School of BioSciences - Theses
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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.