School of BioSciences - Theses

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    Breeding a diverse and resilient reef
    Lamb, Annika Mae ( 2022)
    Coral reef ecosystems are biologically, culturally, and economically valuable but are under threat from a suite of stressors, the most pressing of which are rising sea surface temperatures and the associated increased frequency and severity of summer heat waves. High water temperatures cause corals to lose their algal symbionts (bleach) and may result in mass bleaching events, mass coral mortality, and the degradation of reefs. Managed breeding is an active intervention that involves generating stock for deployment into degraded systems; it has the potential to combat population declines and boost population fitness. Many reef-building corals lend themselves to managed breeding because they produce large numbers of gametes through mass-spawning that can be collected and crossed in controlled ways. The aim of my PhD was to conduct crosses within and amongst mass-spawning coral species to test intervention approaches and their ability to generate genetically diverse and resilient corals for reef restoration. Firstly, I conducted parentage analyses of intraspecific crosses and found that the genetic diversity of coral stock can be optimised by minimising the handling of gametes and combining the egg-sperm bundles of multiple colonies in bulk reactions. Secondly, I conducted interspecific crosses between coral species and tested the performance of interspecific hybrids relative to their purebred counterparts. Under simulated heat waves in aquaria and a natural heat wave in the ocean, coral hybrids performed as well as one of the purebred parental species or intermediately to both parental species in terms of their growth, survivorship, and bleaching resistance. Finally, I demonstrated that F1 interspecific hybrids can reproduce to generate viable F2 hybrid and backcrossed offspring and have greater reproductive fitness than purebreds. The viability of coral hybrids in the field and lack of outbreeding depression in F1 hybrids or the early life stages of F2 hybrids is indicative that interspecific hybridisation could rapidly boost genetic diversity in degraded populations. The reproductive viability of F1 hybrids further demonstrates that introgression of beneficial alleles that boost resilience could occur between purebred species via interspecific hybridisation and that outplanted hybrids could propagate on reefs. Bulk coral crosses and interspecific hybridisation may therefore be useful tools for reef restoration initiatives.
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    Comparative Genetics of Invertebrate Moulting
    Perry, Caitlyn Louise ( 2022)
    The development of molecular phylogenetics in the 1990s led, among many other discoveries, to the finding of a monophyletic clade of moulting invertebrates dubbed Ecdysozoa. Including arthropods and nematodes (alongside numerous smaller groups) Ecdysozoa is unrivalled among living groups for the number of species it contains and for the diversity of these species; moulting is the only non–molecular trait known to be common to all modern ecdysozoans. However, our understanding of how moulting is regulated draws on only a very small number of species, with the vast majority of these being insects of economic importance. Naturally, the sheer size of Ecdysozoa precludes a comprehensive investigation of moulting regulation across this clade; furthermore, many ecdysozoan groups (for instance, aquatic taxa) would be far less suited to laboratory culture and manipulation than the average insect. However, ecdysozoan genome sequencing, once largely restricted to genetic model organisms, pest species and disease vectors, has begun to extend across a far broader range of taxa. This thesis reports my exploration of molecular data in the context of the evolution of moulting. The Ecdysozoa hypothesis was accepted quickly by most workers in arthropod taxonomics, but this should not lead us to underestimate just how significant a change the embrace of Ecdysozoa represented. The better part of a century of investigations into the molecular basis of moulting had been conducted under the assumption that the non–moulting annelid worms were the sister taxon to arthropods, and thus that moulting had either evolved repeatedly or had been lost repeatedly among invertebrates. In order to explain this historical context, I have produced a review of the history of arthropod taxonomy in the Western tradition. This account runs from Aristotle to the present day, but has a particular focus on the development of the Ecdysozoa hypothesis. My experimental work on the genetics of moulting regulation began with an examination of the cytochrome P450 oxidase family CYP307 in the fruit fly genus Drosophila. Although the precise catalytic role of these enzymes has proved elusive, they are required for the synthesis of ecdysteroids, the master moulting regulators of arthropods. It has been known for around fifteen years that multiple duplications in the CYP307 family have occurred in Drosophila, but the functional significance of these duplications has been unclear. I have attempted to address this question by transgenic substitution of CYP307 paralogs, finding that even enzymes which are closely related at the level of amino acid sequence are not functionally interchangeable. The CYP307 family is only one of many acting in the synthesis of ecdysteroids from dietary sterols; almost all of these were first identified in D. melanogaster, and although comparative analyses have generally suggested good conservation of these enzymes across arthropods, exceptions are known. Building on the CYP307 investigation and its suggestion of rapid functional differentiation following duplications in this family, I sought to identify all duplications and losses of ecdysteroid synthesis genes across all available arthropod genomes (at the time I ceased collecting data, 923 genera were represented by at least one genome assembly). In some cases, I was able to connect observed copy number changes to ecological factors (e.g. whether the predominant dietary sterol was of animal, plant or fungal origin), but many duplications and deletions were entirely unexpected and suggestive of additional changes which require further investigation (e.g. replacement of one enzyme with another having similar activity). In some cases, I went on to analyse selective pressures acting on relevant components of the ecdysteroid synthesis and signalling pathways to determine the effects of loss or duplication of ecdysteroidogenic genes. Despite the numerous copy number changes I observed, my findings were generally consistent with the strong conservation of ecdysteroid synthesis genes described by earlier researchers. As a consequence of this conservation, arthropods provide limited evidence concerning the early evolution of ecdysteroid synthesis. This limitation makes the recent release of genome assemblies from lineages closely related to Arthropoda (namely velvet worms and tardigrades) particularly important for understanding ecdysteroid evolution. I used a transgenic approach similar to that previously applied to Drosophila CYP307 enzymes to investigate the function of four cytochrome P450s from the tardigrade Hypsibius exemplaris which showed strong similarity to ecdysteroidogenic enzymes at the amino acid level. I found that H. exemplaris CYP315A1 does not rescue D. melanogaster Cyp315a1 nulls (despite their clear orthology) and that H. exemplaris CYP18K1 overexpression produces a phenotype quite distinct from that associated with D. melanogaster CYP18A1 overexpression. Tardigrades are unlikely to synthesise ecdysteroids, but they, along with representatives of other ecdysozoan taxa such as the priapulids (penis worms), may provide insights into the diverse steroid metabolic pathways of Ecdysozoa. My research has focused on the potential for integration of large–scale molecular analysis with experimentation in tractable model organisms as a means of understanding ancient evolutionary events. While my cross–arthropod screen revealed many changes to the ecdysteroid synthesis pathway which remain to be fully examined, the results of my transgenic experiments demonstrate the use of bioinformatic approaches in identifying promising targets for more direct examination. The moulting process is one of considerable interest both because of its practical importance (disruption of moulting is potentially a potent insecticidal technique) and because of the intrinsic fascination of the morphological changes it enables. However, the combined bioinformatic and transgenic approach I have used could be applied to any process occurring in a taxon from which a model organism has been established.
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    Environmental DNA sampling as a tool for monitoring freshwater vertebrates
    McColl-Gausden, Emily Frances ( 2022)
    To monitor biodiversity effectively, accurate and sensitive detection methods that can be implemented over large spatial scales are required. It has been demonstrated that environmental DNA (eDNA) sampling can be a valid alternative to many traditional sampling methods. This thesis explored the data and conclusions which could be drawn from eDNA sampling regimes across large spatial scales for individual species and communities, answering key questions on species occupancy and detection method sensitivity. Adaptations of site occupancy-detection models (SODM) are used throughout. First, I investigated the relative sensitivity of single- and multi-species detection methods using a consistent methodological framework across multiple datasets with different study designs. I used SODM to evaluate how the molecular method used impacted the probability that a species was detected. I found that qPCR was generally more sensitive at detecting target species but that different methodological decisions impacted its sensitivity. Second, I used eDNA sampling to conduct an extensive platypus occupancy survey across 37% of the species’ estimated distribution. Using a systematic study design, I demonstrated that correlates of platypus occupancy could be revealed using this efficient survey method. I found that platypuses were less likely to occupy sites in areas with a high proportion of surrounding agricultural land or grasslands and were more likely to occupy sites with increased runoff, less zero-flow days, and suitable banks for burrowing. Third, the 2019/2020 mega-fires impacted south-eastern Australia severely. My pre-fire eDNA data provided the opportunity to investigate if platypus occupancy was impacted by this large-scale event. I developed a SODM extension to incorporate a Before-After Control-impact (BACI) design. After surveying 118 sites in three time periods (pre-fire, 2020 and 2021), I found that platypus occupancy was not significantly impacted by the presence of fire. However, I did find a significant interaction between the proportion of a watershed that was burnt at high severity and rainfall post-fire: platypus occupancy was lower in watersheds with a high proportion of high severity fire that had high rainfall post-fire. This finding is consistent with previous work on the impact of fires on aquatic species. Lastly, leading on from my work on large-scale eDNA surveys for an individual species, I used eDNA metabarcoding to investigate occupancy patterns among fish communities. I used a stratified study design across the state of Victoria to investigate how native and introduced fish responded to environmental factors. I found that for the water availability covariates I considered, native and introduced fishes responded similarly. However, for the land use covariates such as the proportion of a contracted catchment covered by agriculture, urban or forested land, different patterns emerged. Native fish responded more positively to these categories, whereas introduced species responded more negatively. This thesis demonstrated that eDNA sampling is an ideal monitoring tool for individual species or communities over large spatial scales. I showed that SODM and eDNA data can be used to estimate correlates of occupancy efficiently and that eDNA sampling over time enables species responses to major disturbances to be determined across large areas.
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    Role of phosphorylation in regulating secondary cell wall cellulose synthesis in Arabidopsis
    XU, HUIZHEN ( 2022)
    Plant secondary cell walls (SCWs) are important for plant growth and development as the vascular tissues and fibers support plants with water and mineral transport. Cellulose is the major component of SCWs, and its synthesis is a highly complex process regulated by transcription factors as well as post-translational modifications. Cellulose synthase (CESA) 4, 7 and 8 are essential enzymes that catalyze the synthesis of SCW cellulose and form a cellulose synthase complex (CSC) that is active at the plasma membrane. The CSCs move at the plasma membrane; a process driven by the catalytic activity of the CESAs. The behaviour of the CSC is an important character of cellulose synthesis and SCW patterning. Protein phosphorylation is arguably the most common post-translational modification in many cells and affects CESA behaviour during primary wall synthesis. However, how SCW CESA phosphorylation contributes to secondary wall production is not understood well. Chapter 1 provides a brief overview about plant cell wall cellulose synthesis, especially secondary cell wall biosynthesis. There are five main aspects discussed, including secondary cell wall patterns, transcriptional regulation during SCW formation, CESA structures and the function of each domain, the effects of phosphorylation on cellulose synthesis, and environmental effects on SCW production. In Chapter 2, proteomic and phospho-proteomic changes were characterized during the transition from primary to secondary wall synthesis using the VASCULAR-RELATED NAC-DOMAIN7 (VND7)-inducible system. A vast number of phosphorylation sites, especially in SCW-related proteins, were detected. The phosphorylation changes of putative and selected phosphorylation sites in primary and secondary cell wall CESAs were analyzed in detail. This phospho-proteomic dataset provides more insights into phospho-protein changes during the process of SCW biosynthesis. In Chapter 3, phosphorylation sites in each SCW CESA were analyzed and mutated to examine if and how phosphorylation regulates SCW biosynthesis. Most of the selected phospho-mutants, either phospho-null or phospho-mimic versions, restored the phenotype of SCW cesa mutants, and did not show significant differences from wild type control. However, one conserved phosphorylation sites in CESA4, S374, did affect SCW biosynthesis, as single-site phospho-null mutant (CESA4S374A) showed dwarf phenotype with deformed xylem vessels, similar to cesa4 mutant. Sequencing and qRT-PCR confirmed the correct amino acid substitutions and gene expression, respectively. Further, both bioinformatic analysis of protein structure and sequence alignments indicated that S374 in CESA4 was likely to be externally exposed and phosphorylated. Thus, phosphorylation in the position of S374 in CESA4 potentially works to positively regulate SCW cellulose biosynthesis. In Chapter 4, an immunoprecipitation approach of a YFP tagged CESAS7 in the VND7-inducible system was used to pull out potential proteins interacting with SCW CESAs, focusing on protein kinases. Thirteen highly enriched kinases were in this way found to potentially associate with the CESAs. One interesting but unknown receptor-like kinase, AT1G09440, may potentially play a role in SCW formation. Subcellular localization analysis further showed that this protein kinase was secreted from the Golgi to the plasma membrane where it is likely to have its main function. In Chapter 5, the conclusion for this research and some future work directions are proposed.
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    Manipulating key developmental and regulatory pathways in the Queensland fruit fly to develop improved strategies for pest control
    Nguyen, Thu Ngoc Minh ( 2022)
    Sterile insect technique (SIT) is an efficient and species-specific biocontrol strategy involving mass-releases of factory bred, sterilised males that seek and mate with wild females. Fertilized embryos or zygotes die due to dominant lethal chromosome aberrations caused by sterilization processes, which ultimately leads to localised population suppression. This approach is currently being implemented to help control outbreaks of the invasive Queensland fruit fly (Bactrocera tryoni), which is a major insect pest of fruit and vegetable crops in Australia. Sterilised B. tryoni SIT flies are marked with fluorescent powder dyes to distinguish them from wild flies when recaptured in monitoring traps. However, this powder-based marking strategy can cause dehydration and death, fail to thoroughly coat flies, be washed off or potentially passed on to wild flies through contact. Current B. tryoni SIT practices involve sterile releases of males plus females, although increased efficiency could be achieved if high-throughput sex separation methods were available. Here, targeted CRISPR/Cas9 gene editing and insect transgenesis helped create resources or prioritise genes with desirable qualities, for improving SIT pest control applications. Strains with diagnostic body pigmentation mutations were generated to potentially differentiate between wild flies and SIT flies captured in monitoring traps, or to act as sex-linked pupal markers for mechanical separation of females prior to release. Second, a single amino acid substitution in the gene RNA polymerase II 215 (R977C, RpII215ts) was confirmed as a promising temperature sensitive candidate for developing a male-only SIT strain via conditional removal of female embryos. However, an amino acid substitution in the gene shibire (G268D, shits1) carried strong fitness costs and is not suitable for developing a male selecting strain for SIT, but other shibire temperature sensitive mutations with milder effects in Drosophila melanogaster still hold potential.
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    Towards Strategies for the Control of Invasive Liriomyza Pests in Australia: Dispersal Pathways, Parasitoid Wasps and Endosymbionts
    Xu, Xuefen ( 2022)
    The Agromyzidae are a family of small, morphologically similar flies whose larvae feed internally on living plant tissue, often as stem and leaf-miners. The genus Liriomyza is the most well-known and more than 20 species are recorded as pests or potential pests of crops worldwide. They are highly polyphagous and have a strong propensity to colonize new plant hosts. Among them, three polyphagous Liriomyza species - Liriomyza sativae, L. huidobrensis, and L. trifolii - are recognized as the most damaging leafmining pests that have spread to many new areas in the world including Australia, imposing additional financial burdens to the agricultural and horticultural industries. Unfortunately, systematic strategies for the management of Liriomyza pests in Australia have been largely unexplored. Therefore, this thesis focuses on the identification of leafmining species, the dispersal pathway of one of the exotic Liriomyza species, and an exploration of biological control methods that can help control the outbreak of Liriomyza in Australia. Firstly, molecular methods based on cytochrome c oxidase subunit 1 (COI) DNA barcodes were deployed to character 13 dipteran leafminer species collected from Australia and overseas. In Australia, there already exist some leafmining species that look similar to exotic Liriomyza pests but with little economic importance. The taxonomy of leafmining flies is often challenging due to the complicated morphological keys as well as putative cryptic species. We here provide a baseline for DNA-based identification of pest Liriomyza incursions spreading across the Australian east coast and other species already present in Australia. Secondly, genome-wide single nucleotide polymorphisms (SNPs) were generated by double-digest restriction-site associated DNA sequencing (ddRAD-seq) to reveal the potential origin(s) of Liriomyza sativae into Australia and contribute to reconstruct its global invasion history, showing that the Torres Strait population is the source of the mainland population and highlighting the mixed origins of the Torres Strait population. Thirdly, 13 dipteran leaf-mining species were examined for Wolbachia infections and the potential for this endosymbiont in biocontrol. The comparison of Wolbachia relatedness relied on the wsp/MLST genes. A colony of Liriomyza brassicae was established and treated with tetracycline for reciprocal crosses and cytoplasmic incompatibility was demonstrated. These findings highlight the potential of Wolbachia to impact the Liriomyza pests based on approaches such as the incompatible insect technique. Fourthly, natural enemies like parasitoid wasps are recognized as effective biological agents worldwide to suppress the outbreak of polyphagous leafmining pests. However, the parasitoid complexes in Australia are poorly studied due to their small body size and challenging morphological diagnostic characteristics. To address the knowledge gap of parasitoid wasps in Australia, molecular methods based on cytochrome c oxidase subunit 1 (COI) DNA barcodes as well as traditional morphological approaches were employed to identify 13 parasitoid species. The status of endosymbionts was also checked in these parasitoid wasps given the limited information available in the literature, and two endosymbionts were identified. These findings provide the foundation for employing parasitoid wasps as biological agents for the management of exotic leafmining pests in the future. Fifthly, thelytokous parasitoid wasps are preferred for mass rearing due to an absence of male wastage, and the possible existence of thelytoky locally was explored. In Japan and China, Neochrysocharis formosa infected with Rickettsia are thelytokous and are suggested to be effective to control the polyphagous Liriomyza pests. In Australia, Neochrysocharis specimens have only been identified to genus but not formally identified. Using five DNA barcodes, we identified N. formosa from Australia and established the presence of Proacrias, a genus of leafminer parasitoid not previously detected in Australia. 16S rRNA sequencing indicated that both species were infected with a Rickettsia bacterium. These findings expand records of parasitoids to attack leafminers in Australia and highlight the potential of applying endosymbionts to produce thelytokous strains to increase the efficiency of biocontrol.
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    Genetic Variation Within an Indigenous Australian Cohort and its Implications for Future Studies of Genomics, Health and Disease
    Silcocks, Matthew ( 2022)
    Gaining an understanding of the genetic characteristics of human populations is important for establishing approaches to and expectations from future studies of genomics, health and disease involving these groups. While genomic studies have recently expanded in scope to sample from a wide range of human ancestry groups, the Indigenous communities of Australia remain poorly characterised, and under-represented in global reference panels. Our failure to gain an understanding of patterns of genetic variation within these communities, and how they differ from other human groups, may widen the already considerable gap in health outcomes between Indigenous Australians and the general Australian population. To address this issue, the National Centre for Indigenous Genomics (NCIG) has collected genomic data from four Indigenous Australian communities from across a wide expanse of the continent. This thesis will describe the analysis of the patterns of genetic variation and diversity within this dataset, and will emphasise how they underpin future research of genomics, health and disease for Indigenous Australians. Firstly, this thesis will describe various forms of analysis aiming to identify the source and quantify the degree of non-Indigenous admixture within the NCIG dataset, and detail approaches to ‘mask’ these regions, and analyse exclusively the Indigenous component of each genome. After producing this masked dataset, subsequent analysis will explore various aspects of Indigenous Australian population variation and diversity relevant to future studies of genomics, health and disease. In particular, it will explore patterns of ‘population structure’ within Indigenous Australian groups, and compare these to patterns observed within human cohorts separated by comparable distances within other regions of the world. The medical and genomic implications of the immense degree of structure, haplotype and rare allele sharing within the Indigenous Australian communities will then be discussed. Subsequently, this thesis will analyse Indigenous Australian genomic variation within the context of worldwide human populations. By analysing the NCIG dataset alongside a diverse global cohort, this investigation will show the high abundance of novel genetic variation within these communities, and will emphasise additional genetic characteristics relevant to the design of future studies involving Indigenous Australian genomes. More demographically oriented analysis, involving Indigenous Australian communities, and groups from the surrounding Oceanic region, will provide context for the key findings presented within this thesis. This data will reveal a previously undocumented history of genetic interaction between the populations of Melanesia and northern Australia, and will show Indigenous Australian communities to have sustained small, yet stable population sizes over recent millennia. This thesis will close with an analysis of patterns of uniparental genetic variation within the dataset, and will assess the ways this data supports autosomal based inferences. In particular, this chapter will highlight the presence of a globally rare, and presumably deleterious Y-chromosome variant, which is present at near fixation within the Tiwi Island community.
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    The evolution of salmon lice (Lepeophtheirus salmonis) in response to parasite management strategies in salmon aquaculture
    Coates, Andrew Lindsay ( 2022)
    Parasites, pathogens and other pests have a major impact on global agriculture. Pest populations are able to adapt to novel selection pressures on farms, making long-term pest management a challenge. This is exemplified by the repeated evolution of pesticide resistance over the last century. Resistance can be slowed or avoided by applying evolutionary principles to pest management. This first requires understanding the evolutionary dynamics of the pest under contemporary farming conditions. Such knowledge is especially needed in rapidly emerging and changing farm systems, such as aquaculture. Salmon farming – among the biggest aquaculture industries – is significantly affected by parasitic sea lice (especially the salmon louse Lepeophtheirus salmonis). Efforts to control this parasite have been undermined by salmon lice evolving resistance to most chemical pesticides. An abundance of non-chemical technologies have been introduced as alternatives, but there has been little research into whether they may also drive louse evolution. This thesis explores the potential for L. salmonis to adapt to the pressures faced in salmon aquaculture today; in particular, possible resistance to non-chemical management strategies. Chapter 1 outlines the need for evolutionary perspectives in pest management, and provides an overview of the thesis. In Chapter 2, I review the literature and conclude that salmon lice could, theoretically, adapt to non-chemical methods. This chapter explores how non-chemical resistance might arise, how it might have ripple effects on wild salmon, and how it could be prevented using evolutionarily-enlightened pest management. Chapters 3 and 4 focus on one non-chemical strategy: using physical barriers to prevent lice from entering salmon cages. In Chapter 3 I describe an empirical study that identified significant inter-family variation in louse swimming behaviour, with some families preferring deeper water than others. Chapter 4 addresses whether preventative cage barriers could impose selection on this behaviour, by inadvertently allowing deeper-swimming lice to pass underneath barriers to infest salmon. This was tested using a particle-tracking model, which predicted strong directional selection on swimming behaviour. Taken together, Chapters 3 and 4 offer the first evidence that lice could adapt to physical barriers. Chapter 5 describes a metapopulation model designed to predict evolutionary dynamics of lice across the larger, interconnected farm network. This model will be a versatile tool for future research exploring how various management regimes could limit the spread of resistance through the population. Finally, Chapter 6 ties the threads of this thesis together into a research framework that can be applied to the wider aquaculture sector to assist with managing resistance. This thesis combines theoretical, empirical and modelling techniques to predict the trajectory of L. salmonis evolution in response to different pest management strategies. Key areas for future study are highlighted – more research is crucial if the salmon industry wishes to find ways to stymie louse resistance. More broadly, this thesis illustrates that pests can adapt to management strategies – especially non-chemical methods – in surprising ways. By looking at the world from the parasite’s point of view, we can discover new techniques for safeguarding farms against undesirable evolutionary processes.
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    Effects of temporally heterogeneous stress on individual marine invertebrates
    Hull, Rebecca Barnes ( 2021)
    Some environmental conditions may be stressful, adversely affecting the growth and reproduction of organisms. The impact of stress, however, may vary with its (1) timing, relative to an individual’s stage of life, (2) duration, or the total length of stress, and (3) frequency, or the number of times an individual is exposed. I used the colonial bryozoan Watersipora subatra and the solitary ascidian Styela plicata to study the response of individuals to stress that varies in timing, duration and frequency. I exposed these invertebrates to stress using elevated concentrations of trace metals, W. subatra to copper in the field, and S. plicata to cadmium and zinc via seawater or food in the laboratory. To expose W. subatra to variable stress, and assess their growth and reproduction, I applied stress (1) at different times within a single life-history stage (early reproductive maturity), (2) that varied in duration at different life-history stages (juvenile, young adult or mature adult), and (3) that differed in duration and frequency for adults at two field locations. To assess the uptake and loss of metals with repeated exposure, I exposed S. plicata twice to cadmium and/or zinc in seawater. Individuals performed differently within a life-history stage. When stressed at the onset of embryo production, fewer adult colonies reproduced, but when stressed as adults in the later stages of brooding or producing subsequent bouts of offspring, colonies decreased in size (fragmented) and released fewer larvae compared with unstressed colonies. Generally, juveniles, young adults and mature adults responded according to life-history stage, not the nature of the stress. Young adults produced more embryos than mature adults, whilst mature adults produced smaller larvae with greater settlement success than young adults. Once reproductive, juveniles produced fewer embryos and were less likely to release larvae than both young and mature adults. The duration of stress affected individuals’ reproduction and, sometimes, growth. Regardless of life-history stage, colonies grew little or fragmented when stressed for longer (2 weeks). Adult colonies at one field location produced fewer larvae when stressed for a shorter period (1, 2 weeks), whilst colonies at a second field location were less likely to release offspring and produced fewer larvae when stressed for longer (2, 3 weeks). When the frequency of exposure differed, colonies altered the quality of larvae. Colonies stressed once (cf. multiply) for two weeks, produced smaller larvae with lower settlement success at one field location, whilst similarly sized, yet better settling, larvae at a second field location. S. plicata accumulated more metal after the second (cf. first) dissolved exposure, and more Cd in the presence of Zn than when exposed singly via sea water. This thesis demonstrates that the temporal nature of stress is important for determining an individual’s response – their growth and reproduction, and future success of their offspring. However, the effects of stress for a given individual may depend on the stress’ temporality – timing, duration, frequency – and conditions at a specific location and time.
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    Exploring cis-regulatory convergence between the extinct thylacine and the gray wolf
    Cook, Laura Emily ( 2021)
    Models of convergent evolution allow us to examine the evolutionary processes that drive phenotypic repetition in nature. Investigating the way convergent traits evolve at the genome level is important for identifying the molecular mechanisms that regulate this kind of adaptation. One of the best models for morphological convergent evolution in mammals is that of the marsupial Tasmanian tiger (thylacine; Thylacinus cynocephalus; Thylacinidae) and the eutherian group Canidae (Carnivora), in particular the gray wolf (Canis lupus). These species have evolved striking similarities in craniofacial morphology as a result of a shared ecological niche. However, the precise regions of the genome controlling the evolution and development of convergent skull shape in the thylacine and canid species are unknown. Clues can be found in the non-coding portion of the genome, where regions under accelerated evolution for both species have previously been described (thylacine-wolf ARs). It is hypothesised that the molecular landscape underpinning this remarkable convergence in skull shape can be found here, in the cis-regulatory elements of the non-coding genome. In this thesis, I use computational, developmental and molecular methods to investigate the role of candidate loci in convergent craniofacial evolution for the thylacine and the gray wolf. Because the thylacine is extinct, my first steps were to develop the fat-tailed dunnart (Sminthopsis crassicaudata) as an alternative model for investigating the evolution and development of craniofacial structures in carnivorous marsupials. Importantly, I showed that ossification has not yet begun for newborn dunnart pouch young, highlighting shifts in the developmental timing of craniofacial structures when compared to eutherian models such as the mouse. I then defined the epigenetic landscape of craniofacial tissue for dunnart pouch young at birth, for the purposes of exploring putative cis-regulatory elements as potential drivers of craniofacial development. Comparisons with epigenetic data for mouse embryonic craniofacial tissues showed that the dunnart overlapped to some extent with all murine stages, further emphasising the rapid development of craniofacial structures in the dunnart compared to the mouse. I next assessed whether thylacine-wolf ARs show the same active marks of cis-regulatory activity that I identified in the dunnart. Interestingly, many thylacine-wolf ARs can be seen to overlap with H3K4me3 and H3K27ac enriched regions in the dunnart, providing further evidence for their potential role in convergent craniofacial evolution. Taking a candidate loci approach, I tested a single thylacine-wolf AR with active markers in both the dunnart and mouse in a lacZ reporter study. This revealed that a putative thylacine enhancer of Satb2 drove strong reporter signalling in key craniofacial prominences throughout embryonic development in the mouse. Finally, in order to assess the regulatory capacity of all thylacine-wolf ARs, I designed and optimised a Massively Parallel Reporter Assay (MPRA). My pilot study informed the feasibility of using this high-throughput approach, but also revealed convergent changes in reporter expression for the thylacine-wolf AR upstream of Satb2. Altogether, this thesis deepens our understanding of the role of cis-regulatory convergence in driving phenotypic convergence and contributes fundamental resources for future comparative studies between marsupials and eutherians.