Ovotestes suggest cryptic genetic influence in a reptile model for temperature-dependent sex determination

Sex determination and differentiation in reptiles is complex. Temperature-dependent sex determination (TSD), genetic sex determination (GSD) and the interaction of both environmental and genetic cues (sex reversal) can drive the development of sexual phenotypes. The jacky dragon (Amphibolurus muricatus) is an attractive model species for the study of gene–environment interactions because it displays a form of Type II TSD, where female-biased sex ratios are observed at extreme incubation temperatures and approximately 50 : 50 sex ratios occur at intermediate temperatures. This response to temperature has been proposed to occur due to underlying sex determining loci, the influence of which is overridden at extreme temperatures. Thus, sex reversal at extreme temperatures is predicted to produce the female-biased sex ratios observed in A. muricatus. The occurrence of ovotestes during development is a cellular marker of temperature sex reversal in a closely related species Pogona vitticeps. Here, we present the first developmental data for A. muricatus, and show that ovotestes occur at frequencies consistent with a mode of sex determination that is intermediate between GSD and TSD. This is the first evidence suggestive of underlying unidentified sex determining loci in a species that has long been used as a model for TSD.     

Effect of daily water treatment on hatchling sex ratios in a turtle with temperature-dependent sex determination

Some previous studies indicate that the local hydric environment may influence sex determination in turtles with temperature-dependent sex determination. In this study, the effect of a daily application of 0.77 mL of ddH(2)0 per egg using an incubation temperature of 29.1 degrees C was examined during the temperature-sensitive period for two consecutive nesting seasons. This regimen yielded sex ratios of 11.8 and 11.1% male in control groups not receiving water supplementation, whereas daily water treatments resulted in sex ratios of 86.7 and 45.7% male during the 2006 and 2007 nesting seasons, respectively. The results indicate that daily water treatments significantly influenced sex ratios (P<0.001). In addition to providing insight on the physiology of sex determination, these results could have implications for studies predicting sex ratios from nests on natural nesting beaches that are periodically exposed to rain.     

Population sex ratios under differing local climates in a reptile with environmental sex determination

Populations that experience different local climates, such as those along a latitudinal gradient, must match life history traits to local environmental conditions. In species with temperature-dependent sex determination, such as many reptiles, population sex ratio is strongly influenced by local climate, yet local climate differs substantially among populations in geographically-widespread species. We studied the painted turtle at three sites across the species’ geographic range to gain a mechanistic understanding of how sex ratios are produced under different local climates. We combined data on maternal nest-site choice, nest incubation temperature, and the resultant offspring sex ratio of populations across a climatic gradient, to demonstrate how geographic variation in behavior and physiology translates into sex ratios among populations of a widely-distributed species. We found that populations across the species’ geographic range match incubation conditions with local climatic conditions through population-specific adjustment of maternal nest-site choice. Incubation temperatures during the thermosensitive period were cooler and clutches were more male-biased in the south, with populations farther north having warmer incubation temperatures and more female-biased sex ratios, yet adult sex ratios were not strongly biased in any population. Most components of maternal nest-site choice varied latitudinally among populations, suggesting that the species may have a considerable repertoire for responding to climate change through adjustment of nest-site choice.     

Gonadal sex differentiation in Alligator mississippiensis,a species with temperature-dependent sex determination

Temperature of egg incubation determines sex in Alligator mississippiensis hatchlings. To define the timing and morphology of sexual differentiation, alligator gonads were examined histologically and ultrastructurally throughout embryogenesis. At the male-producing temperature (33° C), the onset of testis differentiation occurred in most embryos during developmental stages 21–22, when a number of somatic cells in the medulla of the gonad became enlarged, forming presumptive Sertoli cells. Some enlarged somatic cells were also observed at the female-producing temperature (30° C) during gonadogenesis, but they were less widespread than at 33° C. Ovarian differentiation at 30° C began slighlty later, during stage 22–23, and was characterised by proliferation of germs cells in the cortex of the gonad. Testis formation in alligators may depend upon presumptive Sertoli cells differentiating prior to a critical event in embryogenesis, such as germ cell proliferation and meiosis. If follows that ovary formation occurs if this requirement is not met, as at lower incubation temperatures.     

Environmental sex determination in a reptile varies seasonally and with yolk hormones

Most hypotheses that have been put forward in order to explain the persistence of environmental sex determination (ESD) in reptiles assume a relatively fixed association of sex with temperature-induced phenotype and no maternal influence on offspring sex. Here we demonstrate the association of maternally derived yolk hormone levels with the offspring sex ratio and describe two new aspects of temperature-dependent sex determination (TSD), i.e. seasonal variation in both thermal response and yolk steroid levels. Eggs from painted turtles (Chrysemys picta) were incubated at 28 degrees C. The hatchling sex ratio at 28 degrees C (i.e. the phenotypic reaction norm for sex at 28 degrees C) shifted seasonally from ca. 72% male to ca. 76% female. Yolk oestradiol (E2) increased seasonally while testosterone (T) decreased. The proportion of males in a clutch decreased as E2 levels increased and the E2:T ratio increased. These new findings are discussed in relation to heritability and adaptive explanations for the persistence of ESD in reptiles. Maternally derived yolk hormones may provide a mechanism for the seasonal shift in the sex ratio which in turn may help explain the persistence of ESD in reptiles. They may also explain those clutches of other reptiles with TSD that fail to yield only males at maximally masculinizing conditions.     

Segregating variation for temperature-dependent sex determination in a lizard

Temperature-dependent sex determination (TSD) was first reported in 1966 in an African lizard. It has since been shown that TSD occurs in some fish, several lizards, tuataras, numerous turtles and all crocodilians. Extreme temperatures can also cause sex reversal in several amphibians and lizards with genotypic sex determination. Research in TSD species indicates that estrogen signaling is important for ovary development and that orthologs of mammalian genes have a function in gonad differentiation. Nevertheless, the mechanism that actually transduces temperature into a biological signal for ovary versus testis development is not known in any species. Classical genetics could be used to identify the loci underlying TSD, but only if there is segregating variation for TSD. Here, we use the ‘animal model'' to analyze inheritance of sexual phenotype in a 13-generation pedigree of captive leopard geckos, Eublepharis macularius, a TSD reptile. We directly show genetic variance and genotype-by-temperature interactions for sex determination. Additive genetic variation was significant at a temperature that produces a female-biased sex ratio (30 °C), but not at a temperature that produces a male-biased sex ratio (32.5 °C). Conversely, dominance variance was significant at the male-biased temperature (32.5 °C), but not at the female-biased temperature (30 °C). Non-genetic maternal effects on sex determination were negligible in comparison with additive genetic variance, dominance variance and the primary effect of temperature. These data show for the first time that there is segregating variation for TSD in a reptile and consequently that a quantitative trait locus analysis would be practicable for identifying the genes underlying TSD.     

Disentangling sex allocation in a viviparous reptile with temperature‐dependent sex determination: a multifactorial approach

Females are predicted to alter sex allocation when ecological, physiological and behavioural variables have different consequences on the fitness of male and female offspring. Traditionally, tests of sex allocation have examined single causative factors, often ignoring possible interactions between multiple factors. Here, we used a multifactorial approach to examine sex allocation in the viviparous skink, Niveoscincus ocellatus. We integrated a 16‐year observational field study with a manipulative laboratory experiment to explore whether the effects of the maternal thermal environment interact with the resources available to females for reproduction to affect sex allocation decisions. We found strong effects of temperature on sex allocation in the field, with females born in warm conditions and males in cold conditions; however, this was not replicated in the laboratory. In contrast, we found no effect of female resource availability on sex allocation, either independently, or in interaction with temperature. These results corresponded with an overall lack of an effect of resource availability on any of the life history traits that we predicted would mediate the benefits of differential sex allocation in this system, suggesting that selection for sex allocation in response to resource availability may be relatively weak. Combined, these results suggest that temperature may be the predominant factor driving sex allocation in this system.     

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.     

Mutual information reveals variation in temperature-dependent sex determination in response to environmental fluctuation, lifespan and selection

Quantifying the degree to which sex determination depends on the environment can yield insight into the evolution, ecological dynamics, and functional aspects of sex determination. In temperature-dependent sex determination (TSD), theory often predicts a complete dependence of sex on temperature, with a switch-like reaction norm. However, empirical data suggest more shallow relationships between sex and temperature. Here, we demonstrate the usefulness of an index, mutual information (MI), to reflect the degree of temperature dependence in sex. MI depends on both the shape of a reaction norm and the natural temperature variation, thus providing a measure of TSD that is ecologically dependent. We demonstrate that increased lifespan and decreased environmental fluctuation predict reaction norms with high MI (switch-like). However, mutation and weaker selection on sex-specific performance reduce average MI in a population, suggesting that mutation-selection balance can resolve some of the conflict between theoretical predictions of individual-based optimality and population-based empirical results. The MI index allows clear comparison of TSD across life histories and habitats and reveals functional similarities between reaction norms that may appear different. The model provides testable predictions for TSD across populations, namely that MI should increase with lifespan and decrease with historical environmental fluctuations.     

Molecular players involved in temperature-dependent sex determination and sex differentiation in Teleost fish

The molecular mechanisms that underlie sex determination and differentiation are conserved and diversified. In fish species, temperature-dependent sex determination and differentiation seem to be ubiquitous and molecular players involved in these mechanisms may be conserved. Although how the ambient temperature transduces signals to the undifferentiated gonads remains to be elucidated, the genes downstream in the sex differentiation pathway are shared between sex-determining mechanisms. In this paper, we review recent advances on the molecular players that participate in the sex determination and differentiation in fish species, by putting emphasis on temperature-dependent sex determination and differentiation, which include temperature-dependent sex determination and genetic sex determination plus temperature effects. Application of temperature-dependent sex differentiation in farmed fish and the consequences of temperature-induced sex reversal are discussed.     

Inheritance of nesting behaviour across natural environmental variation in a turtle with temperature-dependent sex determination

Nesting behaviour is critical for reproductive success in oviparous organisms with no parental care. In organisms where sex is determined by incubation temperature, nesting behaviour may be a prime target of selection in response to unbalanced sex ratios. To produce an evolutionary change in response to sex-ratio selection, components of nesting behaviour must be heritable. We estimated the field heritability of two key components of nesting behaviour in a population of painted turtles (Chrysemys picta) with temperature-dependent sex determination by applying the ‘animal model’ to a pedigree reconstructed from genotype data. We obtained estimates of low to non-detectable heritability using repeated records across all environments. We then determined environment-specific heritability by grouping records with similar temperatures for the winter preceding the nesting season, a variable known to be highly associated with our two traits of interest, nest vegetation cover and Julian date of nesting. The heritability estimates of nest vegetation cover and Julian date of nesting were qualitatively highest and significant, or nearly so, after hot winters. Additive genetic variance for these traits was not detectable after cold winters. Our analysis suggests that the potential for evolutionary change of nesting behaviour may be dependent on the thermal conditions of the preceding winter, a season that is predicted to be especially subject to climate change.