School of BioSciences - Theses
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ItemGenomically informed gene drive modelling( 2022)CRISPR/Cas gene drives are a focus of genetic biocontrol for pest species. They have the potential to radically affect pest species, by making them more manageable or by eradicating them. However, it is not yet fully understood how the elements of a gene drive interact to guide the progression of a gene drive. We explored how we can design gene drives that are safer, either by being temporally limiting or spatially limiting, through a modelling framework. Our modelling included a range of variables, with the addition of genomic information to infer the homing efficiency of the gene drive. We showed that there was no single variable that differentiated between the outcomes of a gene drive. Granted some variables were more influential in determining the outcome than others. The degree of dominance of the selection coefficient was shown to be strongly influential on the equilibrium outcome. While the interaction between conversion efficiency and resistance was shown to strongly influence the Temporary outcome. Furthermore, we showed that internal dynamics of a gene drive can be regulated by the variables of the gene drive. This provided insight into where effort should be directed in gene drive design to achieve the intended outcome of a gene drive, as well as controlling the progression to that outcome. The inclusion of genomic data in CRISPR gene drive modelling allowed for localisation of the gene drive due to genetic variation alone. Finding loci in the genome where there were allele frequencies differences allowed us to model gene drives that were highly efficient in the target population and poorly efficient in off-target populations. This conversion efficiency differential allowed for sustained gene drive localisation in spite of migration and selection. Population suppression was explored in our modelling to better understand how we could create sustained localised suppression. We showed sustained population suppression was possible through incomplete distortion of the sex ratio of the progeny. A deterministic gene drive model was developed to solve for equilibrium points for a range of migration rates and selection coefficients. These equilibria can be used as thresholds for gene drive design and monitoring. This work aims to further develop our understanding of how gene drives are likely to progress when released. We focussed on characterising which aspects of a gene drive were most important in determining both their progression and outcome. The inclusion of genetic information in our modelling revealed a new avenue that can be exploited to achieve gene drive localisation. This modelling work will aid in the design process of gene drives to increase our confidence that gene drives will work as intended.
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ItemThe evolutionary and functional characterisation of the ecdysteroid kinase-like (EcKL) gene family in insects( 2020)Many thousands of gene families across the tree of life still lack robust functional characterisation, and thousands more may be under-characterised, with additional unknown functions not represented in official annotations. Here, I aim to characterise the evolution and functions of the poorly characterised ecdysteroid kinase-like (EcKL) gene family, which has a peculiar taxonomic distribution and is largely known for containing an ecdysteroid 22-kinase gene in the silkworm, Bombyx mori. I hypothesised that EcKLs may also be responsible for insect-specific ‘detoxification-by-phosphorylation’, as well as ecdysteroid hormone metabolism. My first approach was to explore the evolution of the EcKLs in the genus Drosophila (Diptera: Drosophilidae), which contains the well-studied model insect Drosophila melanogaster. Drosophila EcKLs have evolutionary and transcriptional similarities to the cytochrome P450s, a classical detoxification family, and an integrative ‘detoxification score’, benchmarked against the known functions of P450 genes, predicted nearly half of D. melanogaster EcKLs are candidate detoxification genes. A targeted PheWAS approach in D. melanogaster also identified novel toxic stress phenotypes associated with genomic and transcriptomic variation in EcKL and P450 genes. These results suggest many Drosophila EcKLs function in detoxification, or at least have key functions in the metabolism of xenobiotics, and additionally identify a number of novel P450 detoxification candidate genes in D. melanogaster. I then broadened the phylogenomic analysis of EcKLs to a manually annotated dataset containing an additional 128 insect genomes and three other arthropod genomes, as well as a number of transcriptome assemblies. Phylogenetic inference suggested insect EcKLs can be grouped into 13 subfamilies that are differentially conserved between insect lineages, and order-specific analyses for Diptera, Lepidoptera and Hymenoptera revealed both highly conserved and highly variable EcKL clades within these taxa. Using phylogenetic comparative methods, EcKL gene family size was found to vary with detoxification-related traits, such as the sizes of classical detoxification gene families, insect diet, and two estimations of ‘detoxification breadth’ (DB), one qualitative and one quantitative. Additionally, the rate of EcKL duplication was found to be low in lineages with small DB—bees and tsetse flies. These results suggest the EcKL gene family functions in detoxification across insects. Building on my previous ‘detoxification score’ analysis, I used the powerful genetic toolkit in D. melanogaster and developmental toxicology assays to test the hypothesis that EcKL genes in the highly dynamic Dro5 clade are involved in the detoxification of selected plant and fungal toxins. Knockout or misexpression of Dro5 genes, particularly CG13659 (Dro5-7), modulated susceptibility to the methylxanthine alkaloid caffeine, and Dro5 knockout also increased susceptibility to kojic acid, a fungal secondary metabolite. These results validate my evolutionary and integrative analyses, and provide the first experimental evidence for the involvement of EcKLs in detoxification processes. Finally, I aimed to find genes encoding ecdysteroid kinases in D. melanogaster, focusing on Wallflower (Wall/CG13813) and Pinkman (pkm/CG1561), orthologs of a known ecdysteroid 22-kinase gene. Wall and pkm null mutant animals developed normally, but misexpression of Wall caused tissue-specific developmental defects, albeit not those consistent with inactivation of the main ecdysteroid hormones, ecdysone and 20-hydroxyecdysone. In addition, my hypothesis that Wall encodes an ecdysteroid 26-kinase was not supported by hypostasis experiments with a loss-of-function allele of the ecdysteroid 26-hydroxylase/carboxylase gene Cyp18a1. Combined with existing expression and regulatory data, these results suggest Wall encodes an ecdysteroid kinase with an unknown substrate, and hint at previously unknown complexity in ecdysteroid signalling and metabolism in D. melanogaster. Overall, this thesis provides a detailed exploration of the functions of the EcKL gene family in insects, showing that these genes comprise a novel detoxification gene family in multiple taxa, and that they may also contribute to understudied aspects of ecdysteroid metabolism in a model insect. This work also demonstrates the power and potential of integrating evolutionary, genomic, transcriptomic and experimental data when characterising genes of unknown function.
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ItemPhylogenomics, molecular evolution and extinction in the adaptive radiation of murine rodents( 2020)Adaptive radiation plays a significant role in the generation of biological diversity, and the advent of modern sequencing approaches has unlocked a new genomic perspective on this process. Genomic-scale data from the across the diversity of adaptive radiations can provide unprecedented resolution of the phylogenetic, biogeographic and molecular context of diversification. Murine rodents (Murinae: Rodentia) are a recent and rapid adaptive radiation that make up > 10% of mammal species. Murines have repeatedly colonised new geographic areas and island systems in the Eastern Hemisphere, frequently as a result of overwater transitions. Recurring adaptive radiation, ecological character displacement, and convergent evolution across Murinae make them an ideal model for studying adaptive radiation, especially in the Indo-Australian region. Within broader Murinae, the Hydromyini are a speciose Australo-Papuan radiation that diversified following an overwater colonisation from Sunda to Sahul ca. 8 Ma. Previous multilocus studies did not provide sufficient phylogenetic resolution of the rapid diversification of Hydromyini, and did not adequately sample taxa to reconstruct their complex biogeographic history. In addition to unresolved biogeography, the endemic Australian clade within Hydromyini has suffered the highest rate of recent mammalian extinction in the world. The rapid decline of Australian rodents is thought to be primarily the result of predation by feral cats, combined with other factors such as anthropogenic land clearing. There is little information about the pace of decline in eight species that went extinct on the Australian mainland in the last 150 years, and it is unclear whether these species had suffered longer term declines that predate the arrival of Europeans into Australia in 1788. To resolve these outstanding issues, I develop a novel exon capture approach for murine rodents. Firstly, I investigate the degree of congruent and conflicting phylogenomic signal in a rapid radiation, using genus-level relationships in the Hydromyini as a model example. My results show that in a number of cases, strong conflict is not reflected in branch support metrics obtained using either maximum likelihood or summary coalescent approaches. This result is significant, as it suggests that approaches commonly used to estimate support in phylogenomic data can fail to detect uncertainty in the face of underlying genealogical heterogeneity. Further leveraging this novel exon capture design, I generate a robust phylogenomic tree based on > 350 samples across the Australo-Papuan continent, including extant and recently extinct species in Hydromyini. With these data, I reconstruct the species-level evolutionary and biogeographic history of the Hydromyini across Sahul, recovering numerous examples of overwater colonisation between regions. Consistent with the geomorphological hypothesis that the New Guinea lowlands emerged after the orogeny of the Central Cordillera, I find evidence for increasing ecological opportunity in the Hydromyini from approximately 5 Ma. This first species-level phylogenomic study spanning the entire Sahul region provides a baseline example for future comparative studies that seek to reconstruct the biogeographic drivers of diversification in Sahul at a continental scale. Using exon capture and whole-exome sequencing data from extinct and extant species, I place recently extinct Australian rodents in a phylogenomic context for the first time. I recover no marked evidence of genetic erosion in five extinct species at the time of specimen collection, in comparison to extant species with present-day low allelic diversity. This indicates that the decline of recently extinct Australian rodents occurred extremely rapidly, and its onset likely did not predate European settlement. Additionally, my results taxonomically resurrect a species from extinction, Gould’s mouse (Pseudomys gouldii), which survived as a single island population in Shark Bay, Western Australia (currently classified as P. fieldi). Finally, I generate whole exome data from 38 species in the global radiation of Murinae to examine patterns of positive selection and convergent evolution. I uncovered pervasive positive selection across genes associated with diet, digestion and taste across Murinae, and increased rates of adaptive evolution in carnivores compared to omnivores. Limited evidence for molecular convergence in worm-eating specialists Paucidentomys and Rhynchomys suggests a role for developmental phenotypic control in this striking example of ecological convergence. Broadly, my results indicate that the pronounced ecological and phenotypic shifts that are hallmarks of adaptive radiations may also drive corresponding shifts in the pace and pattern of molecular evolution across the genome. Together, the work in this thesis is fundamental to the understanding of diversification, adaptation and extinction in the Australo-Papuan region, and provides an extensive genomic resource for future studies.
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ItemA systems approach to understanding allergy, asthma and childhood wheeze( 2019)In Australia, asthma is a common respiratory disease with a significant health burden. Our current understanding of the biological mechanisms behind asthma is incomplete. It is not clear what makes a person more susceptible compared to another, nor is it clear how determinants of asthma susceptibility interact to cause disease. Childhood wheeze does not necessarily progress to asthma, and asthma itself is a heterogeneous condition that encompasses many different phenotypes, each with potentially different biology. However, we suspect that, for most affected individuals, the origins of asthma arise in early childhood, as embodied by the “hygiene hypothesis”. Events like microbial and allergen exposure in early life, as well as frequency and severity of respiratory infections, may steer the child on a course towards asthma and disease. Early prediction of disease susceptibility or severity is important because it may permit early intervention in young children, which may then limit the progression of asthma or prevent it altogether. My research thesis had three general aims: 1. To uncover hidden subgroups or “clusters” of children who share similar trajectories of immune function and susceptibility to respiratory infection; and determine how these relate to asthma and other related phenotypes. 2. To describe microbial communities in the upper respiratory tract of infants, specifically distinct patterns of change or trajectories in the microbiome that emerge as the child ages; and to determine how these relate to respiratory health, asthma, and related phenotypes. 3. To identify novel genetic determinants of asthma and related phenotypes in early childhood (including immunorespiratory clusters and microbiome trajectories), and determine how these relate to each other. Through this research, I hope to shed light on the complexity that is asthma pathogenesis. In particular, it may explain how the determinants of asthma are similar or different between individuals. With my research, it may be possible to better characterise the interlocking events that lead from disruption of normal physiology to eventual disease. Future studies can focus on the origins of asthma in specific subpopulations, as well as potential treatment targets within each subgroup. The results of this research may open up the potential for developing therapeutic and preventative measures for asthma, as well as allow earlier intervention for infants at risk of developing asthma later in life.
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ItemClimate adaptation in Eucalyptus microcarpa (Grey Box) and implications for conservation( 2017)Restoration is an important component of conservation management, especially in highly modified landscapes. In the face of rapid environmental change, the mere presence of vegetation doesn’t necessarily equate to the long term sustainability of populations. Rather, there is a need to consider evolutionary potential in conservation planning, including restoration. This thesis investigates two key components of evolutionary potential pertinent to restoration – namely genetic diversity and climate adaptation – in an important restoration tree species in south-eastern Australia, Eucalyptus microcarpa. This thesis aims to understand how genetic diversity and local adaptation are distributed across the range of E. microcarpa and how this knowledge may help inform seed sourcing and enhance resilience of restoration plantings under climate change. To begin, I use a landscape genomic approach to explore genomic diversity in E. microcarpa and how diversity in small habitat remnants and revegetation (restored) sites compare to large remnants. This work found that small, habitat remnants and revegetation sites largely, but not completely captured patterns of genomic diversity across the landscape. Whilst overall genomic diversity was similar between site types, patterns of diversity across the genome varied between site types. These results suggest important genomic differences between site types that may influence future adaptive potential of revegetation sites and small habitat remnants. I then investigated adaptation to climate in E. microcarpa using multiple approaches. Firstly, using a landscape genomic approach, I found evidence of genomic climate adaptation in E. microcarpa. These results suggest climate adaptation to be a genome-wide phenomenon, involving many genes and genomic regions. Exploration of genomic changes that may be required to match projected climate change suggest adaptation to be via shifts in allele frequency from standing variation. In addition to suggesting a number of climate variables associated with adaptation in E. microcarpa, these results highlight the importance of genetic diversity and standing variation for maintaining adaptive potential. Utilising existing genetic resources for this species, I found evidence of heritable, genetic variation in growth and leaf traits of E. microcarpa growing in a provenance trial. Furthermore, significant trait variation between provenances and associations with climate variables suggest climate as a driver of adaptive differences. Finally, I combined the independent genomic and phenotypic analyses to provide stronger support for climate adaptation in E. microcarpa, including links between genomic variants and adaptive traits. Associations between traits and single nucleotide polymorphisms (SNPs) using putatively adaptive SNPs genotyped in provenance trial trees validated genomic results, suggesting some trait variation could be explained by these SNPs. Furthermore, links between all three sources of variation relevant to local adaptation – genotype, phenotype and climate – corroborated findings of the two independent analyses. This approach therefore provides greater support for adaptation to climate in E. microcarpa. Together these analyses address the current genetic state of restoration in E. microcarpa as well as the structure of genetic diversity and climate adaptation across its distribution. These results suggest adaptive differences within E. microcarpa that could be utilised to enhance evolutionary potential within restoration plantings.
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ItemThe evolution of pathogenicity and isolate variation in Talaromyces marneffei( 2017)The opportunistic fungal pathogen of humans, Talaromyces marneffei, is one of very few pathogens in an order of over a thousand species and the only species that has the capacity to switch between two morphologically distinct growth forms (known as dimorphism). Growth at 25°C results in a saprophytic multicellular, hyphal form while infectious growth in a host occurs as a uninucleate unicellular yeast that resides within phagocytic cells of the immune system. The intracellular niche of T. marneffei differs significantly from the niches of other Talaromycetes. The identification of the mechanisms by which T. marneffei can survive and grow in this intracellular niche is a major aim of this study. Comparisons of the genomes of three closely related non-dimorphic, non-pathogenic species with the T. marneffei genome identified unique features that contribute to niche specific growth and the ability to cause disease. Most significant of these were an overall reduction in genome size and gene number in T. marneffei with substantial gene losses in families responsible for environmental interaction. These and other findings strongly indicate that T. marneffei has adapted to an intracellular host niche distinct from its saprophytic relatives. Against this background of gene loss three gene families were identified that had been significantly expanded in T. marneffei. These expanded gene families showed putative extracellular and cell surface localisation and consisted of cell wall galactomannoproteins (mpl family), aspartyl proteases (pop family) and a family of small proteins with very little functional characterisation in any species (mib family). Genes in the pop, mpl and mib families were over-represented in subtelomeric regions, under positive selection, had copy number variation in T. marneffei isolates and many had high levels of repetitive adjacent sequences including several transposon families. In the host T. marneffei grows as an intracellular pathogen within phagocytes and as such extracellular proteins interact directly with the host. Therefore another aim of this study was to characterise these expanded gene families and their role in pathogenesis. Deletion studies in pop genes revealed roles in yeast cell formation during intracellular growth, while high variability in cell-to-cell protein production for two mib genes suggested a role in cell surface variation when interacting with the host. Understanding the type and degree of variation within the population of a fungal pathogen can reveal its population structure and potential to adapt to stressors such as antifungal compounds. Genome wide variation in the T. marneffei population had yet to be examined therefore an aim of this study was to characterise the degree and type of this variation. To this end several clinical and environmental isolates of T. marneffei were examined for variation in chromosomal structure, which is a common means of generating phenotypic variation in other fungi. While no obvious abnormalities were observed, gene copy number variation in subtelomeric regions was widespread and several strains showed specific small mutations with impacts in antifungal resistance and phenotypic instability. Overall this study has revealed the genomic and genetic changes within T. marneffei and between it and other Talaromycetes. Many of these changes help to explain its unique niche as an intracellular pathogen within an almost entirely non-pathogenic clade. This research also highlights specific genes and gene families with roles in this pathogenesis and identifies potential therapeutic targets and genes involved in host interactions for future investigation.
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ItemThe genomic basis of climate and host adaptation( 2017)Many species are currently threatened by the direct and indirect effects of anthropogenically driven climate change. The elevation of global temperatures and increase in variability in both temperature and precipitation pose a risk to biodiversity as species are pushed close to their thermal safety margins. Current predictions suggest a dramatic loss of species diversity and the contraction of geographical ranges of many species. Many ectothermic insects that cannot regulate their body temperature are likely to be threatened, particularly ecologically- restricted herbivorous insects that depend for on plants for food and that are often in phenological synchrony with their plant hosts. However, adaptive shifts in these species in response to host loss and climatic extremes may counter the effects of climate change to some extent. This highlights the importance of studying species-specific adaptation mechanisms including host interactions. This dissertation contributes to this overall aim by studying the genomic basis of climatic and host adaptation. I use Drosophila melanogaster as a model system at the intraspecific level, and Drosophila species from the repleta group as a model system for the comparative level. In assessing the genomic basis of host responses, I consider a much broader range of insect taxa. This dissertation begins with a study on the use of chromosome level sequencing of D. melanogaster populations from two ends of a thermal cline. I present genomic evidence for the role of the inversion 3R Payne in capturing alleles favourable to local climatic conditions in the non-inverted form, and therefore driving adaptation to climate change. The study further elucidates the impact of climatically important chromosomal inversions in driving higher linkage disequilibrium on the non-inverted form - potentially benefiting both karyotypes. In the second chapter, I develop a new pipeline, Orthonome, and tools for multi-species comparisons for prediction of orthologues and inparalogues with the highest accuracy and recall. Using Orthonome, I was able to identify a much greater level of conservation across Drosophilid lineages than earlier thought, amounting to nearly 33% better resolution than industry-accepted methods. I then use Orthonome in the third chapter to compare the genomes of 58 insect species – most of which are known to be agricultural pests. Testing across eight gene families, I present evidence for genomic patterns in only four gene families (P450s, CCEs, GSTs and ABCs) as being associated with polyphagy or particular host ranges. While three of them have been reported before, I find that ABC transporters present much stronger evidence than reflected in earlier studies, with feeding behaviour as well as host tissue displaying an effect on gene gain in more voracious pest species. Finally, in the last study, I use novel genomic data and evidence from the repleta group of drosophilids to carry out phylogenetically constrained analyses of genes potentially associated with host and thermal stress adaptation. My aim here is to find mutually exclusive evolutionary pathways to neofunctionalisation between stress tolerant cactophilic specialists and less tolerant generalists in the group. I also find a different adaptive response in the cactophilic species compared to the generalist species; these species show little lineage specific gene gain, suggesting an exception to current standing theories on neofunctionalisation for adaptation. I further discuss the applicability the species level and order level analyses for an overall detailed and systematic approach to identify the genomic basis of climatic and host adaptation.
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ItemComparative phylogeography and diversity of Australian Monsoonal Tropics lizards( 2016)Tropical savannah biomes cover ~20% of the world’s landmass, however the biodiversity encompassed within these environments and the underlying processes that have shaped it remain poorly understood. Recent increased research to address this knowledge gap have begun to reveal surprisingly high amounts of deep, geographically-structured diversity, much of which is cryptic or hidden within morphologically similar species complexes. These patterns are especially emphasized in vertebrate taxa which are intrinsically linked to rock escarpments and ranges that dissect the savannah woodlands and grasslands of many of these biomes, hinting at a role of heterogeneous topography in structuring diversity. The remote Australian Monsoonal Tropics (AMT) spanning the north of the Australian continent is a particularly vast, and relatively undisturbed, tropical savannah region. Recent increased surveys are revealing numerous new species and endemism hotspots, indicating we are only just beginning to uncover the true biodiversity levels within this biome. Not only is there a relative paucity of knowledge regarding the present diversity within this region, but there is also limited understanding of how this diversity came to be. Phylogeographic studies can assist us in establishing current patterns of diversity and their evolutionary significance within regions and biomes. Furthermore, by comparing and contrasting the patterns and timing of diversification within and between biomes for multiple ecologically diverse taxa, we can begin to elucidate the history of these biomes and the environmental processes that have shaped the diversity we observe today. In this dissertation I aimed to better assess and establish true patterns of biodiversity and endemism within the Kimberley region of the AMT (Western Australia), and to place these patterns within a broader continental context using intra- and inter-biome comparisons in related taxa. Using geckos as a model system I took a comparative phylogeographic approach, integrating advanced next-generation genetics and morphology to establish patterns and timing of diversification across ecologically variable taxa. Within all Kimberley taxa I studied, I uncovered high levels of cryptic diversity. Much of this diversity involves especially short-range endemic lineages concentrated in key regions typically with one or more of the following factors: highly mesic conditions, island or insular environments, and unique or complex geological formations. In recognising these areas I have provided evidence of novel biodiversity hotspots and emphasised the significance of others as representing important “refugia” within the Kimberley that allow persistence and facilitate divergence of lineages through harsh periods of environmental change. These findings indicate diversification patterns are shaped by complex interactions of climatic variation, topography, and species’ ecology, allowing inference of biogeographic history and a greater ability to predict impacts of future environmental change.