Is there sex-specific DNA in the tuatara, a reptile with temperature-dependent sex determination?: a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Ecology at Massey University
It is widely viewed that there is a dichotomy of sex-determining mechanisms within the reptiles: species either exhibit genotypic sex determination or temperature-dependent sex determination (TSD). However, very few species have been examined for both modes. Although it is often considered that the two mechanisms are mutually exclusive, there is evidence that there may be a weak genetic sex-determining mechanism in species in which the primary sex-determining mode is temperature-dependent sex determination. This infers that some TSD individuals may be sex-reversed; that is, their sexual genotype is discordant with their sexual phenotype. This hypothesis of an underlying genotypic system may also be linked to the question of the evolution of sex-determination within the reptiles. The discovery of sex-specific DNA within a TSD reptile could suggest that genotypic sex determination is ancestral and TSD has evolved many times over within independent reptile lineages. This study tested the hypothesis that there is a genetic component to sex determination in TSD species. This was accomplished by searching for sex-specific DNA in the tuatara, a reptile with temperature-dependent sex determination, using two different molecular genetic techniques. The major undertaking of the experimental programme was the completion of a comprehensive minisatellite DNA profiling survey. This incorporated 14 restriction enzymes and five different polycore DNA probes; in total, 66 different probe/enzyme combinations were tested for tuatara genomic DNA. None of the DNA profiles revealed sex-specific fragments. Furthermore, a significant difference in mean fragment numbers for males and females was not detected for any of the probe/enzyme combinations. In addition, a RAPD analysis was conducted in a search for a molecular sex marker in the tuatara. A total of 27 random-sequence oligonucleotide primers were used to successfully amplify anonymous products from the genomic DNA of male and female tuatara. Again, no sex-specific fragments were detected. Thus, evidence of sex-specific genetic differences in the tuatara was not found. This result fails to refute the null hypothesis that there are underlying sexual genotypes in the tuatara. This finding may reflect the absence of genetic sex differences in the tuatara. Alternatively, it might also be the result of accidental inclusion of sex-reversed individuals within the analyses, a situation which could have obscured the sex-specific nature of any sex-linked fragments. It would appear that the key to solving the question of sex-specific DNA within TSD reptiles such as the tuatara lies with the problem of ensuring sex-reversed individuals are excluded from molecular analysis.