Reptiles exhibit marked diversity in sex-determining mechanisms. Many species exhibit
genotypic sex determination (GSD) with male heterogamety (XX females/XY males),
others have GSD with female heterogamety (ZW females/ZZ males), and still others
exhibit temperature-dependent sex determination (TSD). The distribution of these
mechanisms throughout the reptile phylogeny implies evolutionary lability in sex
determination, and in some lineages there has been a number of transitions between
GSD and TSD. Despite this diversity, GSD and TSD have traditionally been viewed as
mutually-exclusive mechanisms of sex determination in reptiles, since there is little
evidence for their co-occurrence. Considerable empirical and theoretical effort has been
directed towards understanding the adaptive significance of TSD in reptiles. In
comparison, there has been little focus on understanding how evolutionary transitions
between GSD and TSD occur at a genetic and mechanistic level. I addressed this
question by applying both empirical and theoretical approaches to investigate
interaction of genotypic and temperature influences in the sex determination of two
endemic species of Australian lizards.
The three-lined skink, Bassiana duperreyi, has XX/XY chromosomal sex
determination, yet a previous investigation reported a significant male bias in the sex
ratio of eggs incubated at low temperatures. To enable an explicit test for temperature induced
sex reversal in this species, a 185 bp Y chromosome marker was isolated by
Amplified Fragment Length Polymorphism (AFLP) analysis. The marker was
subsequently converted into a duplex PCR assay that co-amplified a 185 bp (or 92 bp)
Y chromosome fragment and a 356 bp fragment of the single-copy nuclear gene C-mos
(from both sexes) as a positive control. The accuracy of the PCR sex assay was tested
on 78 individuals for which sex reversal was not expected. PCR genotype and sex
phenotype were concordant for 96% of the animals. This is one of the very few sex tests
developed for a reptile, and the first report of Y chromosome sequence from a reptile.
The PCR assay was subsequently applied to genotype hatchlings from both cool
(16-7.5C) and warm (22-7.5C) cyclical incubation temperature treatments, and
identified sex reversal in 15% of genotypically female (XX) embryos (n=26) from the
cool treatment, but no sex reversal in eggs from the warmer treatment (n=35). Thus, low
incubation temperatures can over-ride genotypic sex determination in B. duperreyi,
indicating that GSD and TSD co-occur in this species.
The Central bearded dragon, Pogona vitticeps (Agamidae), has ZZ/ZW chromosomal
sex determination, and is a member of a lizard family in which GSD and TSD are both
widespread, indicating evolutionary lability in sex determination. AFLP analysis was
applied to isolate homologous Z and W chromosome-linked markers (71 bp and 72 bp,
respectively) from this species. The AFLP sequences were subsequently extended into
larger genomic fragments by a reiterated genome walking procedure, producing three
non-overlapping contigs of 1.7 kb, 2.2 kb and 4.5 kb. The latter two fragments were
verified as distinct, homologous Z/W chromosome fragments by PCR analyses. An
amplified 3 kb fragment of the 4.5 kb contig was physically mapped to metaphase
spreads, identifying the W microchromosome, and for the first time in this species, the
Z microchromosome. PCR analyses indicated the presence of homologous sequences in
other Australian agamid species, including both GSD and TSD species. The isolated
sequences should therefore prove useful as a comparative genomic tool for investigating
the genomic changes that have occurred in evolutionary transitions between sexdetermining
mechanisms in agamids, by enabling the identification of chromosomes in
TSD species that are homologous to the sex chromosomes of P. vitticeps. The isolated
sequences were further converted into a duplex DNA sex assay that co-amplified a 224
bp W chromosome fragment and a 963 bp positive control fragment in both sexes. This
PCR assay diagnosed chromosomal sex in three Pogona species, but was not effective
outside the genus.
Incubation treatment of P. vitticeps eggs revealed a strong and increasing female bias at
high constant temperatures (34-36C), but an unbiased sex ratio between 22-32C.
Hatchlings from three clutches split between 28C and 34 or 36C incubation treatments
were genotyped with the W chromosome AFLP marker. At 28C, the sex ratio was 1:1
but the high temperature treatments produced 2 males and 33 females. All but one of the
30 lizards (97%) incubated at 28C had concordant sex phenotype and genotype, but
only 18 of 35 animals (51%) from the high temperature treatment were concordant. All
discordant animals were genotypic males (ZZ) that developed as females. Thus,
temperature and genotypic influences can interact to determine sex in P. vitticeps.
These empirical findings for B. duperreyi and P. vitticeps were extended into a novel
theory for the evolution of sex-determining mechanisms in reptiles, working within the
framework that species with temperature-induced reversal of chromosomal sex
determination are a window to transitional stages of evolution between GSD and TSD.
A model was derived from the observation that in both lizards, an extreme of incubation
temperature causes sex reversal of the homogametic genotype. In this model, the
strength of a genetic regulatory signal for sex determination must exceed a threshold for
development of the homogametic sex to occur (male in Pogona, female in Bassiana).
The strength of this signal is also temperature-sensitive, so diminishes at extremes of
temperature. Simulation modelling demonstrated that increasing the relative magnitude
of the threshold for sexual development can cause evolutionary transitions between
GSD and TSD. Even more remarkably, decreasing the relative magnitude of the
threshold value causes an evolutionary transition between female and male
heterogametic GSD. Quantitative adjustment of a single model parameter (the threshold
value) thus charts a continuous evolutionary pathway between the three principal
mechanisms of sex determination in reptiles (XX/XY-ZZ/ZW-TSD), which were
previously considered to be qualitatively distinct mechanisms.
The experimental demonstration of temperature-induced reversal of chromosomal sex
determination in both B. duperreyi and P. vitticeps presents a challenge to the traditional
view that reptilian sex determination is strictly dichotomous (GSD or TSD), and
suggests instead that sex determination in reptiles consists of a continuum of systems of
interaction between genotypic and temperature influences. Simulation modelling
provided solid theoretical support for this proposition, demonstrating that transitions
along this continuum are effected simply through shifts in the mean population value for
the sex-determining threshold, without requiring substantial genotypic innovation. An
important implication of this theory is that transitions between XX/XY and ZZ/ZW
modes of GSD may retain the same sex chromosome pair, and the same primary sexdetermining
gene, in contrast to previous models for heterogametic transitions. A more
immediate implication of these findings is that many reptile species believed to have
strict TSD (in particular, lizards and crocodilians), may in fact have a sex-determining
system of GSD-TSD interaction, where there is an equilibrium between GSD and TSD
individuals within the population.
| Identifer | oai:union.ndltd.org:ADTP/211443 |
| Date | January 2008 |
| Creators | Quinn, Alexander E., n/a |
| Publisher | University of Canberra. Institute for Applied Ecology |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | ), Copyright Alexander E. Quinn |
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