School of BioSciences - Theses
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ItemSystematics and biogeography of Spyridium with a focus on Spyridium parvifolium and its hybrids( 2022)Spyridium is a genus of c. 45 species endemic to south-western and south-eastern Australia, with a disjunct distribution across the Nullarbor Plain and Bass Strait. The genus also includes several morphologically distinct phrase name taxa. Spyridium parvifolium is a widespread and morphologically variable shrub from south-eastern Australia. Several varieties and forms of this species have been recognised, but there is disagreement on the accepted taxonomy between Australian states. Spyridium parvifolium is known to hybridise with S. daltonii in the Grampians and is thought to hybridise with S. vexilliferum in locations where these taxa co-occur in western Victoria and south-eastern South Australia. The aim of this research project was to develop a comprehensive molecular systematic understanding of Spyridium, and S. parvifolium and its hybrids, to inform the treatment of Rhamnaceae in the Flora of Australia (Kellermann et al. 2022-). The objectives were to investigate: the species circumscription and biogeographic history of Spyridium, the infraspecific taxa and phylogeographic patterns of S. parvifolium and introgression associated with this species (i.e. S. xramosissimum and S. parvifolium x S. vexilliferum). Entire chloroplast genomes (c. 160k base pairs) and the nuclear ribosomal array (18S–5.8S–26S; c. 6k base pairs) were analysed using both Bayesian and Maximum Likelihood phylogenetic methods. In total sequences from 230 samples were analysed across these phylogenies, including representatives of all recognised species of Spyridium, six phrase name taxa, seventy-two accessions of S. parvifolium, eight putative hybrids and four outgroup taxa. This study provides the most comprehensive phylogenies of Spyridium and S. parvifolium to date. For Spyridium, several biogeographic patterns were identified, including deep diverging clades of taxa endemic to Western Australia, New South Wales and Tasmania. Several taxa were identified as polyphyletic (e.g. S. eriocephalum and S. phylicoides), warranting taxonomical review. For S. parvifolium, early divergence of individuals from west of the Murray Darling Depression, isolation on the inland side of the Great Dividing Range and recent seed-mediated gene-flow across Bass Strait were identified in the chloroplast genome phylogeny. The variants of S. parvifolium were not supported as genetically distinct suggesting the infraspecific recognition of var. parvifolium and var. molle in Tasmania is not warranted. Molecular evidence of introgression between S. parvifolium and both S. daltonii and S. vexilliferum was identified, providing molecular support for hybrids also inferred from intermediate morphology. Other findings include inferred parentage, unidirectional introgression and recombination of the nuclear ribosomal array for some hybrid accessions.
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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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ItemAlternative splicing and stage differentiation in apicomplexan parasites( 2017)Alternative splicing is the phenomenon by which single genes code for multiple mRNA isoforms. This is common in metazoans, with alternative splicing observed in over 90% of human genes (Wang et al., 2008). However, the full extent of alternative splicing in apicomplexans has been previously under-reported. Here, I address this deficiency by transcriptomic analysis of two apicomplexan parasites: Toxoplasma gondii, which causes toxoplasmosis; and Plasmodium berghei, which is a murine model for human malaria. I identified apicomplexan homologues to SR (serine-arginine–rich) proteins, which are alternative-splicing factors in humans. I then localised a homologue, which I named TgSR3, to a subnuclear compartment in T. gondii. Conditional overexpression of TgSR3 was deleterious to growth. I detected perturbation of alternative splicing by qRT-PCR. Parasites were sequenced with RNA-seq, and 2000 genes were identified as constitutively alternatively spliced. Overexpression of TgSR3 perturbed alternative splicing in over 1000 genes. Previously, computational tools were poorly suited to compacted parasite genomes, making these analyses difficult. I alleviated this by writing a program, GeneGuillotine, which deconvolutes RNA-seq reads mapped to these genomes. I wrote another program, JunctionJuror, which estimates the amount of constitutive alternative splicing in single samples. Most alternative splicing in humans is tissue specific (Wang et al., 2008; Pan et al., 2008). However, unicellular parasites including Apicomplexa lack tissue. Nevertheless, I have shown that alternative splicing can still be common. I hypothesised that the tissue-specific alternative splicing of metazoans is analogous to stage-specific alternative splicing in unicellular organisms. I purified female and male gametocytes of P. berghei and sequenced these stages, with the aim of investigating alternative splicing and its relationship to stage differentiation. As a reference point, I first established the wild-type differences between female and male gametocytes. I detected a trend towards downregulation of transcripts in gametocytes compared to asexual erythrocytic stages, with this phenomenon more marked in female gametocytes. I was also able to identify many female- and male-specific genes, some previously-characterised, and some novel. My findings were further placed in an evolutionary context. Sex-specific genes were well conserved within the Plasmodium genus, but relatively poorly conserved outside this clade, suggesting that many Plasmodium sex-related genes evolved within this genus. This trend is least pronounced in male-specific genes, which suggests that sexual development of male gametocytes may have preferentially evolved from genes already present in organisms outside this genus. I then analysed these transcriptomes, now focusing on changes in alternative splicing. While non-gendered gametocyte differentiation is modulated by known transcription factors such as AP2-G (Sinha et al., 2014), I provide evidence that alternative splicing adds another level of regulation, which is required for differentiation into specific genders. I ablated a Plasmodium SR-protein homologue, which I named PbSR-MG. By transcriptomic analysis, I show that it regulates alternative splicing, predominantly in male gametocytes. Ablation was also associated with a drastic reduction in the viability of male gametocytes. Hence, I have shown that alternative splicing is common in apicomplexan parasites, is regulated by specific genes, and acts on specific targets. Alternative splicing is important for parasite viability and fundamental to stage differentiation in Plasmodium.
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ItemPhylogenomics of the pulmonate land snails( 2017)The pulmonates are the most speciose gastropod lineage and are highly diverse in morphological form and habitat. The evolutionary relationships among the pulmonates have remained controversial despite a long history of scientific study. Recent molecular studies have placed traditionally pulmonate (air-breathing) and non-pulmonate taxa into Panpulmonata; however, the relationships within this new group are still poorly understood. Incongruence between molecular studies has generally resulted from a lack of informative loci but the advent of next generation sequencing technologies means it is now feasible to produce large genetic datasets for non-model organisms. The main aim of my thesis was to investigate the timing and pattern of evolutionary relationships within the Panpulmonata, at multiple taxonomic scales, using phylogenomic datasets. The qualification of orthology is a significant challenge when developing large, multi-locus datasets for phylogenetics from transcriptome assemblies. In Chapter 2, I identified 500 orthologous single-copy genes from 21 transcriptome assemblies across the Eupulmonata (mostly terrestrial land snails and slugs) using a thorough approach to orthology determination, involving manual alignment curation and gene tree assessment. I further qualified orthology by sequencing the genes from the genomic DNA of 22 representatives of the Australian land snail family Camaenidae using exon capture. Through comparison, I also found that automated orthology determination approaches can be susceptible to transcriptome assembly errors. I then used the orthologous genes identified in Chapter 2 to investigate the pattern and timing of evolution across Panpulmonata in Chapter 3. My dataset included representatives of all major clades within Panpulmonata including a wide representation of the stylommatophoran land snails, the most successful lineage of terrestrial molluscs. Maximum likelihood and Bayesian analyses confirm that Panpulmonata is monophyletic, and that Pulmonata is not monophyletic, implying that air-breathing has likely evolved more than once. Within Panpulmonata I show strong support for relationships previously unsupported or weakly supported in molecular analyses, including the Geophila, and the Pylopulmonata, a clade that unites the operculate panpulmonates. Molecular dating suggests a Permian or Early Triassic origin for Panpulmonata and a Triassic/Jurassic boundary origin for Eupulmonata and the freshwater Hygrophila. In addition to investigating deep relationships within Panpulmonata, I used a similar approach to investigate phylogenetic relationships on a shallower scale within a land snail family, the Rhytididae. Australia has the highest taxonomic diversity of the Rhytididae, a carnivorous family of land snails with a Gondwanan distribution. Previous higher classifications of the Australian Rhytididae are based on limited morphological characters and have not been assessed with molecular evidence. I present a molecular phylogeny of the Australian Rhytididae based on a large multi-locus dataset comprising nuclear exons sequenced using exon capture. I identified four major monophyletic lineages within the Australian Rhytididae. I also show that there is a high amount of unrecognised diversity, particularly in the smaller rhytidids. Contrary to shell morphology, on which the current taxonomy is based, a number of currently recognised genera are either polyphyletic or paraphyletic. The Australian lineages all resulted from an apparent pulse of diversification approx. 45-30 Ma. Given the South African Nata and the New Zealand Delos and Schizoglossa also belong to this clade, this date suggests that cross-water dispersal has played a role in the evolution of this group.
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ItemA genetic approach to the conservation of holly leaf grevilleas (Proteaceae)( 2016)The holly leaf grevilleas consist of an informal aggregate of 15 species found in south-eastern Australia. The group exhibits high levels of morphological variation and the most widespread species, Grevillea aquifoilum Lindl., is also the most variable. Most species are restricted endemics and their geographic limits make them vulnerable to the effects of fragmentation and environmental change. In some species, production of viable seed is unknown or has not been confirmed. Identifying factors that contribute to the persistence of species when fecundity is low is of critical importance to their conservation. Here, a phylogenetic analysis is used to clarify the evolutionary relationships among lineages within the holly leaf grevilleas. The lineages identified are then the basis of chapters 4 – 6 that address questions of what constitutes the units of conservation and how clonal plants should be assessed. Analysis of 12 cpDNA regions strongly supported the more southerly distributed holly leaf grevilleas as a monophyletic group comprising four clades (‘G. aquifolium’, ‘G. dryophylla’, ‘G. repens’, ‘G. ilicifolia’). The two northern holly leaf grevillea species, G. renwickiana and G. scortechinii, found in New South Wales and southern Queensland, were not positioned with the southern species but their relationship with outgroup species G. willisii from northeastern Victoria and G. acanthifolia and G. laurifolia from New South Wales could not be ascertained with confidence. Two nuclear regions, PHYA and waxy1, were less variable and not analysed in combination with cpDNA. PHYA was largely uninformative with most species forming a polytomy. Two major variants were identified in waxy1 and consisted of one functional and one non-functional copy based on DNA translation. Minor alleles of functional and non-functional copies were present in some accessions. Using only the functional copy (including multiple alleles when present), the southern ‘G. ilicifolia’ clade, as identified from cpDNA, was clearly differentiated from the northern clade and the remaining species. Within the southern species, those not belonging to the ‘G. ilicifolia’ clade were grouped together but clades identified in the cpDNA phylogeny were not recovered in the waxy1 analysis. Incongruence between the phylogenetic placement of some taxa and current species assignment based on morphology, including apparent paraphyly of G. aquifolium, may indicate an evolutionary history of hybridisation, introgression and incomplete lineage sorting and/or the use of morphological characters that are not lineage-specific. For example, the two subspecies of G. montis-cole differentiated morphologically on the basis of style-length are positioned in different clades and warrant specific rank if supported with nuclear data, and G. steiglitziana is split between two lineages within the southern ‘G. dryophylla’ clade. The phylogenetic placement of G. ‘williamsonii’, a taxon no longer recognised, with sympatric G. aquifolium, coupled with microsatellite analysis supports the current taxonomic view of its synonymy with G. aquifolium. The cpDNA phylogeney also raises questions about the taxonomy of G. microstegia and G. bedggoodiana, taxa that are also positioned with G. aquifolium in the ‘G. aquifolium’ clade. Population genetic analyses of G. infecunda and G. renwickiana found both species to be comprised of a small number of clonal lineages with no evidence of contemporary sexual reproduction. Within species, no clones were found at multiple locations and cpDNA haplotypes were derived from single lineages. In G. infecunda, 38 clonal lineages were identified from a microsatellite analysis of 280 samples from 11 populations. The number of clones present per population ranged from 1 to 11 and clone size varied from a single stem to several hectares. In G. renwickiana, analysis of 197 samples revealed that all but one of seven populations were monoclonal. Clones were distributed over a minimum area of one hectare. Sequencing of microsatellite alleles showed that variation in allele size profiles among clonemates could be interpreted as somatic mutation. The genetic patterns evident in the two species are likely to be the result of a loss of sexual reproduction, due to pollen sterility in G. infecunda and the effects of triploidy in G. renwickiana. For the clonal taxa, G. infecunda and G. renwickiana, lack of sexual reproduction leaves little opportunity for adaptation or migration in response to changing conditions. However, to facilitate the adaptive responses of ecological communities rather than individual species, conservation should encompass obligately clonal species because of their role, albeit finite, in mitigating ecological instability as floras respond to rapidly changing environments.
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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.