her phenotype, optimal egg size may vary among maternal phenotypes. This concept can explain correlations among egg size and body size in many taxa, as well as the patterns observed in the present study. In this paradigm, a tight coupling of aperture width (or other ‘constraints’) and egg size may occur in small females, even when such morphological features are not causally related to variation in egg size. In this spirit, we question validity of invoking morphological constraints to explain covariation among egg size and female body size.

In the painted turtle (Chrysemys picta) and red-eared slider turtle (Trachemys scripta), the temperature that eggs are exposed to during incubation determines the sex of the developing embryo. Constant temperature incubation experiments have shown that for each of these species there is a pivotal temperature that produces a 1:1 sex ratio; higher temperatures bias sex ratios toward females, and lower temperatures toward males. Few studies have examined how fluctuating temperatures, as would be experienced in natural nests, affect hatchling phenotype. Models predict that under fluctuating temperatures sex determination depends on the proportion of development that occurs above or below the pivotal temperature. We tested the effect of fluctuating versus constant temperature incubation regimes on sex ratios and other hatchling traits for both painted and red-eared slider turtles. Eggs were divided into two treatments with half of the eggs from each species incubated at a constant intermediate temperature, 28.5°C, and half incubated under temperatures that fluctuated 3°C above and below 28.5°C. We converted the fluctuating temperature data into a constant temperature equivalent (CTE) so that we could directly compare constant and fluctuating incubation regimes. The CTE for the fluctuating regime for both species was higher than the constant temperature, which would predict an increase in the production of females. The fluctuating regime did produce a higher proportion of females, but also resulted in increased developmental time and increased hatchling mass, indicating that fluctuating temperatures produce complex effects on hatchling phenotype. J. Exp. Zool. 307A:274–280, 2007. © 2007 Wiley-Liss, Inc.

Summary 1 Hatchlings of the North American Painted Turtle, Chrysemys picta (Schneider 1783) typically spend their first winter of life inside a shallow, terrestrial hibernaculum (the natal nest) where they commonly are exposed for extended periods to ice and cold. Current evidence indicates that turtles withstand such exposure by resisting freezing and becoming supercooled. 2 Supercooled hatchlings held at constant temperatures in the laboratory experience circulatory impairment and stagnant hypoxia, and consequently rely on anaerobic metabolism to meet a portion of their energy needs. As a result, lactate accumulates in bodies of supercooled animals. 3 The first experiment in the current investigation demonstrated that unfrozen hatchlings exposed to subzero temperatures like those recorded in a natural hibernaculum contained elevated quantities of lactic acid (and free glucose). This finding validates the widespread reliance on laboratory studies to gain insights regarding the physiology of animals overwintering in the field. 4 In a second study, hatchling Painted Turtles held for 20 days at −6 °C contained nearly twice as much lactate as turtles sampled after 10 days at that temperature. Hatchlings held for 10 days at −6 °C and then for another 10 days at −3 °C also contained more lactate than turtles sampled after 10 days at −6 °C, but not as much as the hatchlings that spent 20 days at −6 °C. Thus, animals held for part of the time at the higher subzero temperature still relied on anaerobic metabolism, but not to the same extent as turtles held continuously at the lower temperature. In contrast, hatchlings held for 10 days at −6 °C and then for another 10 days at either 0 °C or +3 °C contained no more lactate than control animals that never were exposed to subzero temperatures. Hatchlings exposed for the second 10 days to either 0 °C or +3 °C apparently were able to catabolize or otherwise process all the lactate that was accumulated during the first 10 days of their treatment. 5 Free glucose in bodies of hatchlings was elevated in all animals exposed to subzero temperatures, even when the initial exposure was followed by 10 days at temperatures as high as +3 °C. This finding has important implications with regard to the substrate that is used to support intermediary metabolism in supercooled turtles as well as to the metabolic pathways that are used to remove accumulated lactate once body temperature of the turtles rises at least to 0 °C.

Painted turtles (Chrysemys picta) are commercially harvested in large numbers in Minnesota for sale to biological supply companies and the pet trade. We investigated the possible effects of this harvest by comparing size, demography, and catch rates of painted turtles in 12 harvested and 10 nonharvested painted turtle populations in 2001 and 2002. We correlated turtle catch rates to harvest status, and harvested lakes had a lower catch-per-unit-effort than nonharvested lakes. Harvest had minimal effect on the size of turtles captured, and we found no significant differences in the count of male:female:juvenile turtles among lakes of different harvest status. We suggest that painted turtle populations likely have been impacted by harvester activities, but it was unclear whether the current harvest is sustainable. Further work is needed to determine whether there are any long-term effects on painted turtle populations.

Road mortality has the potential to alter the structure of turtle populations because turtle popula- tions are highly sensitive to additive sources of adult mortality. To address the issue, we captured painted turtles ( Chrysemys picta; n = 174) and snapping turtles ( Chelydra serpentina; n = 56) in 18 wetlands surrounded by low road density (≤1.5 km roads/km 2 of landscape) and 17 wetlands surrounded by high road density (>1.5 km/km 2 ) in central New York in 2002. High road density was associated with male-biased sex ratios in painted turtles (74% vs. 54% males; p = 0.01) and snapping turtles (95% vs. 74% males; p = 0.08), whereas turtle morphology and abundance were not associated with road density. Disproportionate road mortality of females on nesting migrations is the most likely cause of skewed sex ratios.

Summary 1 Optimal egg size theory predicts that females should produce propagules of a size and number that maximize maternal fitness. However, studies of the allocation of resources to eggs have rarely provided evidence of such optimization. This is presumably because of constraints that limit reproductive allocation. One such example is that of pelvic aperture morphology constraining egg size in turtles. 2 Growing evidence suggests that even this classic example is incomplete. Hormones that regulate both the reproductive cycle and vitellogenic activity in turtles may provide a novel physiological mechanism for control of egg size. 3 This physiological constraint hypothesis was explored by examining the relationship between maternally derived yolk hormones and egg mass in the Painted Turtle (Chrysemys picta, Schneider). 4 Eggs from younger females contained significantly more testosterone in the yolk than did eggs from older females. Younger females laid eggs nearly 20% smaller than those of older females even where body sizes overlapped. 5 Elevated testosterone levels in younger females may thus constrain egg size physiologically, beyond the well-known physical constraints imposed by pelvic aperture morphology. The endocrine system may play an important but previously unrecognized role in the evolution of egg size in this model system.

Summary 1Baby Painted Turtles, Chrysemys picta (Schneider 1783), ingest soil and fragments of eggshell as they are hatching from their eggs. The ingested soil typically contains bacterial nucleating agents that can cause water in the gut to freeze at high subzero temperatures. If ice forms in the gut, however, the crystals penetrate the wall of that organ and cause water in body compartments to freeze. Thus, newly hatched turtles freeze spontaneously at high subzero temperatures owing to the nucleation of ice by bacteria in the gut. 2Painted Turtles purge their gut of soil in the weeks after hatching, but removing particulate material from the GI tract does not, by itself, result in elimination of the bacterial nucleating agents. The purging of nucleating agents requires that hatchlings also be exposed to low (but non-freezing) temperatures. The response to low temperature is completed in little more than 1 month; a longer exposure does not elicit a demonstrable improvement in the capacity for supercooling. 3The limit of supercooling does not vary significantly among turtles hatching on different kinds of soil, despite the fact that different soils typically harbour different concentrations of nucleating agents. Thus, the risk of heterogeneous nucleation probably does not vary appreciably among turtles hatching and overwintering in different kinds of soil in the field. 4Hatchling Painted Turtles that have become acclimatized to low temperatures in the field presumably have acquired a high resistance to freezing by inoculation (as shown by prior study) and an enhanced capacity for supercooling (current study), thereby enabling many animals to withstand exposure to ice and cold during winter by remaining unfrozen and supercooled.

Summary 1Neonatal Painted Turtles, Chrysemys picta (Schneider 1783), typically spend the first winter of their life inside the shallow, subterranean nest where they completed incubation the preceding summer. This behaviour commonly causes hatchlings in northerly populations to be exposed in mid-winter to life-threatening conditions of ice and cold. Neonates apparently withstand such exposure by remaining unfrozen and becoming supercooled. 2Recently hatched turtles are unable to resist freezing by inoculation (i.e. freezing that is induced by the penetration of ice crystals through the integument and into body compartments from frozen soil). Consequently, newly hatched animals that make contact with ice are highly susceptible to freezing when temperature goes below the equilibrium freezing point for their body fluids (c. −0·7 °C). Freezing is fatal, even when temperature does not go below −2 °C, so newly hatched turtles have only a very limited tolerance for exposure to ice and cold. 3Acclimation (or acclimatization) by neonatal turtles to low temperatures in the interval between hatching and the start of winter elicits an increase in effectiveness of the cutaneous barrier to the penetration of ice into body compartments from the environment. Acclimated hatchlings coming into contact with ice in frozen soil usually remain unfrozen and supercooled, and survival by supercooled turtles is uniformly high. This thermally induced enhancement of the cutaneous barrier to inoculation is key to an overwintering strategy based on supercooling.

Western painted turtles (Chrysemys picta bellii) are well-known for being able to survive anoxia for 120 days at 3°C and tolerate plasma lactate concentrations approaching 200 mM. Given that the turtle becomes hyperglycemic and accumulates lactate during anoxia, the potential to exceed the renal transport maxima for these metabolites exists. Here we assessed the role of the kidney during six hours of anoxia at 20°C and 39 hours of recovery from anoxia. Turtles received both subclavian artery and cloacal catheters for continual monitoring of plasma and urine metabolites. Our hypothesis that kidney function would decline in tandem with arterial PO2 was supported, as urinary flow rate decreased significantly by more than 90%. During the first half of recovery, there was significant appearance of lactate and glucose in the urine; the renal transport maxima for these metabolites were likely exceeded. The excretion of lactate during recovery, though energetically wasteful, may help to relieve the lactic acidosi...

Molecular polymorphisms have been used in a variety of ways to estimate both effective and local census population sizes in nature. A related approach for estimating the current size of a breeding population, explored here for the first time, is the use of genetic 'marks' reconstructed for otherwise unknown parents in paternity or maternity analyses of progeny arrays. This method provides interesting similarities and contrasts to traditional mark-recapture methods based on physical tags. To illustrate, this genetic method is applied to a population of painted turtles on the Mississippi River to estimate the number of successfully breeding males. Non-genetic mark-recapture approaches were also applied to animals trapped at this location. Results demonstrate that such genetic data on parentage can be helpful not only in estimating contemporary population sizes, but also in providing additional information, not present in customary mark-recapture data, about possible extended movements of breeding individuals and the size of the pool of mates which they encounter.

Summary 1The contents of newly constructed nests of Painted Turtles, Chrysemys picta (Schneider 1783), were manipulated by reciprocal transplant so that each of several nests received a complement of eggs from each of several females. The eggs were recovered from nests after 8 weeks and allowed to complete their incubation under standard conditions in the laboratory. The design of the experiment enabled us to distinguish between environmental and maternal effects on attributes of hatchlings. 2Several measures of body size and energy reserve varied among turtles hatching from eggs that incubated in different nests, and certain of these measures varied also among turtles hatching from eggs that incubated in different layers within nests. The effects of nest and layer were substantial. For example, fat-free carcasses of hatchlings from one nest weighed 17% more than those of neonates from a second nest, but fat-free yolks from the former weighed only 53% as much as yolks from the latter. 3Stepwise linear regression indicated that the size of hatchlings and the hydration and fat content of their carcasses were positively correlated with the net change in mass of eggs (which is an index to net water-exchange) while they incubated in the field. In contrast, both the fat and fat-free components of unused yolk were negatively correlated with change in mass of eggs. Although the statistical procedure is only correlative, the findings accord well with results of laboratory studies documenting a relationship between uptake of water by eggs, metabolism and growth by embryos, and size and condition of hatchlings. 4Variation among hatchlings representative of different nests accounted for 24% of the statistical variance in mass of dry, fat-free carcasses; 29% of the variance in mass of dry, fat-free yolks; 19% of the variance in mass of storage fat in yolks; and 11% of the variance in mass of storage fat in carcasses. Additional variation was detected between the upper and lower layers of nests. Such environmentally induced variation probably affects survival of neonatal animals in the field.

1. The contents of newly constructed nests of Painted Turtles, Chrysemys picta, were manipulated by reciprocal transplant, so that each of several nests received a complement of eggs from each of several females. The eggs were recovered from nests after 8 weeks and allowed to complete incubation under standard conditions in the laboratory. 2. Live mass, dry mass and water content of carcasses, and dry mass of unused yolk, varied significantly among hatchlings from eggs that incubated in different nests. Indeed, hatchlings from different nests sometimes differed as much in size or physiological condition as animals from different clutches. 3. Stepwise linear regression indicated that size of hatchlings and water content of their carcasses were positively correlated with water exchanges by eggs whereas mass of the unused yolk was negatively correlated with water exchanges. Although the statistical procedure is only correlative, the findings accord well with results of laboratory studies documenting a relationship between uptake of water by eggs, metabolism and growth by embryos, and size and condition of hatchlings. 4. Developmental plasticity, coupled with variation in the physical environment within and among nests, probably accounts (minimally) for 19% of the variation in live mass of hatchlings in the field; 24% of the variation in dry mass of carcasses; 11% of the variation in hydration of carcasses; and 26% of the variation in dry mass of unused yolk. Such variation may impact survival by neonatal animals, and needs to be addressed explicitly by models for the evolution of life history in Painted Turtles.

Hatchling painted turtles (Chrysemys picta) commonly overwinter within their natal nests and survive exposure to temperatures as low as −12°C by supercooling. We report that the supercooling capacity of hatchling C. picta is reduced by direct contact with nest soil which, in samples from northwestern and north-central Nebraska, Indiana, and Ontario, contained potent ice nuclei active in the range of −3.5° to −5°C. These nuclei were sensitive to autoclaving and extractable in water. The supercooling capacity of C. picta hatched in native nest soil, or hatched in sterilized vermiculite (which lacks water-extractable nuclei), and subsequently exposed to nest soil, was reduced by ∼10°C relative to control turtles that were hatched and reared in sterilized vermiculite. The effect of these nuclei was potentiated by the presence of environmental moisture, although even transient exposure to dry nest soil markedly reduced supercooling capacity in ∼50% of the turtles. Unlike turtle species that hibernate underwater (Sternotherus odoratus, Chelydra serpentina, Apalone spinifera), hatchlings of C. picta exhibited an extraordinary capacity for supercooling (temperature of crystallization, −16° to −20°C) when cooled in isolation from external ice nuclei. However, hatchlings of these four species were equally susceptible to inoculation by suspensions of the ice-nucleating bacterium, Pseudomonas syringae. Indirect evidence suggests that the soil nuclei are associated with such microbes. Nucleating activity was higher in soil collected within nests than in soil collected at the same depth, adjacent to these nests. Differences in the activities of ice nuclei in nesting soils may account for geographic and local variation in winter survival of hatchling C. picta. Our finding that similar agents occur in various other terrestrial habitats in central North America suggests that such nuclei may pose a formidable challenge to the overwintering survival of ectothermic animals that rely on supercooling to withstand frost exposure.

Hatchlings of the North American painted turtle (Chrysemys picta) spend their first winter of life inside a shallow, subterranean hibernaculum (the natal nest) where they may be exposed for extended periods to ice and cold. Hatchlings seemingly survive exposure to such conditions by becoming supercooled (i.e., by remaining unfrozen at temperatures below the equilibrium freezing point for body fluids), so we investigated the role of their integument in preventing ice from penetrating into body compartments from surrounding soil. We first showed that hatchlings whose epidermis has been damaged are more likely to be penetrated by growing crystals of ice than are turtles whose cutaneous barrier is intact. We next studied integument from a forelimb by light microscopy and discovered that the basal part of the alpha-keratin layer of the epidermis contains a dense layer of lipid. Skin from the forelimb of other neonatal turtles lacks such a layer of lipid in the epidermis, and these other turtles also are highly susceptible to inoculative freezing. Moreover, epidermis from the neck of hatchling painted turtles lacks the lipid layer, and this region of the skin is readily penetrated by growing crystals of ice. We therefore conclude that the resistance to inoculation imposed by skin on the limbs of hatchling painted turtles results from the presence of lipids in the alpha-keratin layer of the epidermis. Neonates apparently are able to avoid freezing during winter by drawing much of the body inside the shell, leaving only the ice-resistant integument of the limbs exposed to ice in the environment. The combination of behavior and skin morphology enables overwintering hatchlings to exploit an adaptive strategy based on supercooling.

1. Laboratory experiments have documented substantial temperature effects on the physiological ecology of reptilian eggs, embryos and offspring. However, functional links between important habitat characteristics, nest microenvironments and fitness-related traits of neonates in natural nests have rarely been studied. 2. A field study of 11 Painted Turtle (Chrysemys picta) nests was conducted to quantify the relationships between a habitat characteristic (i.e. vegetational cover around nests at oviposition) and (1) developmental temperature and its effect on offspring sex ratio and (2) hibernation temperature and its effect on offspring survivorship. 3. Vegetational cover was negatively correlated with nest temperatures in July, the period when offspring sex is determined. However, neither vegetational cover nor mean nest temperature predicted nest sex ratios, although correlations among these variables were consistent with causal relationships derived from laboratory studies. 4. Summer vegetational cover was also negatively correlated with measures of winter nest temperatures. Of the three nests exhibiting overwinter mortality, two were surrounded by thick vegetation and all experienced temperatures below – 8 °C. The remaining nests reached temperatures as low as – 6 °C without mortality, indicating that hatchlings in these nests exhibited remarkable supercooling ability. 5. The results suggest that habitat characteristics and nest microenvironments are functionally linked and have fitness consequences for both embryos and offspring, implying that nest-site choice by female turtles could have considerable utility.

Unlike birds and mammals, in many reptiles the temperature experienced by a developing embryo determines its gonadal sex. To understand how temperature-dependent sex determination (TSD) evolves, we must first determine the nature of genetic variation for sex ratio. Here, we analyze among-family variation for sex ratio in three TSD species: the American alligator (Alligator mississipiensis), the common snapping turtle (Chelydra serpentina) and the painted turtle (Chrysemys picta). Significant family effects and significant temperature effects were detected in all three species. In addition, family-by-temperature interactions were evident in the alligator and the snapping turtle, but not in the painted turtle. Overall, the among-family variation detected in this study indicates potential for sex-ratio evolution in at least three reptiles with TSD. Consequently, climate change scenarios that are posited on the presumption that sex-ratio evolution in TSD reptiles is genetically constrained may require reevaluation.