School of Botany - Theses
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ItemDefining the roles of essential genes in the malaria parasite life cycle( 2017)The combination of drug resistance, lack of an effective vaccine and ongoing conflict and poverty mean that malaria remains a major global health crisis. Understanding metabolic pathways at all parasite life stages is important in prioritising and targeting novel anti-parasitic compounds. To overcome limitations of existing genetic tools to investigate all the parasite life stages, new approaches are vital. This project aimed to develop a novel genetic approach using post meiotic segregation to separate genes and bridge parasites through crucial life stages. The unusual heme synthesis pathway of the rodent malaria parasite, Plasmodium berghei, requires eight enzymes distributed across the mitochondrion, apicoplast and cytoplasm. Deletion of the ferrochelatase (FC) gene, the final enzyme in the pathway, confirms that heme synthesis is not essential in the red blood cell stages of the life cycle but is required to complete oocyst development in mosquitoes. The lethality of FC deletions in the mosquito stage makes it difficult to study the impact of these mutations in the subsequent liver stage. To overcome this, I combined locus-specific fluorophore expression with a genetic complementation approach to generate viable, heterozygous oocysts able to produce a mix of FC expressing and FC deficient sporozoites. In the liver stage, FC deficient parasites can be distinguished by fluorescence and phenotyped. Parasites lacking FC exhibited a severe growth defect from early to mid-stages of liver development in-vitro and could not infect naïve mice, confirming liver stage arrest. These results validate the heme pathway as a potential target for prophylactic drugs targeting liver stage parasites. Energy metabolism in malaria parasites varies remarkably over the parasite life cycle. Parasites depend solely on anaerobic glycolysis at blood stage but need Krebs cycle, the electron transport chain, and mitochondrial ATP synthase during mosquito stage development. Again, reverse genetic approaches to study the hepatic stage of Plasmodium have been thwarted because parasites with defects in energy pathways are unable to complete the mosquito stage. I used the genetic complementation approach established to study heme biosynthesis to bridge parasites lacking the β subunit of mitochondrial ATP synthase through mosquito stage and studied their development in the liver stage. ATPase knockouts were indistinguishable from wildtype in in-vitro liver stage assays of size, nuclear content, and merosome production. Robust progression to blood stage confirmed the dispensability of mitochondrial ATP synthesis in liver stages. I extended this approach to explore the essentiality of upstream mitochondrial electron transport and Krebs cycle during the liver stage. I speculate that energy metabolism in the liver stage resembles that in the blood stage, relying predominantly on glycolysis for ATP production. There are numerous genetic tools to manipulate the blood stage malaria parasite genome in general, but existing genetic tools to generate viable parasites with defects in blood stage essential genes are limited. To overcome this limitation, I have developed a novel strategy in which I first insert a complementary copy of the essential gene-of-interest, and then delete the endogenous gene, and then take advantage of meiosis and segregation during the mosquito stage to create haploid knockout sporozoites. I genotype the parasites along the way by fluorescence microscopy. As proof of principle, I created complemented knockouts of the blood stage essential 1-deoxy-D-xylulose-5- phosphate reductoisomerase (DXR) gene, crossed these with wildtype parasites, and then tracked the progeny through in-vitro and in-vivo liver development. Precomplementation proved difficult, perhaps due to inappropriate expression of important metabolic genes. Additionally, problems with apparent silencing of the fluorophore tags compromised my ability to genotype cross progeny preventing any firm conclusion on the function of isoprenoid precursor pathway of liver stage parasites. Nevertheless, my success in generating a blood stage essential gene knockout via precomplementation provides encouragement that this novel reverse genetic strategy can be implemented to investigate the role of blood stage-essential genes in sporozoite and liver stages of malaria parasites.
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ItemPathogenicity genes of Leptosphaeria maculans, the fungus that causes blackleg disease of canola (Brassica napus)( 2017)Blackleg disease caused by the fungus Leptosphaeria maculans is the most serious disease of canola (Brassica napus) worldwide. Current approaches to control blackleg are through the use of agronomic techniques such as crop rotations and sowing disease-resistant canola varieties; however, L. maculans has overcome major gene resistance in commercially released cultivars. Effective control strategies require knowledge of plant defence and fungal pathogenicity mechanisms. Fungal pathogenicity genes are used to establish infection or to avoid plant defences and have the potential to be fungicide targets. The focus of this Ph.D. was to identify and characterise pathogenicity genes in L. maculans. In this thesis five L. maculans mutants were identified by screening L. maculans T-DNA mutants for reduced pathogenicity on B. napus. One of these mutants was identified using TAIL-PCR. Unfortunately, traditional PCR-based techniques such as TAIL-PCR and plasmid rescue are not always successful in identifying T-DNA insertion sites. To overcome this, next generation Illumina sequencing was used to identify the T-DNA insertion sites in the remaining four mutants. The next generation sequencing revealed not only the T-DNA insertion sites but also chromosomal rearrangements, deletions and single nucleotide polymorphisms (SNPs). The decreasing cost of next generation sequencing (NGS) makes this is a cost and time-effective method of identifying T-DNA insertion sites. Two putative pathogenicity genes were identified; one a predicted glutathione synthase gene caused by a T-DNA insertion, the other a transcription factor identified in several of the T-DNA mutants caused by a SNP identified using NGS. The transcription factor identified has homologs that are also involved in pathogenicity in two other ascomycete plant pathogens. This represents a new direction in which to pursue our understanding of the genes required for fungi to cause disease. Transcription factors potentially provide multiple targets for drug or fungicide use. Fungicide targets could also include any of the genes regulated downstream of the transcription factor. The identification and characterisation of pathogenicity genes will ultimately lead to an increased understanding of plant-pathogen interactions and provide new potential targets for the development of antifungals or other molecular-based strategies to control blackleg disease of canola and other plant diseases.
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ItemFunctional analysis of the apical polar ring its role in secretion and motility of Toxoplasma parasites( 2017)Human parasites Toxoplasma and Plasmodium species belong to the phylum Apicomplexa and are some of the most successful groups of human parasites on the planet. Part of this success can be attributed to the cytoskeletal components that afford them structural stability and flexibility required to efficiently attach to and invade host cells. As members of the superfamily Alveolata, they possess a pellicle comprised of a set of flattened vacuoles pressed up against the plasma membrane, with proteinaceous support network and actin actin-based motility system. In addition to this, Toxoplasma also possesses an apical complex which is a tubulin based structure comprised of a set of apical polar rings and a conoid, which is a tight-knit tubulin based structure that is evolutionarily derived from ancestral flagella components. The apical complex is biologically significant because it is the entry point the parasite uses to enter a host cell in order to parasitize it, and this process is conserved in Plasmodium species. However, unlike other organelles, the proteins of the apical complex have no known conserved targeting signals so identification of proteins that target here has been slow to progress. A Toxoplasma protein homologous to a predicted cytoskeletal Tetrahymena thermophila protein was identified and localized to the apical complex, which we call RNG2. RNG2 was functionally characterized by inducible knock down and found that RNG2 played a role in the cGMP signalling pathway upstream of calcium dependent activation of CDPKs, which severely impacted microneme secretion, conoid extrusion and even other downstream processes, particularly internal calcium release. In addition to this, I used various calcium and cyclic di-nucleotide signalling agonists and inhibitors to investigate novel regulation patterns of micronemes and dense granules. RNG2 and other cGMP and calcium signalling proteins, PKG, CDPK1 and CDPK3 all show altered secretion of dense granules showing for the first time a regulatory mechanism of dense granules based on calcium.
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ItemClassification and phylogeny of the plant genus Dianella Lam. ex Juss.( 2017)The global distribution of Dianella Lam. ex Juss. (flax lilies, Asphodelaceae, Asparagales) extends from south-eastern Africa, Madagascar and India, to south-east Asia (north to Japan), Australia, Pacific Islands from Micronesia to Tahiti, Pitcairn Islands, New Zealand, New Caledonia, Norfolk Island and Hawaii. It occurs throughout Australia (excluding the central arid region), where there is the greatest diversity of 42 taxa (19 species and 23 varieties) (Australian Plant Census 2016). Of those, three taxa also extend to south-east Asia, and a further 17 species occur outside Australia (Australian Plant Census 2016; Kew World Checklist of Selected Families, compiled by Govaerts et al. 2016). Plants are characterised by two leaf forms: basal strap-like leaves and cauline leaves on aerial stems (+/- extravaginal branching units). The showy flowers have a characteristic struma between the anther and filament, while the fruit is a fleshy berry, typically in shades of purple. The name Dianella is attributed to the Greek goddess Diana, the mythical goddess of the hunt. A cpDNA phylogeny by Wurdack & Dorr (2009) found Dianella to be monophyletic and sister to the monotypic genus Eccremis from South America. However, there has been no comprehensive phylogenetic analysis of taxa within Dianella, which has the potential to reveal not only taxonomic relationships but biogeographic patterns and the evolutionary history of the group, including the role of polyploidy. Furthermore, species delimitations, including complexes of varieties, have been based only on morphology from field observation and herbarium samples and require further study. Using three chloroplast markers (trnQUUG–5'rps16, 3'rps16–5'trnK(UUU) and rpl14– rps8–infA–rpl36) and two nuclear markers, (ITS 4, ITS 5, 18SE-ETS and DIAN-ETS) molecular phylogenetic analyses using Bayesian and Maximum Parsimony are presented (Chapters 2, 3, 4 and 5). Accessions include the majority of Australian and extra-Australian Dianella. The related outgroup genera Eccremis, Stypandra, Thelionema and Herpolirion were also included. The cpDNA and nrDNA phylograms were relatively congruent and a combined data set produced the most resolution. The combined results (Chapter 5) differed from those of Wurdack & Dorr (2009) in showing Stypandra with a sister relationship to Herpolirion + Thelionema. Within Dianella, resolved clades largely related to biogeographic regions, such as the Hawaiian Islands and New Caledonia, Norfolk Island related to New Zealand and Australian bioregions, revealing for example an early divergence between eastern and Western Australian lineages, congruent with the pattern for other Australian biota. Of the four Australian species complexes described by Rodney Henderson in The Flora of Australia, volume 45, the D. caerulea complex was found to be monophyletic except for two varieties that clustered with other far-north Queensland taxa, and two D. caerulea var. caerulea samples that are morphologically distinct when compared to taxa in the complex. Of the other species complexes, D. revoluta, D. pavopennacea and D. longifolia are each polyphyletic. Their relationships indicate biogeographic patterns, such as for D. longifolia accessions, which were resolved in two separate clades, one clade from the Kimberley and Northern Territory, and one clade from eastern and southern Australia. For extra-Australian Dianella, the widespread D. ensifolia was also polyphyletic occurring in multiple clades with distinct taxonomic units able to be recognised. Chromosome counts available from the literature were plotted on the phylogeny for Dianella and indicated that polyploidy has arisen multiple times, particularly in taxa of some of the Australian species complexes and in D. ensifolia sensu lato. These results indicate the need to recognise new species and to resurrect other taxa for Australian and extra-Australian Dianella. Chapter six is a morphometric, multivariate analysis (using phenetic clustering and ordination methods) of Hawaiian Dianella to determine the number of species on the islands. Field collections were made on Oahu, Maui, Hawaii and Kauai to examine populations in situ, develop species concepts and collect plant material for the dataset. The results indicate that five operational taxonomic units should be recognised including the current taxon D. sandwicensis. Fruit morphology is unique, with distinctive fruit dye colour and fruit surface colour in some taxa. D. lavarum, a narrow endemic that inhabits recent dry lava flows, observed in the Hawaii Volcanoes National Park, is to be resurrected. A review of herbarium specimens confirmed its distributional range extends to Maui, which is in agreement with Otto Degener who originally described the species. The D. caerulea complex was also analysed further in Chapter 7, based on extensive fieldwork in Queensland, New South Wales, Victoria and Western Australia, using multivariate analysis of a morphological data set. Morphometric clusters were largely in agreement with Henderson's varieties, but it is recommended that some be raised to species level. D. caerulea var. assera and D. caerulea var. producta, which appear to be sister taxa based on the shared character extravaginal branching, were each found to include morphological variation. It is recommended that these taxa be recognised as species with three subgroups recognised in D. caerulea var. assera, and five subgroups in D. caerulea var. producta; however, further field sampling is required for taxonomic revision.
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ItemCharacterisation of wall-associated kinases (WAKs) in grasses( 2016)Wall-associated kinases (WAKs) are members of the receptor-like kinase family, an important class of plant-specific plasma membrane proteins considered as potential signalling molecules. WAKs in the model eudicot Arabidopsis thaliana have been proposed to be involved in cell expansion and in response to pathogens and mineral toxicities. WAK proteins also regulate turgor pressure by forming interactions with pectins and other proteins (i.e. glycine-rich protein), suggesting a possible mechanism for WAK involvement in cell wall signalling pathways. In contrast, few reports exist on the role of WAKs in grasses. In this thesis, WAKs from barley (Hordeum vulgare) and Brachypodium distachyon (two model commelinid monocot grass species), were investigated using molecular and biochemical approaches. Selected candidate WAKs were characterised to gain an understanding of their expression patterns, function, and interactions at the cell wall. Chapter III describes the identification of WAK proteins in barley (43) and Brachypodium (115). Through analysis of protein structure and motifs, galacturonan-binding domain, EGF-like domain, and a protein kinase domain were identified as typical motifs of WAKs from barley and Brachypodium. Phylogenetic studies of the identified WAKs revealed that 5 AtWAKs, 32 HvWAKs and 107 BdWAKs clustered into one large clade. Within this large clade, AtWAKs formed an exclusive sub-clade, whereas the HvWAKs and BdWAKs were interspersed amongst several sub-clades. Our result suggest that the grass WAKs likely diverged from the common ancestor after the divergence of monocots and dicots. 10 HvWAKs and 12 BdWAKs were selected for further study based on sequence analysis and technical considerations. Expression profiling studies were performed and described in Chapter III. In several tissues of barley and Brachypodium, various expression levels of the selected WAK genes were observed with a differential expression pattern. In coleoptiles, a rapidly expanding tissue in early development, three WAKs (HvWAK2, BdWAK2, BdWAK7) displayed the highest expression level, with an expression peak at 48 h post-germination. The expression pattern of these genes correlated with the growth of the coleoptile, implying a potential role of these genes in regulating cell expansion. In addition to the expression profiling, experiments were conducted to study the expression of selected WAK genes under stress conditions. As described in Chapter IV, six WAKs (HvWAK14, HvWAK11, BdWAK2, BdWAK7, BdWAK8, BdWAK10) showed significantly increased expression levels upon salicylate (SA) treatment, while four WAKs (HvWAK7, HvWAK14, BdWAK2, BdWAK10) were induced upon salt treatment. In combination with expression profiling results, HvWAK2 and BdWAK2 were chosen as candidate genes for further investigation. Following over-expression of BdWAK2 in Nicotiana leaves, a phenotype reminiscent of hypersensitive cell death was observed (Chapter IV). This phenotype was diminished by either truncation or mutation to the kinase domain of BdWAK2, implicating the kinase activity of BdWAK2 as the cause of the cell death. This result, combined with the fact that expression of BdWAK2 was significantly induced under stressed (both SA and salt treatment) conditions, suggests BdWAK2 may have a significant role in defence responses in Brachypodium. Based on the expression data described in Chapter III, HvWAK2 was thought to be a development-related WAK gene involved in cell expansion. In Chapter V, the sub-cellular location and potential homo-dimerisation of HvWAK2 was investigated. Upon expression with a fluorescent tag in both Nicotiana and onion (Allium sepa), HvWAK2 was observed on the plasma membrane. In addition, using a BiFC approach, homo-dimerisation of HvWAK2 was shown. In order to investigate the nature of the attachment of BdWAK2 and HvWAK2 to the cell wall, an in vitro polysaccharide binding assay was performed (Chapter V). The binding assay indicated that, similar to AtWAK2, both BdWAK2 and HvWAK2 form attachments to pectins, but not other classes of cell wall polysaccharides. Through these findings, WAKs such as BdWAK2 are proposed to have dual intracellular signaling roles modulated via interactions with pectins in the cell wall. Along with a summary of the characteristics of grass WAKs, the final chapter (Chapter VI) discusses how the data obtained for grass WAKs in this study correlates with existing models of WAK signalling mechanisms, and provides a description for more targeted approaches for future work on this large gene family.
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ItemAnalysis of cell division patterns in the shoot apical meristem of gymnosperms( 2016)The structure and organisation of the gymnosperm meristem was investigated by examining 75 species in 35 genera using confocal microscopy of whole mount SAMs. This thesis presents detailed surface, internal and shape patterns that provide an informative and comparative model of meristem growth that is able to account for the diversity and variation of gymnosperm meristem form. On the surface of the meristem, two zones with distinct cell division patterns were recognised. The first is the summit zone that covers the apex of the meristem dome and the second is the peripheral zone that covers the flanks of the meristem. In the summit zone, a single cell divides first in half to create two daughter cells, followed by a perpendicular division in one daughter cell and a similar perpendicular division in the other daughter cell to create a packet of four cells or a tetrad. This division pattern is recognisable as packets of two, three or four daughter cells and the packet grows isotropically. In the peripheral zone, cells divide with repetitive transverse divisions to form long axial chains that create packets with anisotropic growth. Within the cell files, longitudinal and oblique divisions establish new files of cells that widen the peripheral packets. This can be repeated proximally to create distinctly wedge shaped packets in the peripheral zone of some meristems. Within the framework of these two zones with their distinct division patterns, broad categories of meristem structure were recognised. Small and tall apices have a single cell or single packet of two-four cells on the summit, and have distinct, narrow wedge shaped packets in their peripheral zone. Larger, tall and conical meristems commonly have four or more tetrad-type packets in their summit zone, and a complex arrangement of wide, wedge shaped packets in their peripheral zone. The largest and flattest meristems have numerous tetrad-type packets occupying a large summit zone and files of cell are limited to the basal part of the meristem and little or no wedge shaped packets are produced. Only cells in the summit zone divide periclinally to contribute daughter cells to the underlying tissue. Those meristems with a small number of cells in the summit zone have a correspondingly small number of periclinal divisions of apical initial cells, a small mother cell and rib zone and a layered peripheral zone. Those meristems with larger summit zones and many wedge shaped packets have a larger number of apical initials dividing periclinally resulting in a larger zone of mother cells and wider rib zone, as well as wider, distinctly layered peripheral zone. Wide and flat meristems have numerous apical initial cells dividing periclinally and a large zone of mother cells, a wide rib zone and a disordered internal peripheral zone. Within gymnosperms there is a great deal of variation in meristem height, diameter and profile and cellular patterns across the whole apical dome proceed differently depending on the size and shape of the meristem. Meristem profile is a particular important component of meristem shape and determines the location of meristem transition from the summit division type to the peripheral division type. Two homeobox genes WUS and KNOX were investigated in three gymnosperms, Ginkgo biloba, Pseudotsuga taxifolia and Cryptomeria japonica. Histone 4 was used to highlight meristematic activity in the developing leaf primordia of Ginkgo biloba. Homologs of WUS and KNOX genes were identified in all three species, but their role in meristem control was not established. Expression of H4 showed that meristematic activity changed from abaxial expression in the earliest stage of Ginkgo leaf development, to expression in tips of the lobes only during the formation of the lobes, and then back to abaxial during the subsequent stage of leaf development. A model of gymnosperm meristem development is proposed to explain the diversity and variation in gymnosperm meristems. The growth of the meristem operates within a predictable cellular framework that generates a pattern consistent for all gymnosperms and allows meristem structure to be predicted based on size and shape. The model proposed shows that meristem size and shape is a flexible character and that variation in cellular patterns across different gymnosperms appears related to meristem geometry with little correlation to phylogenetic relationships.
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ItemThe Role of DEFECTIVE KERNEL1 (DEK1) in control of mechano-sensitive growth in Arabidopsis thaliana( 2016)During growth, plants experience mechanical stress from external and internal stimuli (Jaffe et al., 2002). These mechanical stimuli have been shown to play a crucial role in plant morphogenesis (Monshausen and Gilroy, 2009). For example, when plants are exposed to external mechanical stimuli, such as, wind, touching or rubbing, they tend to produce shorter and thicker stems, more roots, and increased rigidity of stems and roots (Liu et al., 2007). As plant cells are joined together by their cell walls, cells also experience internal mechanical pressure from their neighbours during growth. There is evidence that plants are able to perceive mechanical stimuli, transduce signals into cells, and respond to modify the cell wall (Braam, 2005). This is thought to be important to maintain coordinated growth and structural integrity during cell expansion. A putative cell wall integrity sensor is the plant specific phytocalpain, DEFECTIVE KERNEL1 (DEK1). DEK1 is a 240kDa modular protein located at the plasma membrane, with a large integral membrane region and a cytoplasmic calpain domain (Lid et al., 2002). In animals, calpains are a family of calcium-dependent cytosolic cysteine proteinases and have been reported to be involved in cell proliferation, apoptosis, differentiation, signal transduction, and also have been implicated in endocytosis, exocytosis, and intracellular membrane fusion (Wang et al., 2003). Previously it has been shown that overexpression of the CALPAIN domain of DEK1 results in plants with increased expression of cell wall-related genes and changes in plant morphology (Johnson et al., 2008). Reduced levels of DEK1 result in loss of adhesion between epidermal cells (Galletti et al., 2015). These features have led to the proposal that DEK1 acts as a putative cell wall mechano-sensor to promote cell wall re-modelling during growth. The general aim of this study was to investigate the signalling events downstream of DEK1 that result in changes to the cell wall and plant growth. Results of this research indicate that DEK1 acts as a novel regulator of plant growth and cell wall re-modelling in rosette leaves (Chapter 3) and stems (Chapter 4). In plants with increased levels of calpain (calpain oe), the timing of the transition from cell division to expansion was altered in rosette leaves, followed by a longer expansion phase resulting in enlarged cells. In contrast, an early exit from cell division occurs in plants with reduced levels of DEK1 (amiRNA-DEK1) contributes to smaller organ size. Changes in cell division and expansion also occurred in the stems of plants with altered DEK1. Increased number and size of cells in the cortex and pith were observed in calpain oe stems whereas a decrease in cortex and pith cell size was seen in amiRNA-DEK1 lines. Investigation of cellular morphology of the epidermal layer revealed defects in cell-cell contact in both calpain oe and amiRNA-DEK1lines. The altered contact zones most likely occur due to irregular size and shape of epidermal cells and changes in cell wall composition. Transmission electron microscopy (TEM) studies of walls in epidermal cells from rosette leaves and stems show increased and decreased wall thickness in calpain oe and amiRNA-DEK1, respectively. These results support previous findings by Johnson et al. (2008) that DEK1 is involved in regulating cell wall-related genes. A detailed analysis of cell wall composition was undertaken to determine if changes occur in calpain oe and amiRNA-DEK1 plants compared to wildtype (Chapter 3 and Appendix 1). No obvious changes were observed in monosaccharide and polysaccharide composition of walls whereas immunofluorescence, light and TEM studies revealed changes in the distribution and proportion of cell wall polysaccharides in the epidermal layer. Cellulose and pectin are the cell wall components most influenced by changes in DEK1, both in leaves and stem and suggest a role for DEK1 in the regulation of primary wall growth. Gene expression studies using an inducible calpain overexpression (ioex-calpain) construct showed genes involved in cell wall loosening and pectin biosynthesis were up-regulated. Based on these results, we suggest that the altered phenotypes observed in DEK1 mutants most likely arise from changes in cell wall composition that impact upon their mechanical properties.
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ItemQuantifying search and control performance during marine invasive surveys: a case study from Asterias amurensis( 2015)Marine invasive species are a global threat to marine biodiversity. Effective management of invasive species depends on accurate population information. To best inform management, surveys of abundance, occupancy and detectability must be carefully designed, and account for uncertainty. However, many marine invasive management programs currently do not record and account for the uncertainty of detectability. In this thesis, I document the limitations of searching for a marine invasive species during eradication programs and examine ways to improve detectability in future survey designs. Specifically, I undertake a series of empirical surveys to test how effective observers are at detecting the northern Pacific seastar, Asterias amurensis; this species poses a serious threat to native and commercial species in southern Australia. I use artificial silicone replicas of A. amurensis during empirical surveys so as to eliminate the risk of spreading the marine invasive species. I use data combined with Bayesian methods to develop a population catch-effort model, which provides insights into what influences detectability and whether eradication at these sites was a viable management goal. Finally, I take a novel approach to testing optimal search theory under field conditions. Optimal search theory has been used to support resource allocation when managing invasive species. This is the first time, to my knowledge, environmental decision theory has been tested empirically by examining applications of search theory for any species in an ecological setting. I found that animal size, target distance from the transect line and group clustering size all affect detectability. I also found that pre-survey training reduced the frequency of incorrect detections of A. amurensis for two native co-occurring species by up to 16.1%. I also demonstrate the amount of search effort required to eradicate populations at a site is often considerably higher than the effort actually invested to completely remove a population of A. amurensis. Lastly, I found that using an optimal search strategy compared to three other routinely used strategies during surveys for A. amurensis can improve the number of seastars removed by upwards of 12% for a 20 minute search budget. Evaluating how well previous removal efforts eliminate marine invasive species from a site, and understanding the uncertainty of survey design are critical to improving future post-border management responses. The northern Pacific seastar A. amurensis will continue to threaten the marine environment in its non-native distribution. Understanding how to improve survey design will continue to be essential for active and successful management.
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ItemNovel methods to account for individual heterogeneity in capture-recapture studies( 2015)A major challenge in ecological analyses is estimating population-level parameters such as survival, births and population size when most individuals are not observed by sampling. Capture-mark-recapture (CMR) models provide the ability to understand what proportion of the population is missing in sampling and to account for this missingness in inference. Yet one of the standard assumptions behind CMR analyses is that the probability of sampling an individual is either constant across all individuals or can be modelled as a function of fully-observed covariates. However, in many cases this is not true, a factor that can seriously bias estimates of population-level demographic processes. This thesis develops novel methods to account for individual heterogeneity in capture probabilities. I first provide a general review of CMR studies and history of their development, as well as the application of Bayesian state-space models to CMR studies. I discuss the motivating sampling scenario in which a number of common sources of bias are present, then describe how CMR sampling approaches were deployed to try and account for these biases. In chapter two, I propose a solution to the problem of bias due to temporary migration. I describe a capture-recapture sampling scenario on a population that is closed to births and deaths but in which capture probabilities are confounded by migration, then develop a model to account for migration using auxiliary radio telemetry data. The telemetry data provide a means to estimate migration rates, which can then be used to account for the bias in estimated capture probabilities. Simulation studies show that this approach allows for unbiased estimates of population sizes and should be applicable to a range of situations. Chapter three then considers the problem of state misclassification in data where mortality of animals can be inferred via remote observations of movement patterns. I show that in some cases, misclassification of individuals as dead can result in biased estimates of survival rates. I employ a state misclassification model in order to correct for such errors and show that in wild populations of native fish such bias can result in a reduction in estimated survival rates of up to 50%. Next I describe a means of estimating age-specific capture and survival rates in CMR scenarios involving animals with well-de ned growth patterns. The approach involves estimating growth parameters from length interval data in a capture-recapture context or from other ageing methods, then using these growth as prior information in a CMR model in order to estimate age at first capture. We show how otolith and CMR-based estimates of age correspond well, and simulation studies show how estimate survival rates closely match true values. Finally, I consider a large-scale sampling scenario in which multiple sources of bias are possible and multiple sources of data are available to help account for these errors. I develop a state-space modelling approach to incorporating multiple sources of data in this context. I show how it is possible to evaluate the relative fit of models with differing types of data on the same population through a combination of root-mean-square error evaluation, simulation modelling and comparison of the magnitude of various model parameters. I show how single sources of CMR data in this context are likely influenced by various kinds of individual heterogeneity, but show through simulations how it is possible to help correct for this source of bias by incorporating multiple data sources. I also show how incorporating latent state data can help correct parameter estimates that would otherwise be heavily biased in a model that otherwise appears to have a relatively good fit.
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ItemDecision analysis for threatened species management across the captive-wild spectrum( 2015)Programs for the recovery of threatened species increasingly involve active management of variable intensity, such as captive breeding, reintroduction and translocation. Managers of such programs thus need to make decisions about whether and how to implement a given type of management, usually in the face of uncertainty and constraints. Structured decision making provides decision makers with a theoretical framework and practical methods to make rational decisions under uncertainty. In this thesis, I investigate how different principles and tools of structured decision making can assist decisions in threatened species management. The fundamental decision problems in threatened species management concern whether and how to implement a given type of management for the target species. In Chapter 2, I examine the case of whether the conservation of a species should incorporate ex-situ management, using the recovery plan for an endangered frog species in south-eastern Australia as an example. I demonstrate that this question can only be answered rationally after determining how the ex-situ component will benefit the overarching objectives of the recovery plan. This logical sequence of decisions (how before whether) is however followed only infrequently in real-world conservation. I illustrate how managers can implement it with the aid of decision trees and multi-criteria decision analysis. Since decisions are aimed at achieving objectives, they necessarily reflect the values and preferences of stakeholders. Using value functions, in Chapter 2 I demonstrate how the optimal decision depends on the relative importance attributed to different objectives (for example, maximising the probability of persistence of the target species and meeting budget limitations). In Chapter 3, I expand this analysis to demonstrate how to account for the attitude of decision makers towards the risk of negative outcomes. I use two case studies of recovery plans for threatened frog species to illustrate the application of stochastic dominance, a useful method to rank alternative actions in the face of uncertainty and risk. Structured decision making provides methods to make decisions under uncertainty. However, in some instances reducing the existing uncertainty by collecting additional information can allow more robust decisions. As a result, managers of threatened species programs almost always advocate further research, in the expectation that it will improve the outcomes of management. In Chapter 4, I apply a formal method to calculate the expected benefit of additional information to two examples in threatened species management. I demonstrate how the value of information is a function of the current knowledge, by the potential to react to new information, and by the effectiveness of the learning process. Value of information analysis can help managers determine whether and how to implement experiments and monitoring programs to improve the ultimate outcomes of management. For several threatened species programs, the scale and speed of the threatening processes often require decisions to be made immediately, leaving no time for formal experimental learning. Adaptive management describes a specific case of structured decision making in which managers learn by monitoring the outcomes of management and adjust actions accordingly. In Chapter 5, I analyse the conditions and challenges that exist to the application of adaptive management in threatened species programs. The temporal scale of such programs is often sufficient to allow managers to collect information and react by updating actions in subsequent time steps. Particularly for programs toward the captive end of the management spectrum, controlled conditions are also favourable for effective learning. Adaptive management requires the ability to clearly structure uncertainty into formal hypotheses, to allow effective and focused monitoring that addresses the most important sources of uncertainty. Most importantly, institutions and stakeholders must be committed and capable of implementing learning. For clearly defined decision problems, structured decision making can draw upon a range of technical approaches to determine optimal management strategies. In Chapter 6, I consider a reintroduction program in which management decisions are complicated by the complex life history of the target species and budget constraints. I combine demographic modelling and cost-effectiveness analysis to identify the optimal rates of translocation between captive and wild populations. This thesis illustrates how the iterative cycle of structured decision making can benefit all stages of the design of management strategies for threatened species conservation. First, it can help managers in thinking clearly about the decision problem, allowing a transparent assessment of subjective preferences and value. It can then ensure an objective evaluation of the available management alternatives, using qualitative or quantitative predictive approaches that explicitly recognise uncertainty. Finally, it can assist in finding solutions to trade-offs and incorporating additional knowledge to allow better decisions.