Temperature-dependent Sex Determination In Reptiles Research Articles

Latitudinal Variation in the Pattern of Temperature-Dependent Sex Determination in the Japanese Gecko, Gekko japonicus.

Simple SummaryIn egg-laying lizards, sex is determined by genetic factors in species with sex chromosomes or egg incubation temperatures in species without sex chromosomes, i.e., temperature-dependent sex determination (TSD). Surprisingly, recent studies find sex chromosomes and TSD co-occur in the same species in some lizards. The Japanese gecko from Japan may be this case. However, Japanese gecko with TSD from a Chinese population does not have sex chromosomes, suggesting that the pattern of TSD in this gecko may vary among populations. We incubated gecko eggs from three populations in China at constant temperatures of 24, 26, 28, 30, and 32 °C to quantify the sex determination pattern. We found that the temperature yielding an equal number of sons and daughters of the low-latitude population was lower than that of the two high-latitude populations. Moreover, the low-latitude population had a narrower temperature range producing mixed sex offspring at lower temperatures, but a wider range at higher temperatures. Sex ratio was almost 1:1 for the low-latitude population when incubated from 26 to 30 °C. Conversely, more male offspring were produced at 28 or 30 °C in the two high-latitude populations. Our study may provide an interesting system to explore the evolution of sex determination mechanisms in animals.Identifying latitudinal variation in the pattern of temperature-dependent sex determination (TSD) may provide insight into the evolution of sex determining system in vertebrates, but such studies remain limited. Here, we quantified TSD patterns of three geographically separated populations of the Japanese gecko (Gekko japonicus) along the latitudinal cline of China. We incubated gecko eggs from the three populations at constant temperatures of 24, 26, 28, 30, and 32 °C to quantify the TSD pattern. Our study demonstrated that G. japonicus exhibited a FMF pattern of TSD, with the low and high incubation temperatures yielding significantly female-biased hatchlings, and the medium temperatures producing male-biased hatchlings. More interestingly, we found latitudinal variations in the TSD pattern in terms of pivotal temperatures (Tpivs), transitional range of temperatures (TRT), and the sex ratios at the medium temperatures. The Tpivs for the low-latitude population were lower than those for the two high-latitude populations. The low-latitude population has a narrower FM TRT, but a wider MF TRT. The sex ratio is almost 50:50 for the low-latitude population when eggs were incubated from 26 to 30 °C. Conversely, the sex ratio is male-biased for the two high-latitude populations at 28 or 30 °C. Therefore, G. japonicus may provide an interesting system to explore the evolution of TSD in reptiles given the diversity of TSD patterns among populations.

Maternal provisioning and fluctuating thermal regimes enhance immune response in a reptile with temperature-dependent sex determination

The Charnov-Bull model of differential fitness is often used to explain the evolution and maintenance of temperature-dependent sex determination (TSD). Most tests of the model focus on morphological proxies of fitness, such as size traits, whereas early life physiological traits that are closely related to lifetime fitness might provide a framework for generalizing the Charnov-Bull model across taxa. One such trait is the strength of the early-life immune response, which is strongly linked to early-life survival and fitness. Here, we manipulated temperature, variance in temperature, and sex to test the Charnov-Bull model using a physiological trait, immune system strength, in the snapping turtle (Chelydra serpentina). We found no evidence of sex-specific differences in bactericidal capacity of hatchling blood, and no evidence that mean temperature influences bactericidal capacity. However, we did find that fluctuating incubation temperature (i.e. a more naturalized incubation regime) is associated with a greater bactericidal capacity compared with constant temperature incubation. We also found that egg mass, a proxy for maternal provisioning, is positively associated with bactericidal capacity. Our findings suggest that the evolution of temperature-dependent sex determination in reptiles is unrelated to our measure of early-life innate immunity. Our study also underlines how immune response is condition dependent in early life, and questions the biological relevance of constant temperature incubation in experimental studies on ectotherm development.

Thyroid hormone modulates offspring sex ratio in a turtle with temperature-dependent sex determination.

The adaptive significance of temperature-dependent sex determination (TSD) has attracted a great deal of research, but the underlying mechanisms by which temperature determines the sex of a developing embryo remain poorly understood. Here, we manipulated the level of a thyroid hormone (TH), triiodothyronine (T3), during embryonic development (by adding excess T3 to the eggs of the red-eared slider turtle Trachemys scripta, a reptile with TSD), to test two competing hypotheses on the proximate basis for TSD: the developmental rate hypothesis versus the hormone hypothesis Exogenous TH accelerated embryonic heart rate (and hence metabolic rate), developmental rate, and rates of early post-hatching growth. More importantly, hyperthyroid conditions depressed expression of Cyp19a1 (the gene encoding for aromatase) and levels of oestradiol, and induced more male offspring. This result is contrary to the direction of sex-ratio shift predicted by the developmental rate hypothesis, but consistent with that predicted by the hormone hypothesis Our results suggest an important role for THs in regulating sex steroid hormones, and therefore, in affecting gonadal sex differentiation in TSD reptiles. Our study has implications for the conservation of TSD reptiles in the context of global change because environmental contaminants may disrupt the activity of THs, and thereby affect offspring sex in TSD reptiles.

Dispersal and climate warming determine range shift in model reptile populations

Average air temperature is predicted to rise by at least 3°C across the 21st century. As individual sex (male or female) is determined by temperature in many reptiles, there are concerns that climate warming will skew offspring sex ratios and local species extinctions will follow. Range shift away from hotter areas through dispersal may prevent species extinctions in many reptiles with temperature-dependent sex determination (TSD), and could be facilitated or impeded by sex-biased populations at the expanding edge. We used a simulation model to examine the role of sex-determining mechanism [TSD and genotypic sex determination (GSD)], climate warming and dispersal in determining range shift and population growth in reptiles. Dispersal influenced range shift (after climate warming) in TSD species to a greater extent than in GSD species. Our novel finding is that biased sex ratios may influence range shift, through the mixing of the rare sex (females) with males located at the colder range edges, as both sexes disperse. However, if faced with climate warming of 3°C over the next 100 years many TSD reptiles will show limited capacity for range shift.

Unexpected consequences of a drier world: evidence that delay in late summer rains biases the population sex ratio of an insect.

The complexity of animal life histories makes it difficult to predict the consequences of climate change on their populations. In this paper, we show, for the first time, that longer summer drought episodes, such as those predicted for the dry Mediterranean region under climate change, may bias insect population sex ratio. Many Mediterranean organisms, like the weevil Curculio elephas, become active again after summer drought. This insect depends on late summer rainfall to soften the soil and allow adult emergence from their underground refuges. We found that, as in many protandric species, more C. elephas females emerged later in the season. Male emergence timing was on average earlier and also more dependent on the beginning of late summer rainfall. When these rains were delayed, the observed weevil sex ratio was biased towards females. So far, the effects of global warming on animal sex ratios has been reported for temperature-dependent sex determination in reptiles. Our results show that rainfall timing can also bias the sex ratio in an insect, and highlight the need for keeping a phenological perspective to predict the consequences of climate change. We must consider not just the magnitude of the predicted changes in temperature and rainfall but also the effects of their timing.

Phylogeny of sex-determining mechanisms in squamate reptiles: are sex chromosomes an evolutionary trap?

Squamate reptiles possess two general modes of sex determination: (1) genotypic sex determination (GSD), where the sex of an individual is determined by sex chromosomes, i.e. by sex-specific differences in genotype; and (2) temperature-dependent sex determination (TSD), where sex chromosomes are absent and sex is determined by nongenetic factors. After gathering information about sex-determining mechanisms for more than 400 species, we employed comparative phylogenetic analyses to reconstruct the evolution of sex determination in Squamata. Our results suggest relative uniformity in sex-determining mechanisms in the majority of the squamate lineages. Well-documented variability is found only in dragon lizards (Agamidae) and geckos (Gekkota). Polarity of the sex-determining mechanisms in outgroups identified TSD as the ancestral mode for Squamata. After extensive review of the literature, we concluded that to date there is no known well-documented transition from GSD to TSD in reptiles, although transitions in the opposite direction are plentiful and well corroborated by cytogenetic evidence. We postulate that the evolution of sex-determining mechanisms in Squamata was probably restricted to the transitions from ancestral TSD to GSD. In other words, transitions were from the absence of sex chromosomes to the emergence of sex chromosomes, which have never disappeared and constitute an evolutionary trap. This evolutionary trap hypothesis could change the understanding of phylogenetic conservatism of sex-determining systems in many large clades such as butterflies, snakes, birds, and mammals. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 156, 168–183.

Constraints on temperature-dependent sex determination in the leopard gecko (Eublepharis macularius): response to Kratochvil et al.

Kratochvil et al. (Naturwissenschaften 95:209-215, 2008) reported recently that in the leopard gecko (Eublepharis macularius) of the family Eublepharidae with temperature-dependent sex determination (TSD), clutches in which eggs were incubated at the same temperature produce only same-sex siblings. Interpreting this result in light of studies of sex steroid hormone involvement in sex determination, they suggested that maternally derived yolk steroid hormones could constrain sex-determining mechanisms in TSD reptiles. We have worked extensively with this species and have routinely incubated clutches at constant temperatures. To test the consistency of high frequency same-sex clutches across different incubation temperatures, we examined our records of clutches at the University of Texas at Austin from 1992 to 2001. We observed that clutches in which eggs were incubated at the same incubation temperature produced mixed-sex clutches as well as same-sex clutches. Furthermore, cases in which eggs within a clutch were separated and incubated at different temperatures produced the expected number of mixed-sex clutches. These results suggest that maternal influences on sex determination are secondary relative to incubation temperature effects.

Gonadal expression of Sf1 and aromatase during sex determination in the red-eared slider turtle ( Trachemys scripta), a reptile with temperature-dependent sex determination

Many egg-laying reptiles have temperature-dependent sex determination (TSD), where the offspring sex is determined by incubation temperature during a temperature-sensitive period (TSP) in the middle third of development. The underlying mechanism transducing a temperature cue into an ovary or testis is unknown, but it is known that steroid hormones play an important role. During the TSP, exogenous application of estrogen can override a temperature cue and produce females, while blocking the activity of aromatase ( Cyp19a1), the enzyme that converts testosterone to estradiol, produces males from a female-biased temperature. The production of estrogen is a key step in ovarian differentiation for many vertebrates, including TSD reptiles, and temperature-based differences in aromatase expression during the TSP may be a critical step in ovarian determination. Steroidogenic factor-1 ( Sf1) is a key gene in vertebrate sex determination and regulates many steroidogenic enzymes, including aromatase. We find that Sf1 and aromatase are differentially expressed during sex determination in the red-eared slider turtle, Trachemys scripta elegans. Sf1 is expressed at higher levels during testis development while aromatase expression increases during ovary determination. We also assayed Sf1 and aromatase response to sex-reversing treatments via temperature or the modulation of estrogen availability. Sf1 expression was redirected to low-level female-specific patterns with feminizing temperature shift or exogenous estradiol application and redirected to more intense male-specific patterns with male-producing temperature shift or inhibition of aromatase activity. Conversely, aromatase expression was redirected to more intense female-specific patterns with female-producing treatment and redirected toward diffuse low-level male-specific patterns with masculinizing sex reversal. Our data do not lend support to a role for Sf1 in the regulation of aromatase expression during slider turtle sex determination, but do support a critical role for estrogen in ovarian development.

Temperature Sex Reversal Implies Sex Gene Dosage in a Reptile

Sex in reptiles is determined by genes on sex chromosomes or by incubation temperature. Previously these two modes were thought to be distinct, yet we show that high incubation temperatures reverse genotypic males (ZZ) to phenotypic females in a lizard with ZZ and ZW sex chromosomes. Thus, the W chromosome is not necessary for female differentiation. Sex determination is probably via a dosage-sensitive male-determining gene on the Z chromosome that is inactivated by extreme temperatures. Our data invite a novel hypothesis for the evolution of temperature-dependent sex determination (TSD) and suggest that sex chromosomes may exist in many TSD reptiles.

Estrogens Induced Male Production at a Female-Producing Temperature in a Reptile (Leopard Gecko, Eublepharis Macularius) with Temperature-dependent Sex Determination

Estrogens and estrogen mimics can affect offspring sex ratios in a wide variety of animal species including Leopard Geckos, Eublepharis macularius, a species with temperature-dependent sex determination (TSD). Estrogens can disrupt effects of temperature on sex determination by feminizing or sex-reversing embryos incubated at male-producing temperatures. Estrogens may have paradoxical effects at different incubation temperatures and in different concentrations. In this study, E. macularius eggs were exposed to ethanol, estradiol benzoate, or estradiol 17b at the beginning of the thermosensitive period when sex determination occurs. Eggs were treated and incubated at each of three incubation temperatures known to produce varying primary sex ratios. At a male-producing incubation temperature, estrogen-treated groups produced more females than negative control groups. This result has been reported in other TSD reptiles. In our study, we showed that at a female-producing incubation temperature, estrogen-treated groups produced significantly more males than negative control groups. This is the first report of its kind in which a TSD reptile was shown to produce significantly more males at a female-producing temperature. Our results suggest a complex feedback relationship between aromatase and cofactors during the thermosensitive period of egg incubation in E. macularius. Expected effects of exogenous estrogens on contaminant-exposed wildlife populations may differ depending on nest-site temperatures. In species with temperature-dependent sex determination (TSD), sex is determined by the temperature to which an embryo is exposed during a thermally sensitive period of incuba- tion. The influence of exogenous estrogens on

From gene networks underlying sex determination and gonadal differentiation to the development of neural networks regulating sociosexual behavior

Genes are not expressed in isolation any more than social behavior has meaning outside of society. Both are in dynamic flux with the immediate environment that the gene/individual finds itself, which in turn establishes the timing, pattern, and conditions of expression. This means that complex behaviors and their genetic underpinnings should be viewed as a cumulative process, or as the result of experiences up to that point in time and, at the same time, as setting the stage for what will follow. The evidence indicates that as experiences accumulate throughout life, early experiences shape how genes/individuals will respond to later experiences, whereas later experiences modify the effects of these earlier experiences. A method of graphically representing and analyzing change in gene and neural networks is presented. Results from several animal model systems will be described to illustrate these methods. First, we will consider the phenomenon of temperature-dependent sex determination in reptiles. We will illustrate how the experience of a particular temperature during a sensitive period of embryogenesis sculpts not only the patterns of expression of genes involved in sex determination and gonadal differentiation but also the morphological, physiological, neuroendocrine, and behavioral traits of the adult phenotype. The second model system concerns the effects of the sex ratio in the litter in rats, and the genotype ratio in the litter of transgenic mice, on the nature and frequency of maternal care and how this in turn influences the patterns of activation of identified neural circuits subserving the offspring's sociosexual behavior when it is an adult.

THE ADAPTIVE SIGNIFICANCE OF TEMPERATURE-DEPENDENT SEX DETERMINATION: EXPERIMENTAL TESTS WITH A SHORT-LIVED LIZARD

Why is the sex of many reptiles determined by the temperatures that these animals experience during embryogenesis, rather than by their genes? The Charnov-Bull model suggests that temperature-dependent sex determination (TSD) can enhance maternal fitness relative to genotypic sex determination (GSD) if offspring traits affect fitness differently for sons versus daughters and nest temperatures either determine or predict those offspring traits. Although potential pathways for such effects have attracted much speculation, empirical tests largely have been precluded by logistical constraints (i.e., long life spans and late maturation of most TSD reptiles). We experimentally tested four differential fitness models within the Charnov-Bull framework, using a short-lived, early-maturing Australian lizard (Amphibolurus muricatus) with TSD. Eggs from wild-caught females were incubated at a range of thermal regimes, and the resultant hatchlings raised in large outdoor enclosures. We applied an aromatase inhibitor to half the eggs to override thermal effects on sex determination, thus decoupling sex and incubation temperature. Based on relationships between incubation temperatures, hatching dates, morphology, growth, and survival of hatchlings in their first season, we were able to reject three of the four differential fitness models. First, matching offspring sex to egg size was not plausible because the relationship between egg (offspring) size and fitness was similar in the two sexes. Second, sex differences in optimal incubation temperatures were not evident, because (1) although incubation temperature influenced offspring phenotypes and growth, it did so in similar ways in sons versus daughters, and (2) the relationship between phenotypic traits and fitness was similar in the two sexes, at least during preadult life. We were unable to reject a fourth model, in which TSD enhances offspring fitness by generating seasonal shifts in offspring sex ratio: that is, TSD allows overproduction of daughters (the sex likely to benefit most from early hatching) early in the nesting season. In keeping with this model, hatching early in the season massively enhanced body size at the beginning of the first winter, albeit with a significant decline in probability of survival. Thus, the timing of hatching is likely to influence reproductive success in this short-lived, early maturing species; and this effect may well differ between the sexes.

Sex determination in mythology and history

The history of ideas on how the sexes became divided spans at least three thousand years. The biblical account of the origin of Eve, and the opinions of the philosophers of classical Greece, have unexpected bearings on present-day ideas. The scientific study of sex determination can be said to have begun in the 17th century with the discovery of spermatozoa, but the origin and function of the "spermatic animalcules" eluded investigators until 1841. The mammalian egg was discovered in 1827, and in the last quarter of the century fertilization was observed. The view current at that time, that sex determination was under environmental control, gave way to the idea of chromosomal determination in the first quarter of the 20th century. The study of human and other mammalian chromosomes during the third quarter of the century, and the discovery of sex-chromosome abnormalities, emphasized the importance of the Y chromosome for male sex determination. The last quarter of the century witnessed a hunt for the "testis-determining" gene, thought to be responsible for the differentiation of Sertoli cells, and culminating in the isolation of SRY (Sry in the mouse). However, an increasing number of additional genes and growth factors were found to be required for the establishment of male sex. During the same period evidence emerged that male development was accompanied by enhanced growth, both of gonads and whole embryos. An unexpected finding was the demonstration of temperature-dependent sex determination in reptiles. With the advent of the 21st century, it was shown that Sry induces cell proliferation in fetal mouse gonads, and it has been suggested that male sex differentiation in mammals requires a higher metabolic rate. These insights could lead to a better understanding and improved treatment of abnormalities of sexual development.

THE ADAPTIVE SIGNIFICANCE OF TEMPERATURE-DEPENDENT SEX DETERMINATION: EXPERIMENTAL TESTS WITH A SHORT-LIVED LIZARD

Why is the sex of many reptiles determined by the temperatures that these animals experience during embryogenesis, rather than by their genes? The Charnov-Bull model suggests that temperature-dependent sex determination (TSD) can enhance maternal fitness relative to genotypic sex determination (GSD) if offspring traits affect fitness differently for sons versus daughters and nest temperatures either determine or predict those offspring traits. Although potential pathways for such effects have attracted much speculation, empirical tests largely have been precluded by logistical constraints (i.e., long life spans and late maturation of most TSD reptiles). We experimentally tested four differential fitness models within the Charnov-Bull framework, using a short-lived, early-maturing Australian lizard (Amphibolurus muricatus) with TSD. Eggs from wild-caught females were incubated at a range of thermal regimes, and the resultant hatchlings raised in large outdoor enclosures. We applied an aromatase inhibitor to half the eggs to override thermal effects on sex determination, thus decoupling sex and incubation temperature. Based on relationships between incubation temperatures, hatching dates, morphology, growth, and survival of hatchlings in their first season, we were able to reject three of the four differential fitness models. First, matching offspring sex to egg size was not plausible because the relationship between egg (offspring) size and fitness was similar in the two sexes. Second, sex differences in optimal incubation temperatures were not evident, because (1) although incubation temperature influenced offspring phenotypes and growth, it did so in similar ways in sons versus daughters, and (2) the relationship between phenotypic traits and fitness was similar in the two sexes, at least during preadult life. We were unable to reject a fourth model, in which TSD enhances offspring fitness by generating seasonal shifts in offspring sex ratio: that is, TSD allows overproduction of daughters (the sex likely to benefit most from early hatching) early in the nesting season. In keeping with this model, hatching early in the season massively enhanced body size at the beginning of the first winter, albeit with a significant decline in probability of survival. Thus, the timing of hatching is likely to influence reproductive success in this short-lived, early maturing species; and this effect may well differ between the sexes.

Oestrogens and temperature-dependent sex determination in reptiles: all is in the gonads.

In many species of oviparous reptiles, the first steps of gonadal sex differentiation depend on the incubation temperature of the eggs. Feminization of gonads by exogenous oestrogens at a male-producing temperature and masculinization of gonads by antioestrogens and aromatase inhibitors at a female-producing temperature have irrefutably demonstrated the involvement of oestrogens in ovarian differentiation. Nevertheless, several studies performed on the entire gonad/adrenal/mesonephros complex failed to find differences between male- and female-producing temperatures in oestrogen content, aromatase activity and aromatase gene expression during the thermosensitive period for sex determination. Thus, the key role of aromatase and oestrogens in the first steps of ovarian differentiation has been questioned, and extragonadal organs or tissues, such as adrenal, mesonephros, brain or yolk, were considered as possible targets of temperature and sources of the oestrogens acting on gonadal sex differentiation. In disagreement with this view, experiments and assays carried out on the gonads alone, i.e. separated from the adrenal/mesonephros, provide evidence that the gonads themselves respond to temperature shifts by modifying their sexual differentiation and are the site of aromatase activity and oestrogen synthesis during the thermosensitive period. Oestrogens act locally on both the cortical and the medullary part of the gonad to direct ovarian differentiation. We have concluded that there is no objective reason to search for the implication of other organs in the phenomenon of temperature-dependent sex determination in reptiles. From the comparison with data obtained in other vertebrates, we propose two main directions for future research: to examine how transcription of the aromatase gene is regulated and to identify molecular and cellular targets of oestrogens in gonads during sex differentiation, in species with strict genotypic sex determination and species with temperature-dependent sex determination.

The significance of predation in nest site selection of turtles: an experimental consideration of macro‐ and microhabitat preferences

Nest selection has important consequences for fitness in freshwater turtles, as nest predation is extremely high in most populations. However, nest predation has rarely been considered a major factor affecting nest site selection. Thermal qualities of a nest are often suggested as a factor influencing maternal choice of nest site because incubation temperature affects offspring performance and growth, as well as the sex ratio of the clutch in species with temperature dependent sex determination (TSD). Yet experimental manipulations of the nesting environment (habitat and threat) to test factors influencing maternal nest site selection in turtles are uncommon. In this paper, we show that nest site selection in an Australian turtle (Emydura macquarii) with genetically determined sex determination (GSD) exists at two spatial scales, with predation as the major factor affecting the location of a nest. Females prefer to nest in areas where nest predation is minimal but when the risk of direct predation is increased, they trade‐off minimising nest predation with survival by locating their nests closer to shore. However, experimentally reducing cover demonstrated that females prefer to nest in open areas, which are more common closer to shore. Hence females are forgoing preferred microhabitat to locate their nests away from shore to minimise nest predation. Despite a preference for open nesting areas, females exhibit similar repeatability of microenvironment selection to a North American species with TSD. Repeatability of nesting overstory vegetation in the North American turtle, Chrysemys picta, suggests that females may exert some control over the thermal qualities of the nests; a key assumption in theoretical models of the microevolution and adaptive significance of TSD in reptiles. Incubation temperature is one factor that may affect microhabitat preferences in both turtles but other factors (e.g. predation and soil moisture) affected by microhabitat may be critical in the evolution of repeatability of overstory cover.

Assessment of patterns of temperature-dependent sex determination using maximum likelihood model selection

Sex determination in some reptiles is independent of egg incubation temperature and is called genotypic sex determination (GSD). In many other reptiles, sexual phenotype is dependent on incubation temperature. This phenomenon is called temperature-dependent sex determination (TSD). TSD is categorized by three patterns, based on the majority sex produced at lower and higher incubation temperatures, named MF for Male-Female, FM for Female-Male, or FMF for Female-Male-Female. When large numbers of eggs are incubated at many different incubation temperatures, the assessment of TSD pattern is unambiguous, but when few eggs or few incubation temperatures are used, the categorization of TSD pattern is less straightforward. We propose a new methodology based on maximum likelihood model selection that evaluates and ranks the performance of four descriptive models of sex determination for discrete datasets. This method has the added benefit of giving standardized definitions of two commonly reported parameters of TSD: the pivotal temperature and the transitional range of temperature. Standardization of analyses will help facilitate cross-species meta-analyses of TSD in reptiles.

Dynamics of yolk steroid hormones during development in a reptile with temperature-dependent sex determination

Many oviparous reptiles exhibit temperature-dependent sex determination (TSD); i.e., the temperature at which the egg is incubated determines the sex of the offspring. In TSD reptiles, yolk steroids not only may influence sex determination, but also may mediate hormonal effects on subsequent growth and behavior, as in some avian species. We investigated changes in the levels of estradiol (E 2) and testosterone (T) during development in yolks of snapping turtle eggs, examined how incubation temperature affects hormone levels, and determined how hormones in turtle eggs are influenced by individual females (=clutch effects). Results indicate significant decreases in both hormones (>50% decline) by the end of the sex-determining period, when two-thirds of the development is complete. The declines in both E 2 and T were significantly affected by incubation temperature, but in different ways. Eggs incubated at female-producing temperatures maintained high levels, those incubated at male-producing temperatures had low E 2 values, and eggs incubated at pivotal temperatures had intermediate levels of E 2. At all three temperatures, T values underwent significant but approximately equal declines, except during the developmental stages just after the sex-determining period, when T levels decreased more at the male-producing temperature than at either of the other two temperatures. Initially, there were significant clutch effects in both hormones, but such differences, attributable to individual females, were maintained only for E 2 later in development. Here we report for the first time that incubation temperature significantly affects the hormonal environment of the developing embryo of a turtle with temperature-dependent sex determination. Based on this and related findings, we propose that yolk sex steroids influence sexual differentiation in these TSD species and play a role in sex determination at pivotal temperatures.

Repeatability of microenvironment-specific nesting behaviour in a turtle with environmental sex determination

Evolutionary thermal adaptation is increasingly being elucidated in a variety of systems. However, one of the most striking examples, temperature-dependent sex determination (TSD) in reptiles, has proven stubbornly difficult to decipher. Theoretical models suggest that selection on and heritable variation in thermal sensitivity of embryonic sex determination and maternal behavioural choice of thermal qualities of nest sites control the microevolutionary potential of TSD. To begin addressing this important issue, we conducted a multiyear field study of nesting behaviour in painted turtles (Chrysemys picta), which display TSD. We detected a significant field repeatability for overstory vegetation cover around nests at oviposition, a trait that is correlated inversely with nest temperature during embryonic sex determination and positively with offspring sex ratio (percentage of male). Neither clustered nesting nor individual spatial repeatability in nest site choice caused this striking pattern. Instead, females consistently preferred nest environments with particular quantities of overstory vegetation cover. These findings confirm a crucial assumption of models concerning the microevolution and adaptive significance of TSD in reptiles. The results also indicate that this system may be able to evolve via long-term maternal nesting behaviour in response to skewed sex ratios caused by environmental perturbations, such as gradual long-term climate change.

Sex differences in optimal incubation temperatures in a scincid lizard species.

Most theoretical models for the evolution of temperature-dependent sex determination (TSD) rely upon differential fitness of male and female offspring incubated under different thermal regimes. However, there are few convincing data on this topic. We studied incubation effects in a lizard species (Bassiana duperreyi, Scincidae) with genotypic sex determination, so that we could separate effects due to incubation temperatures from those due to offspring gender. We incubated eggs under two different fluctuating-temperature regimes that simulated hot and cold natural nest-sites. The effects of our incubation treatments on phenotypes of the hatchling lizards (morphology and locomotor performance) differed between the sexes. Females emerging from eggs exposed to the "hot nest" treatment (diel cycling, 23-31°C) were larger, and ran faster, than did their sisters from the "cold nest" treatment (16-24°C). Males showed a smaller and less consistent phenotypic response than females. These incubation-induced responses were relatively stable during the first few weeks of life post-hatching, at least in captive lizards maintained under laboratory conditions. These kinds of sex differences in the phenotypic responses of hatchling reptiles to incubation conditions provide a plausible basis for the evolution of temperature-dependent sex determination in reptiles.