School of BioSciences - Theses
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Impacts of anthropogenic pollution on cognition and sleep in an urban bird
The world is rapidly urbanizing, transforming Earth’s natural habitats. While the loss of these habitats is the most visible impact of urban expansion, human population growth has also brought associated phenomena that have the capacity to negatively impact non-human animals. Two key anthropogenic phenomena associated with urban environments have become almost inescapable: anthropogenic noise and artificial light at night. Anthropogenic noise can now be heard in some of the most remote protected areas in the world, while, in the US alone, half of all land mass is exposed to unnatural levels of light at night. There is growing evidence that these environmental ‘pollutants’ have negative impacts on the behaviours and fitness of urban wildlife. In this thesis, I focus on the potential of these anthropogenic pollutants to disrupt cognitive processes and sleep in an urban-adapted species, the Australian magpie (Cracticus tibicen). Wild magpies occupying territories across suburban Melbourne that were exposed to varying levels of urban noise performed similarly on cognitive tasks. Variation in cognitive performance was best explained by age rather than the amplitude of noise to which birds were exposed on a daily basis. I found some evidence that higher sound levels may impair cognitive development, but the effect was observed in only one of four cognitive tasks presented. In captivity, the performance of magpies on similar cognitive tasks was compared under two conditions (experimental exposure to realistic traffic noise, compared to a quiet control). I again found no difference in cognitive performance in the presence or absence of noise. They did, however, perform better on tasks the second time they experienced them, regardless of noise. Finally, I explored the effect of urban noise and two different colours (blue-rich and blue-reduced) of artificial light at night on sleep in magpies. Magpies exposed to a 24-hour recording of urban noise spent more time awake and less time in non-rapid eye movement (non-REM) and REM sleep throughout the night. Likewise, magpies exposed to artificial light at night spent less time asleep, and their sleep was more fragmented. In addition, blue-rich lighting had a greater effect on sleep than did blue-reduced lighting. My findings shed light on some of the putative pathways by which anthropogenic noise and artificial light can affect key biological processes in suburban wildlife, but also illustrate that not all processes are necessarily detrimentally affected.
Molecular Tool Development For Gene Characterisation and Phylogenetic Inference in Fungi
Fungal species impact humans in many areas, some positively and others negatively. Some fungi are agents of disease in plants or animals, yet others find diverse beneficial uses in industry and food production. A greater understanding of fungal biology is a pressing requirement because it will allow us to ameliorate the negative effects of certain species and enhance currently beneficial interactions, as well as to identify new uses for fungi. With this in mind, this thesis has focused on the development of new molecular tools and approaches that will improve capabilities for gene characterisation and phylogenetic inference in fungi. This work has extended across several species including the canola pathogen Leptosphaeria maculans, the heat tolerant ascomycete Paecilomyces variotii and the species in the order Mucorales. In L. maculans I utilised novel tools including CRISPR-Cas9 to identify pathogenicity genes. After evaluating the efficiency of identifying pathogenicity genes using a combination of RNA-sequencing based prediction and CRISPR-Cas9 targeted disruption of putative pathogenicity genes, I turned to a more conventional approach using T-DNA mutagenesis and forward genetics to screen for mutants with altered pathogenicity. I paired this with whole genome sequencing to identify T-DNA integration events, and in this way identified three new pathogenicity genes, one encoding a Sit4 Associated Protein (SAP), one encoding a flavoprotein, and one encoding a heat repeat protein. In Paecilomyces variotii I developed all the tools required to work effectively at the genetic level. These include two genome sequences and techniques for transformation, targeted gene disruption and sexual crossing. Using these tools, I then examined interesting aspects of the biology of this organism including discovering Repeat Induced Point (RIP) mutation for the first time in the Eurotiales and uncovering the genetic basis for the biosynthesis of the secondary metabolite viriditoxin. In the final chapters of this thesis I describe some of my work uncovering diversity among Australian Mucorales species. This has included the discovery of a new species of Pilaira, a new species of Syncephalastrum and a comprehensive examination of the genus Backusella.
Speciation and secondary contact in a colourful agamid, Ctenophorus decresii
Colour polymorphism, the co-existence of multiple heritable colour morphs within an interbreeding population, is thought to promote rapid phenotypic evolution and speciation. This is based on the importance of colour signals in reproductive isolation in combination with the underlying genetic architecture of polymorphism, where morphs are predicted to be governed by few genes of major effect. This prediction is supported by empirical data and stems from how colour morphs often differ in suites of co-adapted traits. During secondary contact between populations that differ in morphs, there is expected to be a high probability of genetic incompatibilities between morphs due to a breakdown of adaptive genetic correlations. Furthermore, colour signal divergence may also be accompanied by changes in behaviour and/or mating preferences leading to incompatibilities between populations which differ in morphs. These factors together may facilitate the formation of reproductive isolation and ultimately lead to speciation. In this thesis, I investigated divergence and the outcome of secondary contact between lineages of the tawny dragon, Ctenophorus decresii, which differ in morph number and type. Ctenophorus decresii is a sexually dimorphic agamid lizard endemic to South Australia, and comprises two genetically distinct and geographically structured lineages: northern and southern. I tested for differences in colour vision between the lineages, which differ in a sexual signal, male throat coloration, particularly in the absence or presence of ultraviolet (UV) reflectance. The northern lineage is colour polymorphic with four discrete throat morphs which lack significant UV reflectance: orange, yellow, orange-yellow (orange centre surrounded by yellow), and grey. Southern lineage males are monomorphic with blue throats and a strong UV reflectance peak. Male throat coloration is an important intraspecific sexual signal, as it is emphasised in territorial and courtship displays. I investigated whether lineages differ in visual sensitivity to UV wavelengths by measuring retinal opsin protein expression of four cone opsin genes (SWS1, SWS2, RH2, LWS) using droplet digital PCR. I found that lineages did not differ in gene expression of the four opsins and discussed this in the context of conserved visual sensitives in terrestrial systems. The lineages meet in a contact zone where multi-locus genetic data suggested the presence of hybrids and potential barriers to gene flow. Using extensive field surveys, male phenotype data, genomic single nucleotide polymorphisms (SNPs), and a mitochondrial (mtDNA) marker, I investigated the outcome of secondary contact between the lineages. Furthermore, I captive-bred pure and first generation (F1) hybrid offspring to characterise colour traits independent of exogenous selection. I found that the contact zone is narrow and several generations old with no parental forms or F1 hybrids present. The northern mtDNA haplotype was prevalent in hybrids, and there were high frequencies of backcrossing to the northern lineage but not to the southern lineage, indicating genetic incompatibilities. The northern throat polymorphism was maintained, without any loss of morphs, whereas the southern throat morph was absent. This contrasted with the more intermediate throat phenotype of captive-bred F1 hybrids, particularly in ultraviolet reflectance, suggesting strong selection for the northern throat phenotype within the contact zone. The viability and fitness of F1 hybrids have consequences for contact zone dynamics, and ultimately whether species boundaries are eroded or maintained. I performed pure and reciprocal cross F1 hybrids in a laboratory setting and measured parental reproductive traits and offspring fitness traits. I found that northern females have a higher reproductive output with more, larger clutches per breeding season and lower embryonic mortality. Although pure and hybrid offspring did not differ in individual fitness traits, hybrids produced from a combination of northern females and southern males exhibited higher fitness in more categories (i.e. growth rate, bite force, sprint speed). These factors in combination may contribute to the prevalence of northern lineage mtDNA haplotypes in the contact zone. Finally, I taxonomically separated the northern and southern lineages of C. decresii sensu lato on the basis of differentiation in morphology and male coloration, genetic divergence with restricted gene flow, and geographic structuring. This revision results in C. decresii sensu stricto (previously southern lineage) and C. modestus (previously northern lineage). I evaluated morphological traits of the type specimen of Amphibolurus modestus (Ahl 1926), previously a synonym of C. decresii sensu lato, and determined that it represented a specimen of the northern lineage. Therefore, I formally re-instated and re-described Ctenophorus modestus (Ahl 1926). The addition of this species to the C. decresii species group, which now comprises six species, supports the notion that geographic divergence in male coloration is an important component to speciation in this group.
Profiling the molecular mechanisms underlying negative cross-resistance to insecticides using Drosophila melanogaster
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate neurotransmission at cholinergic synapses. The nAChRs are mainly expressed in the central nervous system and are highly conserved across a wide range of insect species. Neonicotinoids and spinosyns are two classes of insecticide that target nAChR subunits to kill pest insects. Mutations in genes encoding several nAChR subunits in various insect species, as well as in Drosophila melanogaster, have been documented as conferring insecticide resistance. Chemical control including insecticides has been a key tool in controlling pest insects. The cycle of insecticide use, resistance evolution and insecticide replacement has been continuing for the past decade, leading to many pest species carrying resistance to multiple classes of insecticides. This thesis examines the interplay between different insecticides and nAChR mutations that are associated with resistance to one insecticide but result in hypersensitivity to another, a phenomenon called negative cross-resistance. The negative cross-resistance relationship presents insecticides that could complement current rotation strategies for resistance management, and this warrants further analysis to understand the mechanism. Examination of loss-of-function mutations on the nAChR subunits in this thesis, confirmed the previous identification of the Dalpha1 and Dbeta2 subunits as targets for neonicotinoids, as well as the Dalpha6 subunit as a target for spinosyns. This study also identifies the Dalpha2 subunit as an additional target for imidacloprid. Importantly, mutations on these subunits were also associated with insecticide hypersensitivity, suggesting negative cross-resistance. The neonicotinoid-resistant, Dalpha1 mutants were hypersensitive to spinosyn, except for a full knockout allele, while the spinosyn-resistant, Dalpha6 mutants were all hypersensitive to neonicotinoids. Additionally, negative cross-resistance was found between two neonicotinoids, nitenpyram and imidacloprid in the Dalpha2 mutants. Analysis of different allelic variations at the gene encoding these subunits indicates that this is not an allele specific phenotype. Combining the negative cross-resistance relationship and analyses of molecular changes induced in the nAChR subunits mutants, our study initiated to characterise the changes at the synapse that underlie the negative cross-resistance phenotype. A mechanism involving nAChR compensatory changes in levels of another receptor subunit/subtype was hypothesised to cause the phenotype. Following measurement of transcriptional changes and subunit protein changes, the study classified few correlations between nAChR subunit expressions and the negative cross-resistance, and these vary between the mutants suggesting other possible route(s) for the insecticide hypersensitivity. A genome-wide differential gene expression analysis in specific neuronal cell types of larval brain revealed differentially expressed genes in the Dalpha1 and Dalpha6 mutants. Interestingly, gene ontology enrichment analysis indicates dysregulation of cellular processes, including oxidative stress, protein trafficking and proteasomal degradation pathways in the mutants, that may contribute to the insecticide hypersensitivity. Dysregulation of oxidative stress may predispose the nAChR mutants to further insecticide-induced increase in oxidative levels. Finally, blocking dynamin-mediated endocytosis and proteasome activity, using chemical inhibitors, showed protection against larval movement reduction following imidacloprid and/or spinosad exposure. These findings indicate that the relatively straightforward phenotypic observation of insecticide hypersensitivity in response to loss of a receptor subunit is most likely underpinned by several complex changes in neurons, altering the sensitivity of their response to insecticides and their capacity to cope with downstream effects of insecticide exposure.
The beneficial effects of Trichoderma harzianum T-22 in barley (Hordeum vulgare L.) roots under salt stress
Soil salinity is an important problem that impacts agriculture globally. A sustainable approach for improving productivity is to adopt beneficial microorganisms to enhance the supply of soil nutrients to plants in stressful environments. Our work is showing that the fungus Trichoderma harzianum T-22 enhances barley growth and nutrient uptake in saline conditions. The fungus symbiotically lives inside the roots and triggers beneficial biochemical and metabolic changes. This project has broad implications for applying beneficial plant-microbe interactions to improve agricultural productivity. Chapter 1 covers the history and performance of endophytic fungi applied to crops as an alternative or supplement to the use of plant genetics or soil management to alleviate salt stress in crops. We focus primarily on root-associated microorganisms. The root-soil zone is the first point of defence for the plant against salt moving into the plant with the transpiration stream. It has dynamic biogeochemical processes driven by diverse metabolites released by the plant root and associated soil microorganisms. Fungal endophytes associated with some crops not only protect against plant pathogens and pests but also impart strong tolerance against several abiotic stresses in crops, including salinity. This is achieved via inducing systemic resistance, increasing the levels of metabolites such as pathogen protectants and osmolytes, activating antioxidant systems to prevent damage caused by ROS, and modulating plant growth phytohormone levels. Colonization by endophytic fungi improves nutrient uptake and maintains ionic homeostasis by modulating ion accumulation, thereby restricting the transport of Na+ to leaves and ensuring a low cytosolic Na+:K+ ratio in plants. This literature review has been submitted after external review to the journal Plant and Soil for publication. Following there is an addendum (Section 1.6) covering investigations and application of the endophyte-plant interactions using in metabolomics and lipidomics. The addendum aims to provide an overview of the necessity to investigate plant-fungal interactions and its influence on plant metabolism. Lastly, the chapter gives a brief description of the main experimental, technical and instrumental methods employed in this project. Chapter 2 determines the effect of the salt tolerant beneficial endophyte, Trichoderma harzianum strain T-22, on the growth and development of barley under optimal and saline conditions. Two barley genotypes were used in the study, cv. Vlamingh as a salt tolerant cultivar and cv. Gairdner as a salt sensitive cultivar. Barley was chosen as it is not only an agriculturally and industrially important crop but is also the most salt tolerant cereal crop. Two experimental setups were used for this study. In the first experiment, agar was used as the growth medium for plants. This was performed to observe and determine the effect of fungus on the growth of plants under controlled conditions. In the subsequent experiment, sandy loam soil was used as the matrix to grow plants and to emulate agricultural scenarios. Light microscopy was used to confirm the association of the fungus within roots. The results confirm the positive effect of this fungus under controlled and saline conditions in both experiments. Various parameters were measured to confirm the effects of salt and fungus on both genotypes under controlled and saline conditions. This study suggests that inoculation of salt sensitive plants with T. harzianum T-22 may ameliorate the effects of salinity and improve plant growth. Chapter 3 describes the role of Trichoderma harzianum T-22 in alleviating NaCl-induced stress in two barley genotypes (cv. Vlamingh and cv. Gairdner) by mapping metabolites and lipids using GC-MS for polar metabolites and LC-MS for lipids. This was performed to provide insights into the biochemical changes in barley roots treated with fungus during the early stages of interaction. T. harzianum increased the root length of both genotypes under controlled and saline conditions. The fungus reduced sugars in both genotypes and caused no changes in organic acids under saline conditions. Amino acids decreased only in cv. Gairdner in fungal-inoculated roots under saline conditions. Triacylglycerols (TAGs) were the substantially increased lipids in inoculated roots of both genotypes under saline conditions. This study shows that the fungus imparts adaptation or tolerance mechanism to cv. Vlamingh and in cv. Gairdner by remodelling lipids mainly from glycerolipids after salt stress. Chapter 4 examined the role of Trichoderma strain T-22 on two barley genotypes (cv. Vlamingh and cv. Gairdner) grown in saline soil as soil is an important substrate for Trichoderma. Biomass results described in Chapter 2 of this thesis clearly show the positive effect of this fungus on both genotypes under control and saline conditions as measured using several physiological parameters. Here, the aim of this part of study was to determine the metabolites and lipids modified in roots grown in saline soil following endophyte inoculation that are involved in conferring positive effects on barley plants as mentioned in Chapter 2. We employed gas chromatography and liquid chromatography both coupled to mass spectrometry to analyse metabolites and lipids in inoculated and uninoculated roots of both genotypes under control and saline conditions. Chapter 5 explains the application of mass spectrometry imaging (MSI), liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS), inductively coupled plasma mass spectrometry (ICP-MS), and X-ray fluorescence (XRF) to determine the spatial distribution of metabolites, lipids and a range of elements, such as K+ and Na+, in seeds of two barley genotypes with contrasting germination phenology (Australian barley varieties Mundah and Keel). This chapter was published in Front Plant Sci (Gupta et al. 2019). Following this, an addendum (Section 5.10) is added to this chapter where the effect of T. harzianum was measured on germination of four barley genotypes used in this project. The results showed that the fungus improves germination efficiency of the sensitive genotypes, suggesting its role in reducing the adverse effects of salt stress on germination and growth of the plant. The final Chapter 6 describes the application and importance of mapping the biochemical changes involving metabolites and lipids, imparted due to the inoculation of the fungus in roots of barley seedlings and provides a view on future perspectives of research of plant-fungal interactions for plant growth promotion which can useful for sustaining agricultural productivity.
Systemic impacts of low dose insecticide exposures in Drosophila: a mechanism centred on oxidative stress
The plight of insect populations around the world has gained increasing attention. A recent meta-analysis published in Science reported an average decline of terrestrial insect abundance by average 9% per decade since 1925 (van Klink et al. 2020). While this is a lower rate of decline than reported in earlier meta-analyses (Sanchez-Bayo and Wyckhuys 2019) it still suggests that many terrestrial insect species are under threat. The extinction of terrestrial insect species would severely affect agriculture and ecosystems due to the vital role that many species play in pollination, the recycling of organic matter, pest control and other ecosystem services. Insecticide exposure has been proposed to be one of the significant contributing factors for population declines of non-pest species. Insecticide contamination in biomes, resulting from intensive usage on agricultural crops, is likely to lead to exposures for many non-pest insect species. Low doses of insecticides are known to impact the fitness and behaviour of various insect species, but the underlying molecular, cellular, and physiological impacts of such doses in insects are not well defined. The absence of a mechanism that explains how low doses affect insects is an obstacle to ascertaining the extent to which insecticides may contribute to the demise of populations. The aim of this study was to scrutinize the impacts of low insecticide doses on the metabolism and physiology of the model organism Drosophila melanogaster in order to propose a mechanism to explain the impact of such doses on insect biology. Two insecticides were investigated in detail. The first of these is the synthetic neonicotinoid imidacloprid. Having been banned in the EU due to some evidence of a role in collapse in honeybee colonies, imidacloprid remains one of the most widely used insecticides in the world. The second insecticide is spinosad. Composed of two structurally similar natural fermentation products from the soil bacterium Saccharopolyspora spinosa, this insecticide is classified as organic and considered to be less harmful to beneficial insects. Both insecticides target evolutionarily conserved nicotinic acetylcholine receptors (nAChRs) in the Central Nervous System (CNS) of insects. nAChRs are pentameric ligand gated ion channels. Activation by the natural ligand, acetylcholine, leads to a flux of calcium, potassium or sodium ions into neurons, regulating a myriad of responses in the insect brain. The Drosophila genome encodes 10 nAChRs subunits (Dalpha1 to Dalpha7 and Dbeta1 to Dbeta3), meaning that there is a vast number of subunit combinations that could assemble to form functionally distinct receptor subtypes. Imidacloprid targets the Dalpha1, Dalpha2, Dbeta1 and Dbeta2, subunits, whilst spinosad targets the Dalpha6 subunit. Acute exposure to imidacloprid, at doses that do not kill Drosophila larvae, rapidly increased in the levels of reactive oxygen species (ROS) in the brain, most likely due to the sustained calcium flux into neurons caused by the interaction between the insecticide and its nAChR targets. This led to oxidative stress marked by mitochondrial dysfunction that in turn led to a significant decrease in energy (ATP) levels. While this process was initiated in the brain, lipid storage in the metabolic tissues (fat body, Malpighian tubules, and midgut) was affected. Transcriptomic analysis of the larval brain and fat body revealed a significant perturbation in the expression of genes involved in metabolism, oxidative stress, and immune response. Using genetic manipulations to elevate ROS levels exclusively in the brain, lipid storage was shown to be perturbed in the metabolic tissues, indicating that a ROS signal initiated in the brain radiates to other tissues. Severe damage to glial cells and neurons (i.e. neurodegeneration) was observed in the visual system of adults subjected to chronic low-dose exposure to imidacloprid. This precipitated a progressive loss of vision. Spinosad showed a different mode of action, blocking nAChRs and preventing calcium influx. The blocked receptors were shown to be recycled from the neuronal membranes through endocytosis. This mechanism led to an increase in the number and size of lysosomes in the CNS, characteristic of lysosomal storage diseases, which precipitates elevated generation of ROS by impairing mitochondrial activity and neurodegeneration. The high levels of ROS measured in the CNS after spinosad exposure, were associated with a cascade of phenotypes in metabolic tissues similar to the ones observed after imidacloprid exposure. Experiments examining the lipid environment in Dalpha6 knockout mutants (resistant to spinosad) indicated that impacts observed in the metabolic tissues of spinosad-exposed larvae are due to the interaction between Dalpha6 and spinosad. These data corroborate the hypothesis that impairments observed in metabolic tissues are triggered by a chemical signal from the brain, suggested to be a peroxidized lipid. Although there were some differences in the responses observed for the two insecticides (e.g. in transcriptomes and lipidomes), a similar cascade of processes was observed to be initiated following the elevation of ROS levels in the brain. A potent antioxidant, N-Acetylcysteine amide, strongly suppressed a range of phenotypes observed in both larvae and adults, indicating a causal role for ROS and oxidative stress. As the nAChR targets of these insecticides are conserved among insects, it is likely that similar impacts would be precipitated by exposures in other non-pest species, albeit at different doses. As insecticides from a wide range of chemical classes create markers of oxidative damage, the low dose mechanism of action observed for imidacloprid and spinosad may apply more broadly. This requires investigation. Considered together, the low dose impacts of imidacloprid and spinosad severely impair insect biology, without necessarily killing. These impairments could render insect species more vulnerable to the other major threats proposed to contribute to the decline of populations: climate change, habitat loss, pathogens, and parasites.
Phylogenomics, molecular evolution and extinction in the adaptive radiation of murine rodents
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.
Metabolic Engineering Strategies to Increase Ascorbate Concentrations in Rice and Wheat
Ascorbate (ascorbic acid, vitamin C) is essential for both plants and mammals. Ascorbate is a reducing agent capable of donating electrons, enabling it to perform a range of biochemical functions, such as scavenging reactive oxygen species, assisting enzymatic activity, and reducing higher oxidative states of iron (Fe). In plants, ascorbate is the most abundant water-soluble antioxidant and plays a key role in many fundamental processes, such as photosynthesis, stress tolerance, and the transport of Fe. In humans, ascorbate is an essential micronutrient that must be obtained through diet and takes part in a range of important physiological functions, such as collagen synthesis, epigenetic programming, and Fe uptake in human digestion. Several pathways towards ascorbate biosynthesis have been proposed in plants, but there is only definitive evidence for the L-galactose pathway. The GDP-L-galactose phosphorylase (GGP or vtc2/5) gene encodes the first-committed and rate-limiting enzymatic step of the L-galactose pathway and represents the most promising candidate for increasing ascorbate biosynthesis in plants. In addition to transcriptional regulation, the translation of GGP is regulated through a highly conserved, cis-acting upstream open reading frame (uORF) in the 5’ leader sequence of the GGP mRNA. Developing strategies to increase ascorbate biosynthesis in rice (Oryza sativa L.) and wheat (Triticum aestivum L.), two of the world’s most important staple crops, has the potential to improve both food security and crop productivity. As part of this PhD project, two distinct metabolic engineering strategies were employed to increase ascorbate concentrations in rice: (i) constitutive overexpression of the OsGGP coding sequence (35S-OsGGP plants), and (ii) CRISPR/Cas9-targeted mutagenesis of the OsGGP uORF (uorfOsGGP mutants). Ascorbate levels were negligible in both 35S-OsGGP and uorfOsGGP brown rice, likely due to the decline of ascorbate levels in maturing grain reported in cereals; highlighting the challenge of increasing ascorbate levels in cereal species, such as rice. Ascorbate concentrations were significantly increased in germinated brown rice and tissues of 35S-OsGGP plants at the vegetative growth phase. In contrast, foliar ascorbate concentrations were significantly reduced in 35S-OsGGP plants at the reproductive growth phase. This was dependent on homozygosity of the 35S-OsGGP transgene and was associated with a significant reduction in endogenous OsGGP transcript levels, suggesting the occurrence of gene silencing. Foliar ascorbate concentrations were significantly increased in uorfOsGGP mutants, without any changes to OsGGP transcript levels, attributed to alleviated ribosomal stalling on the OsGGP uORF and enhanced translation of the GGP major ORF. Editing the GGP uORF represents an effective transgene-free strategy to increase ascorbate concentrations not only in rice, but other species. Challenging convention, automated imaging revealed that neither the 35S-OsGGP nor the uorfOsGGP plants displayed increased salt tolerance at the vegetative growth phase, despite having elevated ascorbate levels. Ascorbate concentrations were positively correlated with ferritin concentrations in Caco-2 cells—an accurate predictor of Fe uptake in human digestion—exposed to in vitro digests of null segregant and 35S-OsGGP brown rice and germinated brown rice, suggesting that ascorbate-enriched crops may be able to improve Fe bioavailability in human diets. Grain Fe concentrations were not changed in the uorfOsGGP mutants, indicating that ascorbate may not facilitate the transport of Fe into developing rice grain. Next, this PhD project identified six TaGGP genes in the hexaploid bread wheat genome, each with a highly conserved uORF in the 5’ leader sequence. Phylogenetic analyses demonstrated that the TaGGP genes separate into two distinct groups and identified a duplication event of the GGP gene in the ancestor of the Brachypodium/Triticeae lineage. A microsynteny analysis revealed that the TaGGP1 and TaGGP2 subchromosomal regions have no shared synteny suggesting that TaGGP2 may have been duplicated via a transposable element. A transcript analysis of the TaGGP genes identified that the TaGGP1 homoeologs were broadly expressed across different tissues and developmental stages and that the TaGGP2 homoeologs were highly expressed in anthers. Finally, transient transformation of the TaGGP coding sequences in Nicotiana benthamiana significantly increased foliar ascorbate concentrations more than five-fold, confirming their activity toward ascorbate biosynthesis in planta. The six TaGGP genes and uORFs identified in this study present an opportunity to fine-tune ascorbate biosynthesis in this important staple crop.
Symbiosis between arthropod predators
In natural communities, species may evolve characteristics in response to a variety of complex interactions involving heterospecific or conspecific members. Symbioses between arthropod predators, traditionally considered to be predatory and thus antagonistic, provide intriguing opportunities to explore this topic. Recent studies of interspecific interactions among certain spider symbiotic systems reveal outcomes ranging from araneophagy to mutualism. The latter is apparently unusual among any predators, and may reflect the unappreciated role of visual and chemical signalling as prey-luring traits in symbioses. In this thesis, I used a tractable symbiosis allowing long-term trials, which comprises web-building host spiders whose mobility is low and their associates whose behaviour is easily observed, to develop an understanding of the evolutionary significance of symbioses among arthropod predators. First, as orb-weavers are generally the focal species in these symbiotic systems, I investigated the deceptive signal and trait evolution of these web-building spiders. By performing field experiments conducted under contrasting light conditions with paper model spiders, I examined the visual signal design for prey luring. The results show that both the colour (yellow) and pattern (yellow and black mosaic) are essential for luring prey in a high ambient light environment. Subsequently, using a phylogenetic comparative approach, I investigated the association between prey viewing environment and the ventral signal of orb-weavers among 63 species and found that yellow mosaic patterns are more prevalent for web-building spiders living in high ambient light intensity. Combined, my data indicate that high contrast yellow mosaic colour patterns are highly effective in luring prey for orb-weavers occurring under high ambient light conditions, suggesting that prey colour preferences may be important in the evolution of visually mediated prey-luring systems. Second, to highlight the direct and indirect ecological effects in multispecies arthropod symbioses, I conducted field experiments that manipulated the presence of web-building and non-web-building guests to test the synergistic and antagonistic effects of these inquilines on their hosts across a broad latitudinal range. I discovered that Cyrtophora hosts may promote their foraging success by forming heterospecific aggregations with web-building Leucauge guests. Specifically, a Cyrtophora host web complex intercepted more prey when Leucauge guest webs were attached, and thus these hosts experienced higher weight gain. However, non-web building Argyrodes guests imposed fitness cost on their hosts, but only when Leucauge guests were excluded. This pattern was consistent among different host-guest pairs and over an extensive spatial distribution, in which the negative interaction between Cyrtophora host and Argyrodes guest was mitigated by the mutualism between web-building host and its guest – Leucauge orb-weaver. This suggests that the outcome of interspecific interactions between arthropod predators may be more associated with biotic factors – the presence of a third partner species, rather than an abiotic factor – environmental gradients. Lastly, I investigated the underlying mechanism facilitating the establishment of symbioses between arthropod predators. Using a series of field experiments performed on guest species that form distinct interspecific interactions with their host, I evaluated if the settlement pattern of Argyrodes guest spiders is affected by the nature of their relationship with, and the cues from the host and conspecifics. The field surveys confirmed that more guests settled onto the webs with a host and fewer conspecifics, and that the effect of conspecifics was stronger for kleptoparasites than mutualists. This suggests that intraspecific competition may be stronger in kleptoparasitism. Using behavioural assays with y-maze olfactometers, I revealed the role of volatile odours in intra- and interspecific communication. Both A. kumadai from Japan and A. fissifrons from Taiwan preferred host related odours, but klepoparasitic A. kumadai appeared to be repelled by the odours of conspecifics, and the mutualistic A. fissfrons was indifferent to the odours. The differences in cue discrimination ability correspond with the density of sensilla on the front legs of these guest species. These results highlight how the nature of species interactions can act as a selection pressure on not only the strength of cues for partnership recognition but on odorant perception traits.
Reproductive Communication in the Australian Gumleaf Skeletonizer, Uraba lugens (Walker)
Theoretical and empirical studies demonstrate that female moths vary their investment into pheromone-releasing behaviour according to both biotic factors (including age, mating status and competitive signalling) and abiotic factors (including temperature, host plant quality, and photoperiod). This suggests that female moths are capable of sophisticated strategic adjustment of their pheromone production, yet the impact of variation in pheromone output on male arrival rates and male preferences in moths are relatively unexplored. Furthermore, studies documenting sexual chemical communication in moths have focused primarily on the chemical nature of the signal, and have largely ignored female signalling strategies or chemical receiving structures (antennae), despite the costs of signalling and of maintaining signal receiving structures. In Chapter 1, I provide a synthetic review of the results of my research on the gumleaf skeletonizer moth, Uraba lugens, in the context of the broader literature, and argue that while females strategically adjust their sex pheromone signalling behaviour and hence their attractiveness, males strategically balance their investment between longevity and antennal morphology, which reflects their mate searching capacity. In the context of chemical communication in moths, these females calling (pheromone-releasing) strategies reduce the likelihood of mating failure and allow males to maximise their encounter rates with females. In Chapter 2, I explored how adult age influences the calling behaviour of virgin female U. lugens over four continuous ten-hour scotophases (dark periods). I found that female U. lugens alter their calling behaviour with age, but in contrast with theoretical predictions and empirical observations in other species, older females were less likely to call and spent less time calling than younger females. Older females, however, commenced calling earlier in the scotophase, suggesting a strategic shift, potentially to avoid competition from younger females. Behavioural assays with y-maze olfactometers showed that males prefer the pheromones produced by younger females, and that pheromone quality likely plays a role in this choice. In Chapter 3, I explored how juvenile population density influences pheromone output in female U. lugens. I found that female U. lugens facultatively adjust their calling behaviour in response to socio-sexual cues: females that eclosed from high juvenile population densities started calling earlier and spent more time calling than individuals eclosed from low juvenile population densities. Juvenile density also affected female pheromonal attractiveness: males prefer the pheromones produced by females reared at high juvenile densities. Females are likely to benefit from this strategic investment: increased investment into chemical signalling (at high densities) suggests that females compete with conspecific neighbouring signallers in order to avoid mating failure. In Chapter 4, I explore how juvenile diet influences reproductive investment of both female (the quality of female sex pheromone) and male (pre- and post-copulatory) adults. I found that the effect of juvenile diet on adult fitness depended upon adult sex: in females, diet influenced body size, while in males diet influenced longevity. Juvenile diet also affected female pheromonal attractiveness: males tended to prefer the pheromones produced by females reared on host plants which has been supplemented with a fertiliser. Finally, host plant species affected male pre-copulatory investment: males reared on Eucalyptus camaldulensis have longer antennae but less dense sensilla than when reared on a different Eucalypt species, although there was no difference in the testes size of males reared on the two different species. In Chapter 5, I explore how upregulation of immunity affects male antennal functional morphology, female pheromone quality, and other life-history traits. I found that immune activation affected male, but not female signalling investment: immune challenged males had a lower density of sensilla on their antennae, but female pheromonal attractiveness was not affected by their immune status. Nevertheless, immune activation reduced female investment into ovary mass, and the longevity of adult males and females increased following an immune challenge. In conclusion, my different experiments consistently reveal that females alter their sex pheromone production and releasing behaviour for mate attraction in order to avoid mating failure, while males balance the resources allocated to longevity and chemoreception, which improves the likelihood of locating mates.
Non-volatile secondary metabolites in foliar oil glands of Eucalyptus species
Plants synthesise a vast range of secondary metabolites that are stored in specialised cells or organs. The presence of sub-dermal glands rich in volatile terpene essential oils is characteristic of the trees of the genus Eucalyptus (Myrtaceae). Recent studies showed that non-volatile compounds (NVCs), particularly monoterpene acid glucose esters (MAGEs), co-occur with volatile components in Eucalyptus foliar oil glands. The principal aim of this thesis is to characterise MAGEs and other non-volatiles localised to Eucalyptus oil glands and to explore their relationships to the co-housed oil components. Glandular extracts from a range of Eucalyptus species belonging to the two major subgenera, Symphyomyrtus and Eucalyptus, were investigated in Chapter 2. Non-volatiles were extracted from enzymatically isolated glands and analysed using high-performance liquid chromatography (HPLC) and mass spectrometry (LC-MS). MAGEs were identified based largely on diagnostic mass spectral fragmentation patterns. MAGEs were the dominant NVC in Symphyomyrtus species. In contrast, the sampled subgenus Eucalyptus species lacked MAGEs, but were rich in phenolics. Volatile oil components were also analysed using gas chromatography with flame ionisation detection and mass spectrometry (GC-FID and GC-MS). Monoterpenes and sesquiterpenes were identified and quantified for each species. In addition, cell suspensions of E. polybractea were successfully established from leaf-derived callus as a potential tool to investigate the biosynthesis of MAGEs. Glandular extracts from subgenus Eucalyptus species rich in non-volatiles containing phenolic moieties were further analysed in Chapter 3. A suite of unsubstituted B-ring flavanones was identified as the dominant glandular NVCs. In addition, flavones, flavanone-O-glucosides, flavanone-b-triketone conjugates, triketone heterodimers and chromone-C-glucosides were also identified. Flavanones were quantified and species-specific variations were observed. This chapter also showed that flavanones are exclusively localised to the glands rather present throughout leaf tissues. A positive correlation was observed between some monoterpenes and sesquiterpenes with total flavanones and particularly with pinostrobin. Interestingly, b-triketones were also found in the volatile extracts of glands from E. suberea and E. brevistylis. The presence of glandular b-triketones was further explored in Chapter 4. A major discovery of this thesis was the occurrence of two gland types in E. brevistylis that differ in their colour and importantly, in their metabolic contents. ‘Sesquiterpene glands’, which are translucent-white in appearance, contained sesquiterpene alcohols. ‘Triketone glands’, which are golden-brown in appearance, contained mostly the b-triketone conglomerone and sesquiterpene hydrocarbon caryophyllenes in lower abundance. None of the glands contained the NVCs identified from the other species in this thesis. The results were consistent in trees from a natural population of E. brevistylis and in glasshouse-grown seedlings and saplings. In addition, ‘Triketone glands’ seem to develop earlier than ‘sesquiterpene glands’ in leaf ontogeny. This is the first identification of such metabolic differentiation of embedded glands from any tree species. The work presented in this thesis revealed many novel aspects of oil glands, particularly in relation to their non-volatile and volatile constituents and how they are related to one other. Overall, the findings of this thesis contribute significantly to the knowledge on Eucalyptus foliar oil gland chemistry and biology.
A metabolic engineering approach for iron and zinc biofortification of bread wheat (Triticum aestivum L.): impacts on plant growth, grain nutrition and food processing
Human iron (Fe) and zinc (Zn) deficiencies are among the most prevalent nutritional disorders in the world and manifest as a range of health issues including fatigue, impaired cognitive development and increased mortality. Micronutrient supplements and fortificants are frequently used to increase human Fe and Zn intakes yet these strategies require continuous investment and frequently miss rural populations; improving the density and/or bioavailability of Fe and Zn in staple crops (a process known as biofortification) represents a powerful alternative. Bread wheat (Triticum aestivum L.) is cultivated on more land than any other crop and is processed into a range of food products to supply ~20% of the daily calories consumed by humans. The wheat grain is mostly comprised of starch and protein, with micronutrients such as Fe and Zn concentrated in the outer aleurone layer of the grain. In the aleurone layer, Fe and Zn are complexed to compounds that inhibit their absorption (bioavailability) in the human gut and the aleurone layer is removed during grain milling to produce white flour. White flour (representing the inner wheat endosperm) contains low concentrations of dietary Fe and Zn, and high consumption of either wholemeal flour or white wheat flour coincides with high prevalences of human Fe and Zn deficiencies. Generating Fe and Zn biofortified wheat through conventional breeding in modern wheat cultivars is hindered by inherently low grain Fe and Zn concentrations and a lack of genetic variation for these traits. Nicotianamine (NA) is a low-molecular weight metal chelator present in all higher plants with high affinity for Fe2+, Zn2+, and other divalent metal cations. In graminaceous plant species such as wheat, NA serves as the biosynthetic precursor to 2’-deoxymugineic acid (DMA), a root-secreted mugineic acid family phytosiderophore that chelates ferric iron (Fe3+) in the rhizosphere for subsequent uptake by the plant. Both NA and/or DMA are the major chelators of Fe within white wheat flour, and NA is known to enhance Fe bioavailability in cereal grain. For these reasons, increasing the biosynthesis of NA/DMA through upregulation of nicotianamine synthase (NAS) genes has emerged as a popular strategy for Fe and Zn biofortification of cereal crops. In this study we employed constitutive expression (CE) of the rice (Oryza sativa L.) nicotianamine synthase 2 (OsNAS2) gene in bread wheat to upregulate the biosynthesis of NA and DMA, and evaluate plant growth, grain nutrition and food processing properties of CE-OsNAS2 wheat. Our lead CE-OsNAS2 wheat transgenic event (CE-1) demonstrated higher concentrations of Fe, Zn, NA and DMA in wholemeal flour, white flour and white bread, altered distribution of Fe in the grain, and higher white flour Fe bioavailability relative to null segregant (NS) control. Protein composition, dough rheology and breadmaking properties were similar between CE-1 and NS white flour, and a chicken (Gallus gallus) feeding study over a period of six weeks demonstrated that chickens consuming CE-1 white flour had improved Fe status, intestinal morphology and microbial populations relative to chickens that consumed NS white flour. Multi-location confined field trial (CFT) evaluation over three field seasons demonstrated no differences between CE-1 and NS agronomic performance apart from plant height. Throughout all CFTs, grain yield was negatively correlated with grain Fe, Zn, and protein concentrations yet not correlated with grain NA and DMA concentrations. White flour Fe bioavailability was positively correlated with white flour NA concentrations, and we determined NA to be the strongest enhancer of in vitro Fe bioavailability identified to date. Together these results suggest the proportion of Fe that is chelated to enhancers of bioavailability (such as NA and DMA) should be prioritized in future crop biofortification efforts and highlight new strategies for developing Fe and Zn biofortified wheat as a more nutritious staple food.