Rajkumar S. Radder

Genetic evidence for co-occurrence of chromosomal and thermal sex-determining systems in a lizard.

An individuals sex depends upon its genes (genotypic sex determination or GSD) in birds and mammals, but reptiles are more complex: some species have GSD whereas in others, nest temperatures determine offspring sex (temperature-dependent sex determination). Previous studies suggested that montane scincid lizards (Bassiana duperreyi, Scincidae) possess both of these systems simultaneously: offspring sex is determined by heteromorphic sex chromosomes (XX–XY system) in most natural nests, but sex ratio shifts suggest that temperatures override chromosomal sex in cool nests to generate phenotypically male offspring even from XX eggs. We now provide direct evidence that incubation temperatures can sex-reverse genotypically female offspring, using a DNA sex marker. Application of exogenous hormone to eggs also can sex-reverse offspring (oestradiol application produces XY as well as XX females). In conjunction with recent work on a distantly related lizard taxon, our study challenges the notion of a fundamental dichotomy between genetic and thermally determined sex determination, and hence the validity of current classification schemes for sex-determining systems in reptiles.

Corticosterone Exposure during Embryonic Development Affects Offspring Growth and Sex Ratios in Opposing Directions in Two Lizard Species with Environmental Sex Determination

Stress experienced by a reproducing female can substantially affect the morphology, behavior, and physiology (and hence fitness) of her offspring. In addition, recent studies demonstrate that stress hormones (corticosterone) influence sex determination of embryos. To explore these issues, we manipulated corticosterone levels in eggs of two Australian lizard species (Amphibolurus muricatus and Bassiana duperreyi) that exhibit temperature‐dependent sex determination (TSD). Elevated corticosterone levels during embryonic development affected body size, growth rates, and sex ratios of the resultant offspring, but the direction and magnitude of these effects differed between the species. Corticosterone enhanced growth rates of hatchling B. duperreyi but inhibited growth of A. muricatus. Eggs with elevated levels of corticosterone produced more daughters in A. muricatus and more sons in B. duperreyi. The sex‐ratio effect in A. muricatus may have been due to sex‐specific embryonic mortality, but it may represent a direct effect on sex determination in B. duperreyi (because embryonic mortality was not affected by corticosterone manipulation in this species). These results demonstrate the complexity of proximate mechanisms for sex determination among reptiles with TSD and illustrate the potential role of corticosterone in sex‐determining systems.

Determinants of incubation period: do reptilian embryos hatch after a fixed total number of heart beats?

SUMMARY The eggs of birds typically hatch after a fixed (but lineage-specific) cumulative number of heart beats since the initiation of incubation. Is the same true for non-avian reptiles, despite wide intraspecific variation in incubation period generated by variable nest temperatures? Non-invasive monitoring of embryo heart beat rates in one turtle species (Pelodiscus sinensis) and two lizards (Bassiana duperreyi and Takydromus septentrionalis) show that the total number of heart beats during embryogenesis is relatively constant over a wide range of warm incubation conditions. However, incubation at low temperatures increases the total number of heart beats required to complete embryogenesis, because the embryo spends much of its time at temperatures that require maintenance functions but that do not allow embryonic growth or differentiation. Thus, cool-incubated embryos allocate additional metabolic effort to maintenance costs. Under warm conditions, total number of heart beats thus predicts incubation period in non-avian reptiles as well as in birds (the total number of heart beats are also similar); however, under the colder nest conditions often experienced by non-avian reptiles, maintenance costs add significantly to total embryonic metabolic expenditure.

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.

Offspring sex in a lizard depends on egg size

Current paradigms may substantially underestimate the complexity of reptilian sex determination. In previous work, we have shown that the sex of a hatchling lizard (Bassiana duperreyi, Scincidae) does not depend entirely on its genes (XX versus XY sex chromosomes); instead, low nest temperatures can override genotype to produce XX as well as XY males. Our experimental studies now add a third mechanism to this list: sex determination via yolk allocation to the egg. Within each clutch, the eggs that produce daughters are larger than those that produce sons. If (and only if) eggs are incubated at low temperatures, removing yolk from a newly laid egg turns the offspring into a male. Adding yolk from a larger (but not smaller) egg turns the recipient eggs offspring into a female. Remarkably, then, offspring sex in this species is the end result of an interaction between three mechanisms: sex chromosomes, nest temperatures, and yolk allocation.

Maternally derived egg yolk steroid hormones and sex determination: Review of a paradox in reptiles

During the past decade, maternally derived steroid hormones in the egg yolk of oviparous vertebrates have been the focus of attention for their possible role in sex determination and hence, information on the consequences of maternal egg yolk steroids on sex determination has accumulated rapidly in reptiles and birds. Until recently, the observations were dominated by the idea that yolk steroids of maternal origin play an important role in sex determination of oviparous vertebrates. However, more recent studies have cast significant doubt on the above conclusion. These studies suggest instead that steroids may be present in the yolk simply as the byproduct of passive uptake during yolk formation or observed correlations might reflect embryonically produced rather than maternally derived steroids. Thus, the objective of the present review is (i) to provide an overview of such paradoxical observations on the role of maternal yolk steroids in sex determination of reptiles, (ii) to identify and provide brief explanations for the observed paradoxical results, and (iii) to provide some future research directions.

Offspring Sex Is Not Related to Maternal Allocation of Yolk Steroids in the Lizard Bassiana duperreyi (Scincidae)

The eggs of birds and reptiles contain detectable levels of several steroid hormones, and experimental application of such steroids can reverse genetically determined sex of the offspring. However, any causal influence of maternally derived yolk steroids on sex determination in birds and reptiles remains controversial. We measured yolk hormones (dihydrotestosterone, testosterone, and 17 β‐estradiol) in newly laid eggs of the montane scincid lizard Bassiana duperreyi. This species is well suited to such an analysis because (1) offspring sex is influenced by incubation temperatures and egg size as well as by sex chromosomes, suggesting that yolk hormones might somehow be involved in the complex pathways of sex determination, and (2) experimental application of either estradiol or fadrozole to such eggs strongly influences offspring sex. We obtained yolk by biopsy, before incubating the eggs at a temperature that produces a 50:50 sex ratio. Yolk steroid levels varied over a threefold range between eggs from different clutches, but there were no significant differences in yolk steroids, or in relative composition of steroids, between eggs destined to become male versus female. Further, yolk steroid concentrations were not significantly related to egg size. Thus, yolk steroid hormones do not appear to play a critical role in sex determination for B. duperreyi.

Maternal Size Determines Clutch Mass, whereas Breeding Timing Influences Clutch and Egg Sizes in the Tropical Lizard, Calotes versicolor (Agamidae)

Abstract We studied the correlations between body size parameters (SVL and body condition) and clutch parameters (size and mass) as well as between clutch size and egg parameters (mass, volume, length, and width) in a population of Calotes versicolor, a multiclutched lizard that breeds from May to October in southern India. We also analyzed the relationships between early, mid, and late clutches among lizards of different SVL groups. Clutch size and SVL did not vary among years. Clutch size and clutch mass were positively correlated with body size. Egg mass and volume were negatively correlated with clutch size. Clutch size was larger in early and midbreeders than in late breeders. Interestingly, the eggs of late clutches were larger than those of early breeders. A trade-off between clutch size and egg volume was always obvious. The width of eggs did not vary with SVL or clutch timing, perhaps because the width of the pelvic aperture remained virtually constant after maturity. Thus, variation in egg volume in C. versicolor is because of variation in egg length. A wide range of variation in egg volume even among lizards with similar SVL suggests that there is no optimization of egg volume in this species. In contrast, the clutch mass of early, mid, and late clutches did not vary among lizards of comparable SVL. Apparently, SVL acts as a prime factor determining clutch mass in C. versicolor, and the latter is optimized by natural selection.

Thermally induced torpor in fullterm lizard embryos synchronizes hatching with ambient conditions

Eggs inside an underground nest have limited access to information about above-ground conditions that might affect the survival of emerging hatchlings. Our measurements of heart rates of embryos inside the intact eggs of montane lizards (Bassiana duperreyi, Scincidae) show that low temperatures induce torpor in fullterm embryos, but do not do so during earlier embryogenesis or later, post-hatching. Because above-ground conditions affect soil temperatures, this stage-dependent torpor effectively restricts hatching to periods of high ambient temperatures above ground. Torpor thus can function not only to synchronize activity with suitable environmental conditions during post-hatching life (as reported for many species), but also can occur in embryos, to synchronize hatching with above-ground conditions that facilitate successful emergence from the nest.

Isolation and development of a molecular sex marker for Bassiana duperreyi, a lizard with XX/XY sex chromosomes and temperature-induced sex reversal

Sex determination in the endemic Australian lizard Bassiana duperreyi (Scincidae) is influenced by sex chromosomes and incubation temperature, challenging the traditional dichotomy in reptilian sex determination. Analysis of those interactions requires sex chromosome markers to identify temperature-induced sex reversal. Here, we report the isolation of Y chromosome DNA sequence from B. duperreyi using amplified fragment length polymorphism PCR, the conversion of that sequence to a single-locus assay, and its combination with a single-copy nuclear gene (C-mos) to form a duplex PCR test for chromosomal sex. The accuracy of the assay was tested on an independent panel of individuals with known phenotypic sex. When used on offspring from field nests, our test identified the likely occurrence of a low rate of natural sex reversal in this species. This work represents the first report of Y chromosome sequence from a reptile and one of the few reptile sex tests.