How rapidly can maternal behavior affecting primary sex ratio evolve in a reptile with environmental sex determination?

Theoretical models identify maternal behavior as critical for the maintenance and evolution of sex ratios in organisms with environmental sex determination (ESD). Maternal choice of nest site is generally thought to respond more rapidly to sex ratio selection than environmental sensitivity of offspring sex (threshold temperatures) in reptiles with temperature-dependent sex determination (TSD, a form of ESD). However, knowledge of the evolutionary potential for either of these traits in a field setting is limited. I developed a simulation model using local climate data and observed levels of phenotypic variation for nest-site choice and threshold temperatures in painted turtles (Chrysemys picta) with TSD. Both nest-site choice and threshold temperatures, and hence sex ratios, evolved slowly to simulated climate change scenarios. In contrast to expectations from previous models, nest-site choice evolved more slowly than threshold temperatures because of large climatic effects on nest temperatures and indirect selection on maternally expressed traits. A variant of the model, assuming inheritance of nest-site choice through natal imprinting, demonstrated that natal imprinting inhibited adaptive responses in female nest-site choice to climate change. These results predict that females have relatively low potential to adaptively adjust sex ratios through nest-site choice.     

Nest-site selection in two eublepharid gecko species with temperature-dependent sex determination and one with genotypic sex determination

At present, most turtles, all crocodilians, and several lizards are known to have temperature-dependent sex determination (TSD). Due to the dependence of sex determination on incubation temperature, the long-term survival of TSD species may be jeopardized by global climate changes. The current study was designed to assess the degree to which this concern is justified by examining nest-site selection in two species of Pattern II TSD geckos (Eublepharis macularius and Hemitheconyx caudicinctus) and comparing these preferences with those of a species with genotypic sex determination (GSD) (Coleonyx mitratus). Temperature preferences for nest sites were found to be both species-specific and female-specific. While H. caudicinctus females selected a mean nest-site temperature (32.4°) very close to the upper pivotal temperature (32°C) for the species, E. macularius females selected a mean nest-site temperature (28.7°C) well below this species' lower pivotal temperature (30.5°C). Thus, the resultant sex ratios are expected to differ between these two TSD species. Additionally, nest-site temperatures for the GSD species were significantly more variable (SE=+0.37) than were temperatures for either of the TSD species (E. macularius SE=±0.10; H. caudicinctus SE =+ 0.17), diereby further demonstrating temperature preferences within the TSD species.     

Novel evolutionary pathways of sex‐determining mechanisms

Evolutionary transitions between sex‐determining mechanisms (SDMs) are an enigma. Among vertebrates, individual sex (male or female) is primarily determined by either genes (genotypic sex determination, GSD) or embryonic incubation temperature (temperature‐dependent sex determination, TSD), and these mechanisms have undergone repeated evolutionary transitions. Despite this evolutionary lability, transitions from GSD (i.e. from male heterogamety, XX/XY, or female heterogamety, ZZ/ZW) to TSD are an evolutionary conundrum, as they appear to require crossing a fitness valley arising from the production of genotypes with reduced viability owing to being homogametic for degenerated sex chromosomes (YY or WW individuals). Moreover, it is unclear whether alternative (e.g. mixed) forms of sex determination can persist across evolutionary time. It has previously been suggested that transitions would be easy if temperature‐dependent sex reversal (e.g. XX male or XY female) was asymmetrical, occurring only in the homogametic sex. However, only recently has a mechanistic model of sex determination emerged that may allow such asymmetrical sex reversal. We demonstrate that selection for TSD in a realistic sex‐determining system can readily drive evolutionary transitions from GSD to TSD that do not require the production of YY or WW individuals. In XX/XY systems, sex reversal (female to male) occurs in a portion of the XX individuals only, leading to the loss of the Y allele (or chromosome) from the population as XX individuals mate with each other. The outcome is a population of XX individuals whose sex is determined by incubation temperature (TSD). Moreover, our model reveals a novel evolutionarily stable state representing a mixed‐mechanism system that has not been revealed by previous approaches. This study solves two long‐standing puzzles of the evolution of sex‐determining mechanisms by illuminating the evolutionary pathways and endpoints.     

Exploring the evolution of environmental sex determination, especially in reptiles

Environmental sex determination has been documented in a variety of organisms for many decades and the adaptive significance of this unusual sex-determining mechanism has been clarified empirically in most cases. In contrast, temperature-dependent sex determination (TSD) in amniote vertebrates, first noted 40 years ago in a lizard, has defied a general satisfactory evolutionary explanation despite considerable research effort. After briefly reviewing relevant theory and prior empirical work, we draw attention to recent comparative analyses that illuminate the evolutionary history of TSD in amniote vertebrates and point to clear avenues for future research on this challenging topic. To that end, we then highlight the latest empirical findings in lizards and turtles, as well as promising experimental results from a model organism, that portend an exciting future of progress in finally elucidating the evolutionary cause(s) and significance of TSD.     

Evolution of the gene network underlying gonadogenesis in turtles with temperature-dependent and genotypic sex determination

The evolution of sex determination has long fascinated biologists, as it has paramount consequences for the evolution of a multitude of traits, from sex allocation to speciation and extinction. Explaining the diversity of sex-determining systems found in vertebrates (genotypic or GSD and temperature-dependent or TSD) requires a comprehensive and integrative examination from both a functional and an evolutionary perspective. Particularly revealing is the examination of the gene network that regulates gonadogenesis. Here, I review some advances in this field and propose some additional hypotheses about the composition of the gene network underlying sexual development, the functional links among some of its elements and their evolution in turtles. I focus on several pending questions about: (1) What renders TSD systems thermo-sensitive? (2) Is there one developmentally conserved or multiple TSD mechanisms? (3) Have evolutionarily derived GSD species lost all ancestral thermal-sensitivity? New data are presented on embryonic expression of Dax1 (the dosage-sensitive sex-reversal adrenal hypoplasia congenital on the X chromosome gene in the turtles Chrysemys picta (TSD) and Apalone mutica (GSD). No differential Dax1 expression was detected in C. picta at any of the stages examined, consistent with reports on two other TSD turtles and alligators. Notably, significantly higher Dax1 expression was found at 30°C than at 25°C at stage 15 in A. mutica (GSD), likely caused by Wt1's identical expression pattern previously reported. Because Sf1 is an immediate downstream target of Dax1 and its expression is not affected by temperature, it is proposed that Sf1 renders Dax1's differential signal ineffective to induce biased sex ratios in A. mutica, as previously proposed for Wt1's thermosensitive expression. Thus, it is hypothesized that Sf1 plays a major role in the lack of response of sex ratio to temperature of A. mutica, and may function as a sex-determining gene in this GSD species. These and previous data permit formulating several mechanistic hypotheses: (1) the postulation of Wt1 as a candidate thermal master switch alone, or in combination with Sf1, in the TSD turtle C. picta; (2) the proposition of Sf1 as a sex-determining gene in the GSD turtle A. mutica; and (3) the hypothesis that differing patterns of gene expression among TSD taxa reflect multiple traits from a developmental perspective. Moreover, the recent finding of relic differential Wt1 expression in A. mutica and the results for Dax1 in this species provide empirical evidence that GSD taxa can harbor thermal sensitivity at the level of gene expression, potentially co-optable during TSD evolution.     

Evolutionary transitions between mechanisms of sex determination in vertebrates

Sex in many organisms is a dichotomous phenotype--individuals are either male or female. The molecular pathways underlying sex determination are governed by the genetic contribution of parents to the zygote, the environment in which the zygote develops or interaction of the two, depending on the species. Systems in which multiple interacting influences or a continuously varying influence (such as temperature) determines a dichotomous outcome have at least one threshold. We show that when sex is viewed as a threshold trait, evolution in that threshold can permit novel transitions between genotypic and temperature-dependent sex determination (TSD) and remarkably, between male (XX/XY) and female (ZZ/ZW) heterogamety. Transitions are possible without substantive genotypic innovation of novel sex-determining mutations or transpositions, so that the master sex gene and sex chromosome pair can be retained in ZW-XY transitions. We also show that evolution in the threshold can explain all observed patterns in vertebrate TSD, when coupled with evolution in embryonic survivorship limits.     

Environmental sex determination in reptiles: ecology, evolution, and experimental design

Sex-determining mechanisms in reptiles can be divided into two convenient classifications: genotypic (GSD) and environmental (ESD). While a number of types of GSD have been identified in a wide variety of reptilian taxa, the expression of ESD in the form of temperature-dependent sex determination (TSD) in three of the five major reptilian lineages has drawn considerable attention to this area of research. Increasing interest in sex-determining mechanisms in reptiles has resulted in many data, but much of this information is scattered throughout the literature and consequently difficult to interpret. It is known, however, that distinct sex chromosomes are absent in the tuatara and crocodilians, rare in amphisbaenians (worm lizards) and turtles, and common in lizards and snakes (but less than 20% of all species of living reptiles have been karyotyped). With less than 2 percent of all reptilian species examined, TSD apparently is absent in the tuatara, amphisbaenians and snakes; rare in lizards, frequent in turtles, and ubiquitous in crocodilians. Despite considerable inter- and intraspecific variation in the threshold temperature (temperature producing a 1:1 sex ratio) of gonadal sex determination, this variation cannot confidently be assigned a genetic basis owing to uncontrolled environmental factors or to differences in experimental protocol among studies. Laboratory studies have identified the critical period of development during which gonadal sex determination occurs for at least a dozen species. There are striking similarities in this period among the major taxa with TSD. Examination of TSD in the field indicates that sex ratios of hatchlings are affected by location of the nests, because some nests produce both sexes whereas the majority produce only one sex. Still, more information is needed on how TSD operates under natural conditions in order to fully understand its ecological and conservation implications. TSD may be the ancestral sex-determining condition in reptiles, but this result remains tentative. Physiological investigations of TSD have clarified the roles of steroid hormones, various enzymes, and H-Y antigen in sexual differentiation, whereas molecular studies have identified several plausible candidates for sex-determining genes in species with TSD. This area of research promises to elucidate the mechanism of TSD in reptiles and will have obvious implications for understanding the basis of sex determination in other vertebrates. Experimental and comparative investigations of the potential adaptive significance of TSD appear equally promising, although much work remains to be performed. The distribution of TSD within and among the major reptilian lineages may be related to the life span of individuals of a species and to the biogeography of these species.(ABSTRACT TRUNCATED AT 400 WORDS)     

Different optimal offspring sizes for sons versus daughters may favor the evolution of temperature-dependent sex determination in viviparous lizards

Temperature-dependent sex determination (TSD) has evolved independently in at least two lineages of viviparous Australian scincid lizards, but its adaptive significance remains unclear. We studied a montane lizard species (Eulamprus heatwolei) with TSD. Our data suggest that mothers can modify the body sizes of their offspring by selecting specific thermal regimes during pregnancy (mothers with higher and more stable temperatures produced smaller offspring), but cannot influence sons versus daughters differentially in this way. A field mark-recapture study shows that optimal offspring size differs between the sexes: larger body size at birth enhanced the survival of sons but reduced the survival of daughters. Thus, a pregnant female can optimize the fitness of either her sons or her daughters (via yolk allocation and thermoregulation), but cannot simultaneously optimize both. One evolutionary solution to reduce this fitness cost is to modify the sex-determining mechanism so that a single litter consists entirely of either sons or daughters; TSD provides such a mechanism. Previous work has implicated a sex difference in optimal offspring size as a selective force for TSD in turtles. Hence, opposing fitness determinants of sons and daughters may have favored evolutionary transitions from genetic sex determination to TSD in both oviparous turtles and viviparous lizards.     

Heritable Variation for Sex Ratio under Environmental Sex Determination in the Common Snapping Turtle (Chelydra Serpentina)

The magnitude of quantitative genetic variation for primary sex ratio was measured in families extracted from a natural population of the common snapping turtle (Chelydra serpentina), which possesses temperature-dependent sex determination (TSD). Eggs were incubated at three temperatures that produced mixed sex ratios. This experimental design provided estimates of the heritability of sex ratio in multiple environments and a test of the hypothesis that genotype x environment (G x E) interactions may be maintaining genetic variation for sex ratio in this population of C. serpentina. Substantial quantitative genetic variation for primary sex ratio was detected in all experimental treatments. These results in conjunction with the occurrence of TSD in this species provide support for three critical assumptions of Fisher's theory for the microevolution of sex ratio. There were statistically significant effects of family and incubation temperature on sex ratio, but no significant interaction was observed. Estimates of the genetic correlations of sex ratio across environments were highly positive and essentially indistinguishable from + 1. These latter two findings suggest that G x E interaction is not the mechanism maintaining genetic variation for sex ratio in this system. Finally, although substantial heritable variation exists for primary sex ratio of C. serpentina under constant temperatures, estimates of the effective heritability of primary sex ratio in nature are approximately an order of magnitude smaller. Small effective heritability and a long generation time in C. serpentina imply that evolution of sex ratios would be slow even in response to strong selection by, among other potential agents, any rapid and/or substantial shifts in local temperatures, including those produced by changes in the global climate.     

Fitness effects of the timing of hatching may drive the evolution of temperature-dependent sex determination in short-lived lizards

In several species of short-lived Australian agamid lizards, an individual’s sex is determined by the nest temperatures encountered during incubation. The adaptive significance of such systems remains unclear. Here, we explore the hypothesis that (1) the optimal timing of hatching differs between the sexes, and thus (2) temperature-dependent sex determination (TSD) enhances maternal and offspring fitness by generating seasonal shifts in offspring sex ratios. Our model predicts that TSD can indeed enhance maternal fitness returns in short-lived lizards if (1) male–male competition is intense, thus reducing mating success of newly-matured males (but not females), and (2) the nesting season is prolonged, such that seasonal effects become significant. Available data on the distribution of TSD in Australian agamid lizards broadly support these predictions. Because both the level of male–male competition and the length of nesting season can vary at small spatial and temporal scales, selective forces on sex-determining mechanisms also should vary. Hence, our model predicts extensive small-scale (intraspecific) variation in sex-determining systems within agamid lizards, as well as among species.     

Unexpected resilience of species with temperature-dependent sex determination at the Cretaceous-Palaeogene boundary

It has been suggested that climate change at the Cretaceous-Palaeogene (K-Pg) boundary, initiated by a bolide impact or volcanic eruptions, caused species with temperature-dependent sex determination (TSD), including dinosaurs, to go extinct because of a skewed sex ratio towards all males. To test this hypothesis, the sex-determining mechanisms (SDMs) of Cretaceous tetrapods of the Hell Creek Formation (Montana, USA) were inferred using parsimony optimizations of SDMs on a tree, including Hell Creek species and their extant relatives. Although the SDMs of non-avian dinosaurs could not be inferred, we were able to determine the SDMs of 62 species; 46 had genotypic sex determination (GSD) and 16 had TSD. The TSD hypothesis for extinctions performed poorly, predicting between 32 and 34 per cent of survivals and extinctions. Most surprisingly, of the 16 species with TSD, 14 of them survived into the Early Palaeocene. In contrast, 61 per cent of species with GSD went extinct. Possible explanations include minimal climate change at the K-Pg, or if climate change did occur, TSD species that survived had egg-laying behaviour that prevented the skewing of sex ratios, or had a sex ratio skewed towards female rather than male preponderance. Application of molecular clocks may allow the SDMs of non-avian dinosaurs to be inferred, which would be an important test of the pattern discovered here.     

The evolution of sex ratios and sex-determining systems

Sex determination is a fundamental process governed by diverse mechanisms. Sex ratio selection is commonly implicated in the evolution of sex-determining systems, although formal models are rare. Here, we argue that, although sex ratio selection can induce shifts in sex determination, genomic conflicts between parents and offspring can explain why single-factor systems (e.g. XY/XX or ZW/ZZ) are common even in species that experience selection for biased sex ratios. Importantly, evolutionary shifts in sex determination do not always result in the biased production of sons and daughters sensu sex ratio theory. Thus, equal sex ratios might be an emergent character of sex-determining systems even when biased sex ratios are favored by selection.     

Climate change and temperature-dependent sex determination: can individual plasticity in nesting phenology prevent extreme sex ratios?

Under temperature-dependent sex determination (TSD), temperatures experienced by embryos during development determine the sex of the offspring. Consequently, populations of organisms with TSD have the potential to be strongly impacted by climatic warming that could bias offspring sex ratio, a fundamental demographic parameter involved in population dynamics. Moreover, many taxa with TSD are imperiled, so research on this phenomenon, particularly long-term field study, has assumed great urgency. Recently, turtles with TSD have joined the diverse list of taxa that have demonstrated population-level changes in breeding phenology in response to recent climate change. This raises the possibility that any adverse impacts of climate change on populations may be alleviated by individual plasticity in nesting phenology. Here, we examine data from a long-term study on a population of painted turtles (Chrysemys picta) to determine whether changes in phenology are due to individual plasticity and whether individual plasticity in the timing of nesting has the capacity to offset the sex ratio effects of a rise in climatic temperature. We find that individual females show plasticity in the date of first nesting each year, and that this plasticity depends on the climate from the previous winter. First nesting date is not repeatable within individuals, suggesting that it would not respond to selection. Sex ratios of hatchlings within a nest declined nonsignificantly over the nesting season. However, small increases in summer temperature had a much stronger effect on nest sex ratios than did laying nests earlier in the season. For this and other reasons, it seems unlikely that individual plasticity in the timing of nesting will offset the effects of climate change on sex ratios in this population, and we hypothesize that this conclusion applies to other populations with TSD.     

Climate change overruns resilience conferred by temperature‐dependent sex determination in sea turtles and threatens their survival

Temperature‐dependent sex determination (TSD) is the predominant form of environmental sex determination (ESD) in reptiles, but the adaptive significance of TSD in this group remains unclear. Additionally, the viability of species with TSD may be compromised as climate gets warmer. We simulated population responses in a turtle with TSD to increasing nest temperatures and compared the results to those of a virtual population with genotypic sex determination (GSD) and fixed sex ratios. Then, we assessed the effectiveness of TSD as a mechanism to maintain populations under climate change scenarios. TSD populations were more resilient to increased nest temperatures and mitigated the negative effects of high temperatures by increasing production of female offspring and therefore, future fecundity. That buffered the negative effect of temperature on the population growth. TSD provides an evolutionary advantage to sea turtles. However, this mechanism was only effective over a range of temperatures and will become inefficient as temperatures rise to levels projected by current climate change models. Projected global warming threatens survival of sea turtles, and the IPCC high gas concentration scenario may result in extirpation of the studied population in 50 years.     

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.     

Do operational sex ratios influence sex allocation in viviparous lizards with temperature‐dependent sex determination?

Under certain environmental situations, selection may favour the ability of females to adjust the sex ratio of their offspring. Two recent studies have suggested that viviparous scincid lizards can modify the sex ratio of the offspring they produce in response to the operational sex ratio (OSR). Both of the species in question belong to genera that have also recently been shown to exhibit temperature-dependent sex determination (TSD). Here we test whether pregnant montane water skinks (Eulamprus tympanum) utilise TSD to select offspring sex in response to population wide imbalances in the OSR, by means of active thermoregulation. We use a combination of laboratory and field-based experiments, and conduct the first field-based test of this hypothesis by maintaining females in outdoor enclosures of varying OSR treatments throughout pregnancy. Although maternal body temperature during pregnancy was influenced by OSR, the variation in temperature was not great enough to affect litter sex ratios or any other phenotypic traits of the offspring.     

Thermal and fitness-related consequences of nest location in Painted Turtles (Chrysemys picta)

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.     

Temperature-Dependent Sex Determination in Sea Turtles in the Context of Climate Change: Uncovering the Adaptive Significance

The adaptive significance of temperature-dependent sex determination (TSD) in reptiles remains unknown decades after TSD was first identified in this group. Concurrently, there is growing concern about the effect that rising temperatures may have on species with TSD, potentially producing extremely biased sex ratios or offspring of only one sex. The current state-of the-art in TSD research on sea turtles is reviewed here and, against current paradigm, it is proposed that TSD provides an advantage under warming climates. By means of coadaptation between early survival and sex ratios, sea turtles are able to maintain populations. When offspring survival declines at high temperatures, the sex that increases future fecundity (females) is produced, increasing resilience to climate warming. TSD could have helped reptiles to survive mass extinctions in the past via this model. Flaws in research on sex determination in sea turtles are also identified and it is suggested that the development of new techniques will revolutionize the field.