Background: The development of male- or female-specific phenotypes in squamates is typically controlled by either temperature-dependent sex determination (TSD) or chromosome-based genetic sex determination (GSD). However, while sex determination is a major switch in individual phenotypic development, it is unknownhow evolutionary transitions between GSD and TSD might impact on the evolution of squamate phenotypes, particularly the fast-evolving and diverse genitalia. Here, we take the unique opportunity of studying the impact of both sex determination mechanisms on the embryological development of the central bearded dragon (Pogona vitticeps). This is possible because of the transitional sex determination system of this species, in which genetically male individuals reverse sex at high incubation temperatures. This can trigger the evolutionary transition of GSD to TSD in a single generation, making P. vitticeps an ideal model organism for comparing the effects of both sex determination processes in the same species.
Results: We conducted four incubation experiments on 265 P. vitticeps eggs, covering two temperature regimes ("normal" at 28 °C and "sex reversing" at 36 °C) and the two maternal sexual genotypes (concordant ZW females or sex-reversed ZZ females). From this, we provide the first detailed staging system for the species, with a focus on genital and limb development. This was augmented by a new sex chromosome identification methodology for P. vitticeps that is non-destructive to the embryo. We found a strong correlation between embryo age and embryo stage. Aside from faster growth in 36 °C treatments, body and external genital development was entirely unperturbed by temperature, sex reversal or maternal sexual genotype. Unexpectedly, all females developed hemipenes (the genital phenotype of adult male P. vitticeps), which regress close to hatching.
Conclusions: The tight correlation between embryo age and embryo stage allows the precise targeting of specific developmental periods in the emerging field of molecular research on P. vitticeps. The stability of genital development in all treatments suggests that the two sex-determining mechanisms have little impact on genital evolution, despite their known role in triggering genital development. Hemipenis retention in developing female P. vitticeps, together with frequent occurrences of hemipenis-like structures during development in other squamate species, raises the possibility of a bias towards hemipenis formation in the ancestral developmental programme for squamate genitalia.
Keywords: Embryonic development; Genitalia; Sex reversal; Squamates; Staging table.