Sex in vertebrates can be determined by genes on specific chromosomes (genotypic sex-determination, GSD), by environmental factors such as temperature (e.g., temperature-dependent sex determination, TSD) or by the combination of both. Reptiles, especially lizards, exhibit the greatest diversity in sex-determining mechanisms (SDM) among amniotes. This diversity suggests frequent transitions between sex-determining modes. Among the lizards, the Agamidae family (commonly known as agamids) is known to show diverse modes of reproduction as well as modes of sex determination mechanism, possibly even among congenerics. This family contains more than 500 species across Africa, Asia and Australia under six subfamilies and includes species in which both temperature and genes interact to determine sex. The multiple modes of sex determination in agamid lizards have evolved many times, suggesting multiple and independent evolutions of sex-determining modes within the animal kingdom. Most of the studies of sex determination in agamids have focused on the species under one subfamily from one continent, i.e., Australian species of the sub-family Amphibolurinae. Only little is known about the agamids from other subfamilies. As a result, the diversity and evolution of sex determination mechanisms remain unidentified among a significant group of agamid lizards, yet these have the potential to uncover novel sex determination mechanisms, including sex chromosomes. Filling in this knowledge gap would provide insight into the overall understanding of the phylogenetic relationship and evolutionary history of sex-determination mechanisms. In my thesis, I examined aspects of evolution and ecology of sex-determination across the family Agamidae with a combination of incubation experiments, cytogenetics and genomics. As part of this study, I conducted an extensive literature review on the background of the current knowledge of sex determination and sex chromosome in reptiles in the General Introduction chapter (chapter 1). I published a review as a first author (chapter 2) on lizards focussing on what makes this group unique among reptiles in terms of sex determination and sex chromosome evolution. The sex chromosomes in lizards are known for remarkable diversity in terms of morphology and degree of degeneration. The presence of TSD species together with such diversity implies multiple and independent origins of sex chromosomes and, in turn, the lability in sex determination mechanisms within lizard lineages. The review perceived that such lability in sex determination in lizards are largely due to following independent trajectories in sex chromosome evolution coupled with multiple lineage divergences within this group. This may have contributed to the diversified systems ranging from species with sex chromosomes (homomorphic, XY/ZW heteromorphic or multiple sex chromosomes) to species in which sex chromosomes are absent, that is, where there is a weak genetic determinant at most, involving autosomal genes acting differently on sex-determining pathways. I then examined the chromosomes of agamids from multiple lineages within the family Agamidae with the aim of identifying the sex determination mechanisms (i.e. sex-determining modes) across different subfamilies of Agamids lizards. My key findings are as follows: Chapter 3 has been published as a research article. In this chapter, I investigated the possible synteny of the sex chromosomes of P. vitticeps across agamid subfamilies. I used cross-species two-colour FISH with two BAC clones from the pseudo-autosomal regions of this species against representative species from all six subfamilies as well as two species of chameleons, the sister group to agamids. I found that one of the BAC sequences is conserved in macrochromosomes and the other in microchromosomes across the agamid lineages. However, within the subfamily Amphibolurinae, multiple chromosomal rearrangements were evident. No hybridisation signal was observed in chameleons for either BAC. Overall, my study showed lineage-specific evolution of sequences/syntenic blocks and successive rearrangements and revealed a complex history of sequences leading to their association with critical biological processes such as the evolution of sex chromosome and sex determination. In chapter 4, a published article, I identified a pair of microchromosomes as sex chromosomes in the Canberra grassland earless dragon Tympanocryptis lineata, a threatened grassland specialist species endemic to Australia. This brings to five the number of Australian agamid species (Pogona vitticeps, P. barbata, Diporiphora nobbi, Ctenophorus fordi and T. lineata)for which sex chromosomes have been identified. All five species have micro sex chromosomes and female heterogamety (ZZ/ZW). The study included further investigation of the sex determination mode in T. lineata (Chapter 5) through incubation experiments and identification of sex-linked loci (markers) through DArTseqTM, a genome complexity reduction and high throughput sequencing method. Incubation experiments conducted at five different constant temperatures (24°, 26°, 28°, 30° and 32 °C) confirmed this species to be a GSDspecies. DArTseq identified female-biased single nucleotide polymorphism (SNP) and presence-absence (PA) loci in T. lineata, supporting a female heterogamety (ZZ/ZW). Based on the results from Chapter 4 and 5, it can be concluded that T. lineata determine their sex through genotypic sex determination (GSD) with female heterogametic (ZZ/ZW) system but cannot, as yet, rule out higher incubation temperatures than those tested. In Chapter 6, I investigated the evidence of variation in sex determination modes in Oriental Garden lizards, Calotes versicolor, a species with a wide distribution range from Iran through south and southeast Asia. The taxon is considered to be a complex of cryptic species. C.versicolor samples were collected from Bangladesh and Thailand, at three locations in each country. A two-step study was undertaken. First, I used genomic (SNP data from methylation sensitive DArTseq analysis) and mitochondrial (Sanger sequencing) DNA data to test for population and phylogenetic structuring within this species complex and second, I used DArTseq (SNP) and SilicoDArT (PA) data to identify potential sex-linked markers in this species. I showed that the samples collected from different localities were genetically distinct, providing evidence that the taxon currently recognised as C. versicolor is a complex of cryptic species. My analyses of sex-linked markers revealed variation in sex determination modes among these different forms, implying that different sex determination mechanisms have evolved in closely related species and possibly even lineages within this species. The studies conducted under this thesis have expanded the knowledge of labile sex-determination mechanisms in reptiles, keeping agamid lizards as models. The studies on the sex determination in this group were previously concentrated mainly on the Australian clade of Amphibolurinae (subfamily), while this research went beyond this boundary and initiated an investigation including species from other subfamilies. The study identified the sex-determination mode and sex chromosomes in a threatened Australian agamid species, reported variation of sex-determination modes between populations and closely related species and explored chromosomal synteny among the subfamilies of the agamid lizards using P. vitticeps sex-chromosome BACs. The results presented here are still preliminary, and to fully understand the process of sex determination and sex chromosome evolution in the studied species, additional studies using advanced molecular cytogenetic and genomic techniques are needed, with particular priority to gain access to samples where the gonads have been dissected.
|Date of Award
|MD Tariq Ezaz (Supervisor), Arthur Georges (Supervisor), Dianne Gleeson (Supervisor) & Stephen Sarre (Supervisor)