Dragon sex: boys will be girls

The Australian central bearded dragon Pogona vitticeps, a favourite of amateur herpetologists, has a reproductive trick that might have appealed to a mythic female firedrake just a breath away from extinction. Just heat your eggs above 34°C and turn some or all of the male embryos into females.

Like birds, P. vitticeps has a ZW sex-determination system that, all things being equable, produces a 50:50 ratio of male to female offspring. But in torrid times, incubation temperatures between 33 and 37 degrees can override the as yet-unidentified Z-chromosome gene that determines maleness, skewing sex ratios increasingly towards females until, at 36°C, all hatchlings are female.

In the April 20, 2007, issue of Science, University of Canberra molecular ecologist Associate Professor Stephen Sarre, his UC colleagues Professor Arthur Georges and PhD student Alexander Quinn, the Australian National University's Dr Tariq Ezaz and internationally renowned sex-determination expert Professor Jenny Graves, reported temperature-induced sex reversal in P. vitticeps embryos under laboratory conditions.

But they have also found a lone, phenotypically female but karyotypically male (ZZ) central bearded dragon, in the Australian arid zone, confirming that temperature-induced sex reversal can indeed occur in nature.

The Canberra researchers chose P. vitticeps as their model species because it breeds readily in the captivity and because it was known to exhibit genotypic sex determination (GSD). But they are also interested in two sister species of Amphiboluris bearded dragons.

A. norrisi and A. muricatus exhibit GSD and Temperature-dependent Sex Determination (TSD) respectively. Sarre says genetic comparisons of two species may yield a potential entry point into the sex-determination pathway.

In A. muricatus, incubation temperature is the key determinant of sex ratios - at both higher and lower temperatures, the sex ratio skews towards females. Sarre believes that, in the GSD species P.vitticeps, the "maleness" gene - or genes - determine sex at incubation temperatures up to about 32°C, but is overridden by temperatures beyond 32°C. When the heat is on, boys will be girls.

Could the interaction of two modes of sex determination in P.vitticeps confer a survival advantage through the manipulation of sex ratios in Australia's unpredictable climate?

"It's a difficult thing to demonstrate, especially when you've got a long-lived species, because it suggests the females are making judgments about which is going to be the advantageous sex some years down the track," Sarre says. "It would be more likely in a short-lived species.

"Finding a sex-reversed individual in nature throws a spanner in the works, because it shows that some natural P.vitticeps nesting sites have the potential to alter the frequency of sex chromosomes, as well as changing the phenotypic sex ratio."

X, Y, Z

In all birds and some reptiles - particularly lizards - females are the chromosomal "odd couple" with a lone Z chromosome, pigeon-paired with a W chromosome. Males have a ZZ karyotype. This system differs from the XY sex-determination system of eutherian and marsupial mammals, where males are the heterogametic sex, and carry a male-determining gene on their truncated Y chromosome. In XX embryos, development defaults to the female pathway.

All crocodilians - crocodiles, alligators, caimans and gavials - have TSD; all snakes have ZW sex chromosomes, while turtles and lizards have plumbed the full range of possibilities: some species have ZW, others use a mammal-like XY system, while many others have temperature-determined sex determination.

A rare few, like isolated populations of the WA gecko Heteronotia binoei, have even resorted to parthenogenetic procreation, producing female clones of themselves - possibly a consequence of dipping into the gene pool of a closely related species, consigning their male conspecifics to local oblivion, according to Sarre.

In the late 1990s, Sarre worked on temperature-determined sex determination in Sphenodon punctatus, one of New Zealand's two species of tuatara, a "living fossil" of uncertain affinity to lizards and snakes. In the chilly maritime environment of several offshore islands, tuatara embryos normally incubate at temperatures of 20-21°C. At a maximum of 25°C - the minimum incubation temperature for dragons - tuatara sex ratios skew towards female.

He returned to Australia in 2001, enticed by the unrivalled richness of the Australian reptile fauna, and the variety of their sex-determination systems, which had clearly evolved independently on multiple occasions in different taxa.

Sarre reasons that, if natural selection has found essentially the same solutions in both unrelated and closely related taxa, all reptiles must share a highly conserved suite of sex-determining genes and hormones. Both genetic and environmental mechanisms may be superimposed on this underlying system early in embryonic development.

Although some reptiles have a mammal-like XY sex-determination system, and presumably a "maleness" gene, like the mammalian SRY, no SRY homologue has been found in lizards.

"Obviously, some genetic switch other than SRY is operating in reptiles, and that's what we're trying to find," he says.

"We want to understand how sex is primarily determined in reptiles, and how the relationship between temperature and genes work."

---PB--- Micro-chromosomes

There may be multiple points in the developmental cascade where environmental influences intervene to reverse sex - the ultimate target is activation of the aromatase gene, which converts testosterone and androstenedione to estrone and estradiol, directing development into the female pathway.

This year, after a painstaking search, Sarre and his colleagues identified tiny Z and W sex chromosomes - micro-chromosomes, in fact - in P. vitticeps, which has genetic sex determination.

By screening amplified restriction length polymorphisms (AFLPs), they identified a 72 base-pair, female-specific marker on the W chromosome. It differs by only a single base from its two Z homologues, but distinguishes genotypically female (ZW) embryos from genotypically male (ZZ) embryos that have changed sex because of high incubation temperatures.

In their Science paper, Sarre's team notes that for many reptiles with temperature sex determination, the male-to-female switch occurs at both ends of the temperature scale - a balanced sex ratio occurs only in a "Goldilocks zone" between 22 and 34 degrees.

They were unable to determine if all P. vitticeps embryos develop as females at incubation temperatures below 22°C, because no embryos survived.

They believe a dosage mechanism, involving a Z-linked gene, or genes, determines sex. With two active copies of the unknown gene, a ZZ karyotype producing twice the dose of a gene product may repress female development.

The ZW karyotype generates in a female embryo, due to inactivation of the Z-linked gene or its protein - which may act alone, or as a component of a transcription complex.

Sarre and his colleagues doubt that the W chromosome harbours a female equivalent of the mammalian SRY gene that initiates female development but aim to investigate that possibility through the application of genomic approaches.

They propose that when incubated at high or low temperatures, both maleness genes in ZZ embryos are progressively deactivated, and development switches to the female pathway - the "default" pathway, as in the mammalian XY system.

"The big question is how such transitions occur - and how such a diversity of sex-determining modes evolved in this one group of organisms," he says.

Sarre's team has bought a bacterial artificial chromosome (BAC) library to hunt for candidate maleness genes in the regions of the Z chromosome flanking the marker sequence on the Z chromosome.

In a 2004 review article in BioEssays, Sarre and colleagues observed that many reptile researchers consider genetic and environmental sex-determination mechanisms to be fundamentally different, and mutually exclusive.

They argued that genetic and environmental sex determination in reptiles should be seen as a continuum of states between species that rely primarily on genetic sex determination, and those in which sex is determined by environment/temperature.

The continuum would include species like P. vitticeps, in which the two mechanisms coexist and interact in some measure, determined by environmental influences, to bring about sex determination.