Daniel A. Warner

The adaptive significance of temperature-dependent sex determination in a reptile.

Understanding the mechanisms that determine an individual’s sex remains a primary challenge for evolutionary biology. Chromosome-based systems (genotypic sex determination) that generate roughly equal numbers of sons and daughters accord with theory, but the adaptive significance of environmental sex determination (that is, when embryonic environmental conditions determine offspring sex, ESD) is a major unsolved problem. Theoretical models predict that selection should favour ESD over genotypic sex determination when the developmental environment differentially influences male versus female fitness (that is, the Charnov–Bull model), but empirical evidence for this hypothesis remains elusive in amniote vertebrates—the clade in which ESD is most prevalent. Here we provide the first substantial empirical support for this model by showing that incubation temperatures influence reproductive success of males differently than that of females in a short-lived lizard (Amphibolurus muricatus, Agamidae) with temperature-dependent sex determination. We incubated eggs at a variety of temperatures, and de-confounded sex and incubation temperature by using hormonal manipulations to embryos. We then raised lizards in field enclosures and quantified their lifetime reproductive success. Incubation temperature affected reproductive success differently in males versus females in exactly the way predicted by theory: the fitness of each sex was maximized by the incubation temperature that produces that sex. Our results provide unequivocal empirical support for the Charnov–Bull model for the adaptive significance of temperature-dependent sex determination in amniote vertebrates.

Maternal nutrition affects reproductive output and sex allocation in a lizard with environmental sex determination

Life-history traits such as offspring size, number and sex ratio are affected by maternal feeding rates in many kinds of animals, but the consequences of variation in maternal diet quality (rather than quantity) are poorly understood. We manipulated dietary quality of reproducing female lizards (Amphibolurus muricatus; Agamidae), a species with temperature-dependent sex determination, to examine strategies of reproductive allocation. Females maintained on a poor-quality diet produced fewer clutches but massively (twofold) larger eggs with lower concentrations of yolk testosterone than did conspecific females given a high-quality diet. Although all eggs were incubated at the same temperature, and yolk steroid hormone levels were not correlated with offspring sex, the nutrient-deprived females produced highly male-biased sex ratios among their offspring. These responses to maternal nutrition generate a link between sex and offspring size, in a direction likely to enhance maternal fitness if large body size enhances reproductive success more in sons than in daughters (as seems plausible, given the mating system of this species). Overall, our results show that sex determination in these animals is more complex, and responsive to a wider range of environmental cues, than that suggested by the classification of ‘environmental sex determination’.

Maternal nest-site choice in a lizard with temperature-dependent sex determination

In oviparous species, mothers can optimize the incubation environments of their offspring by selecting nest sites with conditions conducive to embryonic development. Hence, maternal nest-site selection can enhance egg survival and offspring fitness. However, the challenge to a nesting female is complex because of trade-offs among relevant parameters (e.g. hotter nests may be drier) and seasonal shifts in ambient conditions. Moreover, the influence of nest conditions on offspring sex ratios adds another level of complexity for species with environmental sex determination. To elucidate these issues, we need field data on nesting behaviour that incorporate the multiple influences that operate under natural conditions. We radiotracked gravid jacky dragons, Amphibolurus muricatus, in open forest habitats of southeastern Australia to document the lizards criteria for nest-site choice. Females selected sites with lower than average canopy cover, resulting in relatively warm nests throughout the prolonged nesting season. Although soil moisture levels decreased over this period and were strongly correlated with mean temperatures, female lizards were able to locate nesting sites that provided consistently moist (and increasingly warm) incubation conditions as the season progressed. Such sites apparently became more difficult to find later in the season, resulting in a higher incidence of test holes prior to nest excavation. Seasonal shifts in diel maximum temperatures also pushed the daily time of digging activity into the evening. Because jacky dragons have temperature-dependent sex determination, the seasonal shift in mean incubation temperatures of natural nests would generate seasonal shifts in offspring sex ratios in a direction likely to enhance maternal fitness.

Lizards combine stored energy and recently acquired nutrients flexibly to fuel reproduction

1. Energy allocation strategies for reproduction are viewed typically as a continuum between reliance on income (recently acquired energy) vs. capital (stored reserves) for fuelling reproduction. Because ectothermy facilitates long-term energy storage and often involves low feeding rates, traditional views suggest that many ectotherms rely heavily on stored reserves for egg production. 2. We explored the temporal relationship between energy intake and expenditure in a multi-clutching lizard (Amphibolurus muricatus) by evaluating the effect of maternal nutrition on reproductive output and by contrasting delta(13)C measurements of the maternal diet and endogenous energy stores with that of the eggs produced. 3. Our experiment revealed that females utilize both endogenous energy stores and recently acquired food to fuel reproduction; this pattern did not shift seasonally from first to second clutches produced. Importantly, however, egg lipid was derived primarily from capital, whereas egg protein was derived about equally from both income and capital. 4. Overall, these results suggest that the energy allocation strategy used for reproduction differs among egg components, and that the use of recently acquired energy for reproduction may be more widespread in ectotherms than thought previously.

WINDOWS OF EMBRYONIC SEXUAL LABILITY IN TWO LIZARD SPECIES WITH ENVIRONMENTAL SEX DETERMINATION

Temperature-dependent sex determination (TSD) occurs in all major reptile lineages, but the selective forces and physiological mechanisms that link sex to incubation temperature may differ among and within those groups. Different models for TSD evolution make different predictions about when offspring sex will respond to environmental cues. Although TSD has evolved in several lizard lineages, there is less detailed information on these taxa than in turtles and crocodilians with TSD. We incubated eggs of an agamid lizard (Amphibolurus muricatus) and a scincid lizard (Bassiana duperreyi), two species with TSD. Rather than manipulate incubation temperature to identify periods of sexual lability (as in most previous studies of this topic), we topically applied the aromatase inhibitor fadrozole to eggs at a variety of times through the incubation period. Fadrozole application sex-reversed the resultant hatchlings if applied from the time of oviposition until at least 60% of the way through incubation. In all of the TSD lizard species studied so far, offspring sex is determined either while the eggs are held inside the mothers body or soon after oviposition, providing substantial maternal control over incubation temperatures at this critical period. Hence, the hypothesis that TSD evolves because it enables offspring sex to be matched to conditions that are unpredictable at the time of laying is less likely to apply to squamates than to turtles, sphenodontians, and (especially) crocodiles, in which the period of sexual lability is delayed until long after oviposition.

Reproducing lizards modify sex allocation in response to operational sex ratios

Sex-allocation theory suggests that selection may favour maternal skewing of offspring sex ratios if the fitness return from producing a son differs from that for producing a daughter. The operational sex ratio (OSR) may provide information about this potential fitness differential. Previous studies have reached conflicting conclusions about whether or not OSR influences sex allocation in viviparous lizards. Our experimental trials with oviparous lizards (Amphibolurus muricatus) showed that OSR influenced offspring sex ratios, but in a direction opposite to that predicted by theory: females kept in male-biased enclosures overproduced sons rather than daughters (i.e. overproduced the more abundant sex). This response may enhance fitness if local OSRs predict survival probabilities of offspring of each sex, rather than the intensity of sexual competition.

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.