Lizard Embryos Prioritize Posthatching Energy Reserves over Increased Hatchling Body Size during Development.

Embryonic development in oviparous organisms is fueled by maternally allocated yolk, and many organisms hatch before that energy store is used completely; the resultant leftover (residual) yolk is internalized and may support early posthatching life. However, embryos that use most, or all, of their yolk supply before hatching should hatch at a larger size than those that do not exhaust those energy reserves, which could also have benefits for posthatching growth and survival. To examine the trade-off between residual yolk and offspring size, we experimentally reduced yolk quantity at oviposition in lizard eggs (Amphibolurus muricatus) and then quantified offspring size and the amount of internalized residual yolk. This design enabled us to determine whether embryos (1) exhaust yolk supply during development (thereby maximizing neonatal size) or (2) reduce neonatal size by retaining yolk reserves at hatching. Our data support the latter scenario. Eggs from the yolk-reduced treatment produced smaller offspring with a proportion of residual yolk similar to that of offspring from unmanipulated eggs, suggesting that the fitness benefits of posthatching energy stores outweigh those of larger neonatal size.

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.

Factors influencing offspring traits in the oviparous multi-clutched lizard, Calotes versicolor (Agamidae).

The determinants of offspring size and number in the tropical oviparous multi-clutched lizard, Calotes versicolor, were examined using both univariate and multivariate (path) analyses. In C. versicolor maternal snout-vent length (SVL) and body condition influence clutch mass and clutch size but have no significant influence on offspring size. The positive effect of maternal SVL and body condition on offspring number is counterbalanced by a negative effect of breeding time on egg mass. In fact, breeding time directly influences the offspring body mass and condition through variation in the egg mass. There is a trade-off between offspring mass and condition with offspring number, and breeding time influences both. Offspring hatched from the eggs of early (May-June) or mid (July-August) breeding periods invariably show lower mass and condition than those hatched from the eggs of late breeding season (September-October). Yet, there is no variation in offspring SVL among early, mid and late clutches. Thus, in C. versicolor offspring SVL is optimized while body mass and condition are not optimized.

Can reptile embryos influence their own rates of heating and cooling?

Previous investigations have assumed that embryos lack the capacity of physiological thermoregulation until they are large enough for their own metabolic heat production to influence nest temperatures. Contrary to intuition, reptile embryos may be capable of physiological thermoregulation. In our experiments, egg-sized objects (dead or infertile eggs, water-filled balloons, glass jars) cooled down more rapidly than they heated up, whereas live snake eggs heated more rapidly than they cooled. In a nest with diel thermal fluctuations, that hysteresis could increase the embryo's effective incubation temperature. The mechanisms for controlling rates of thermal exchange are unclear, but may involve facultative adjustment of blood flow. Heart rates of snake embryos were higher during cooling than during heating, the opposite pattern to that seen in adult reptiles. Our data challenge the view of reptile eggs as thermally passive, and suggest that embryos of reptile species with large eggs can influence their own rates of heating and cooling.

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?

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.

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 egg's 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.

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

An individual's 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.

Are the phenotypic traits of hatchling lizards affected by maternal allocation of steroid hormones to the egg?

In lizards as in many other kinds of animals, strong maternal effects on the phenotypic traits of hatchlings are frequently reported. One plausible non-genetic mechanism that might produce such differences among clutches involves maternal allocation of steroid hormones. Lizard eggs often display considerable inter-clutch variation in the quantities of maternally allocated steroids, and exogenous application of such steroids has been reported to influence the phenotypic traits (especially, sex) of hatchlings. We examined correlations between naturally occurring yolk steroid levels and offspring traits in the scincid lizard Bassiana duperreyi, and also conducted experimental trials (exogenous application of testosterone or 17beta-oestradiol to eggs) to test for causal effects of hormones. Although exogenous hormones readily reversed sex of the hatchling lizards, no other phenotypic traits of the hatchlings (morphology, locomotor performance) were significantly correlated with naturally occurring levels of testosterone, dihydrotestosterone or estrogen, nor were these phenotypic traits significantly affected by exogenous application of hormones. Hence, our results do not support the hypothesis that reproducing female lizards manipulate the phenotypic traits of their offspring by differential allocation of steroid hormones.

Why do female lizards lay their eggs in communal nests?

1. In many reptile species, females oviposit communally (i.e. many clutches are laid within the same nest). This behaviour might result from constraint (scarcity of nest-sites offering suitable incubation conditions) or adaptation (direct fitness benefits accruing from the proximity of other eggs). 2. To test between these alternatives, we gathered field and laboratory data on montane scincid lizards Bassiana duperreyi from south-eastern Australia. Our data support the adaptationist hypothesis. 3. In the field, communal vs. solitary clutches were laid in similar sites, and the relative frequency of communal nesting was not predictable from nest-site availability. Thermal regimes for incubation did not differ between communal vs. solitary nests, nor between eggs at the core vs. periphery of a communal nest. In the laboratory, females selectively oviposited beside existing eggs rather than in otherwise identical potential nesting sites. 4. From cycling-temperature incubation in the laboratory, eggs incubated within a cluster of other eggs took up less water, but produced hatchlings that were larger and faster-running than were hatchlings from eggs incubated alone. 5. Hydric modifications of incubation conditions within a cluster of tightly packed eggs thus may provide a direct fitness benefit to communal oviposition.

Compensating for a bad start: catch-up growth in juvenile lizards (Amphibolurus muricatus, Agamidae).

In most natural environments, food availability varies unpredictably through space and time, and growth rates of individual organisms respond accordingly. However, growth rates are not necessarily a simple function of current nutritional conditions: growth rates can be affected by earlier nutritional experience as well as current circumstances. Thus, even a brief period of dietary restriction early in life might influence growth rates later on: either reducing them (if early experience sets subsequent rates, as in the "silver spoon" effect) or increasing them (if underfed individuals can compensate by growing more rapidly to cancel out the early decrement). Alternatively, later growth may be unaffected by earlier rates of growth. We experimentally manipulated food supply (and thus, growth rates) of hatchling lizards (Amphibolurus muricatus) for 1 month post-hatching, then maintained both high-food and low-food animals under identical nutritional conditions in outdoor enclosures for another 6 months. Low food abundance early in life significantly reduced juvenile growth, but these previously underfed animals exploited the subsequent (common garden) conditions to grow much faster than their larger (initially better-fed) siblings. Thus, the two groups were indistinguishable in body size at 6 months of age. Intriguingly, the compensatory growth occurred in winter, a period that is generally unsuitable for rapid growth in ectotherms.

Yolk partitioning in embryos of the lizard, Calotes versicolor: maximize body size or save energy for later use?

The hatchlings of Calotes versicolor and other congeners retain residual yolk for immediate post-hatching needs. Excision of 8% yolk (approximately equal residual yolk) from the eggs resulted in smaller hatchlings when compared to those emerging from sham operated eggs. However, hatchlings in both groups retained the same amount of residual yolk. The findings suggest that residual yolk in hatchlings of C. versicolor is an important part of the energy budget set aside by developing embryos and that the advantages of large hatchling size are counter balanced by selection for residual yolk.

Lowering body temperature induces embryonic diapause during prolonged egg retention in the lizard, Calotes versicolor.

The lizard Calotes versicolor delays oviposition of oviductal eggs for as long as 6 months or more under unfavourable conditions. During this period of prolonged egg retention, the growth of oviductal embryos is arrested at stage 34. The present study shows for the first time among reptiles that the "embryonic diapause" is manifested by the gravid females by lowering their body temperature ( T(b)) by 3-5 degrees C during the period of egg retention by mechanism(s) presently unknown.