A cryptic haplosporidian parasite was detected infecting rock oysters from the Montebello Islands in north-western Australia using a PCR targeting the parasite’s small ribosomal subunit gene. The PCR products were cloned and sequenced along with the remaining sections of the parasite’s SSU rRNA gene. Phylogenetic analysis of the sequence generated indicated a Minchinia species (Haplosporidia). The SSU sequence generated was used to develop two in situ hybridisation assays to visualise the parasite in H/E sections as well as a PCR assay to detect the parasite. The molecular assays were assessed for specificity and sensitivity and were then used to compare the parasite to previous haplosporidian parasite infections of pearl oysters. Both assays produced positive results from the infected pearl oysters but not from other closely related haplosporidian species. An SEM and TEM electron microscopy analysis was performed on spores from both parasite species. The spores of the pearl oyster parasite had two spore wall filaments wound around the spore originating for a posterior thickening while the spores of the rock oyster parasite were covered in microtubule-like structures. These data suggests pearl oysters where co-infected with both the Haplosporidium sp. and the Minchinia sp. detected in rock oysters. No evidence of a posterior thickening could be found on the spores of the rock oyster parasite. Attempts to detect the parasite at the previous geographic sites of its detection in pearl oysters resulted in detection of the Minchinia species in tropical oysters in the Kimberley region of Western Australia by in-situ hybridisation.

The Aristolochiaceae are one of the largest angiosperm families, the family has been divided into two subfamilies: Asaroideae, which include Asarum and Saruma, and Aristolochioideae, which includes Thottea sensu lato and Aristolochia sensu lato (Kelly and Gonzales, 2003). Aristolochia sensu lato comprise between 450 and 600 species, distributed throughout the world with centers of diversities in the tropical and subtropical regions (Neinhuis et al., 2005, Wanke et al., 2006a, 2007). However, the extended Mediterranean region including Turkey, the Caucasus and the Near East is likely to be the only diversity hotspot of the genus Aristolochia in the northern hemisphere were up to 60 species and subspecies could be observed (Wanke 2007). Most important contributions to the knowledge of these species were published by Nardi (1984, 1988, 1991, 1993) and Davis & Khan (1961, 1964, 1982), all of these studies were based on morphological characters only. In recent years, with the progress of molecular techniques and in light of the systematic chaos, a detailed study was needed to unravel the evolutionary history prior to a taxonomic revision of this group. The first chapter of my thesis should be regarded as the starting point for more detailed investigation on population level. Preliminary molecular phylogenitic analysis recovered the Mediterranean Aristolochia species as monophyletic (de Groot et al 2006). However, only very few members were included in that study. The latest phylogenetic study by Wanke (2007) dealed with west Mediterranean Aristolochia species and sampled also few members belonging to the east Mediterranean and Caucasian species (3 from Greece, 2 from Georgia and 1 from Turkey). This study reported the Mediterranean Aristolochia species as two molecular and morphologically well supported clades, which were sister to each other. Furthermore, the two closely related species A. sempervirens and A. baetica which have an east west vicariance and are known as Aristolochia sempervirens complex has been recovered as sister group to the remaining west Mediterranean species. A detailed investigation of the evolutionary history of this group is the topic of the second chapter of my thesis (Chapter 2). The Aristolochia sempervirens complex is characterized by an unusual growth form and has a circum Mediterranean distribution. The investigation of these species complex seem to be of great importance to understand speciation and colonization of the Mediterranean by the genus Aristolochia and might shade light in historical evolutionary processes of other plant lineages in the Mediterranean. Furthermore, I test applicability and phylogenetic power of a nuclear single copy gene (nSCG) region to reconstruct well resolved and highly supported gene genealogies as a prerequisite to study evolutionary biology questions in general. Furthermore, a comprehensive overview of leaf epicuticular waxes, hairs and trichomes of 54 species from the old and new world taxa of the genus Aristolochia were investigated using scanning electron microscopy (SEM) to clarify taxonomic status of theses species in contrast to their molecular position. Also this study which is the third chapter of this thesis (Chapter 3), has a strong focus on Mediterranean Aristolochia and tries to provide additional support for molecular findings based on epicuticular waxes and to test them as synapomorphies. Each chapter has its own introduction and abstract resulting in a short general introduction here.

Recently, hybridisation has been increasingly recognised as contributing to the extinction of species; with the risk especially high for rare species hybridising with more common species. Such risks have raised concerns for the Banks Peninsula tree weta, Hemideina ricta, which is restricted to the eastern half of Banks Peninsula and in some areas lives in sympatry with the more widespread Canterbury species, H. femorata. A previous genetics study found evidence of hybridisation between these two species. However, conclusions made by this study were likely limited by its small sample size. To further assess the risk hybridisation poses to the conservation of these species, a larger genetic study was undertaken. With hybridisation between H. ricta and H. femorata previously hypothesized to be a rare event, modelling of likely sympatric zones was undertaken to optimize the sampling effort. The results of genetic analysis on the resulting samples were consistent with the previous study, in that they suggest hybridisation does occur but is fairly rare. To help determine what processes are maintaining the distinction between the two species, the current study has expanded to incorporate observations of mating behaviour and egg hatching experiments. As well as potential risks from hybridisation, H. ricta and H. femorata have also suffered habitat loss. The Canterbury region has been transformed by the introduction of exotic plant species, fire and logging, with only small patches of native bush remaining. The loss and fragmentation of the native forest is likely to impact the forest fauna such as the tree weta. A previous study of a closely related species H. maori, in a naturally fragmented habitat, determined that dispersal between suitable habitat patches was fairly limited. Therefore, similar to their habitat, H. ricta populations may be small and isolated. Such populations are prone to the fixation of deleterious alleles as well as a loss of genetic diversity. Deleterious traits not only have a short term negative impact but a lack of genetic variation can prevent adaptation in the long term. In the past, studies of population structure have included the influence of intrinsic factors, such as dispersal capabilities but neglected extrinsic factors, such as the environment. The current study uses microsatellite markers to determine the population structure of both species and where possible, maps of land-cover are analyzed for a correlation with genetic structure.

It has become well known that an evolutionary tree is inadequate to represent fully the history of life. Two possible ways of dealing with this are the rooted subtree prune and regraft distance between a pair of trees, which measures how different they are, and the slightly more biologically sound hybridisation number of a set of trees that attempts to determine the minimum number of hybrid events that must have occurred for a given set of evolutionary trees. When characterised via agreement forests both problems are, although NP hard, fixed parameter tractable---meaning the problem can be converted to a similar problem with a smaller input size. This thesis investigates ways of improving existing algorithms for calculating the minimum rooted subtree prune and regraft distance and hybridisation number for a pair or, in the latter case, set of trees. In both cases a technique is used that allows the problem to be rekernelised during the run of the program. Another, less effective method, is also looked at which finds the rooted subtree prune and regraft distance or hybridisation number solely on what cannot be contained within any agreement forest. Additionally the characterisation of the minimum rooted subtree prune and regraft distance via maximum agreement forests is extended to non-binary trees and the hybridisation number of a set of phylogenetic trees is extended to unrooted trees.

The purpose of this study was to use molecular techniques and morphological measurements to set out to find whether a hybrid zone exists between two coastal populations of the common gecko (Hoplodactylus maculatus), on the Wellington south coast. I collected geckos from five sites in a coastal transect from the population of small geckos to the large geckos. Using four genetic loci, one mitochondrial (16S) and three nuclear (Rag-1, Rag-2, C-mos), I was able to determine that the coastal populations do have geneflow, however each population maintains some unique alleles. Morphological evidence reveals a significant difference in gecko sizes from Turakirae Head and those caught at Ocean Beach, separated by just 15 km. Adult geckos at Turakirae Head are on average 10mm smaller (snout-to-vent) than adult geckos at Ocean Beach, representing almost a doubling in average weight. The centre of the steep frequency clines of four characters is coincident and the widths are concordant. The narrower morphological clines indicate stronger selection on the size of the gecko, than on genetic loci.

Microbial communities associated with gastrointestinal tract of animals play a critical role in gut development, digestion and resistance to disease, thus the prospect of altering these communities beneficially by using probiotics is attractive. In terrestrial animals, the gut provides a stable, moist habitat in an otherwise moisture-limited environment, thus microbial communities tend to be very stable. In contrast, farmed aquatic animals reside within an environment that can support microbes in high densities, and as many marine animals drink continuously for osmoregulation, they are subjected to potential re-inoculation. Consequently, little is known of the stability of gut microbial communities in marine shrimp or whether it is possible to establish beneficial bacteria in the gut. The aims of this thesis were therefore to examine the midgut microbial community associated with farmed black tiger shrimp, Penaeus monodon, and to investigate whether the introduction of potentially probiotic Bacillus could alter the species diversity or abundance of the present microbes. Using culture methods it was found that B. pumilus was able to transfer between animals via the water column and persisted in the midgut for at least 7 days, while B. subtilis was only recovered from animals directly fed the bacteria and persisted for less than 24 h in the midgut. V. parahaemolyticus, a known shrimp pathogen,remained in the tanks it was originally found in, and did not transfer via the water column to other tanks and is therefore tightly associated with its host. A bacterium with apparent probiotic qualities was isolated from control animals in the above study and identified as a strain of B. pumilus. Its safety for food animal use was confirmed due to the absence of B. cereus toxin genes, and the isolate’s pH and salt tolerances were investigated. Moreover, the isolate was highly inhibitory to crustacean pathogens in the family Vibrionaceae. Methods to investigate the gut microbiota using the full cycle 16S rRNA methodology were optimized. Fluorescence in situ hybridization (FISH) probes designed specifically targeting B. pumilus, B. subtilis and B. licheniformis, commercially available probiotics, were validated for specificity and optimal hybridization conditions. For FISH analysis of bacteria in situ in histological sections of shrimp midgut trunks, fixation times in 4 % paraformaldehyde wereoptimizedfor bacterial RNA retention whilst maintaining tissue integrity. Due to the broad range of autofluorescence in the shrimp tissue, spectral imaging is required to adequately differentiate between host tissue and multiple bacterial probes. The richness and diversity of the midgut microbiota of animals treated with the novel strain of B. pumiluswere analyzed using 16S rRNA gene clone libraries and FISH analysis of histological sections. It was confirmed that B. pumilus can enter the midgut via top-coated feed and through water inoculation. In the tanks that were treated with B. pumilus the proportion of Vibrio sp. in the microbial community decreased, however, only in the systems in which B. pumilus was recovered from the shrimp midgut did the proportion of pathogenic Vibrio species decrease. The application of the B. pumilus caused a shift in the shrimp midgut microbiota, but the community returned to its initial diversity over time. The midgut microbiota of P. monodon is relatively stable but can be adjusted using probiotics. The transience or residence of the probiotics is strain-specific and should be tested for any new strains before determining optimum application protocols. The methods designed in this study are applicable to future research in this field.

All animals have evolved mechanisms to recognise and eliminate nonself in order to defend against invading pathogens and to prevent chimerism, the fusion between genetically distinct conspecifics. Like other metazoans, sponges are known to rely on sophisticated systems to maintain their self-integrity. As poriferans are also considered one of the most ancient extant metazoan phyla, they represent a critical comparative model for understanding the early evolution of immunity and self/nonself recognition in animals. The Toll-like receptor (TLR) signalling cascade plays a crucial role in immunity, and recent findings in the sponge Suberites domuncula suggest that its origin could predate eumetazoan cladogenesis. My genome and expressed sequence tag (EST) screens of the demosponge Amphimedon queenslandica detected homologues to most components of this pathway, supporting the notion that a primordial TLR signalling cascade emerged at the dawn of the Metazoa. The sponge also encodes a couple of putative TLR-related proteins (AmqIgTIRs) that consist of at least one extracellular immunoglobulin (Ig) and an intracellular Toll/Interleukin-1 receptor/resistance (TIR) domain. The presence of other unconventional TLRs in S. domuncula and in cnidarian representatives, implies that an ancestral TLR probably existed in the last common ancestor of all living metazoans, and independent duplication and divergence events led to the variety of forms observed in animals. Among the putative transcription factors present in Amphimedon, which are known to be activated by the TLR signalling cascade in other eumetazoans, I detected a single member of the Rel/nuclear factor-kappaB (NF-κB) family, AmqNF-κB, which is also the only Rel homology domain (RHD)-containing gene present in the sponge. This gene encodes a protein that is equipped with both a RHD and ankyrin (ANK) repeats, suggesting that the ancestral metazoan NF-κB was configured identically to contemporary vertebrate and sponge forms, and that the truncated NF-κB found in Nematostella vectensis resulted from the secondary loss of ANK. Aside from immunity, the Toll and TLR pathways contribute to a variety of biological processes in bilaterians, however their functions have only been investigated in detail in a limited number of metazoan model organisms. While studies have tested the immune role of various sponge genes, including components of the TLR cascade, no research has yet established whether they are also involved in development. Therefore, I investigated the expression of some of the immunity-related genes I isolated in Amphimedon in a developmental and immune context to shed light on the potential ancestral function(s) of the proteins they encode. Using in situ hybridisation, I demonstrate that AmqIgTIR2, AmqMyD88, AmqTollip, AmqPellino and AmqNF-ĸB are expressed during A. queenslandica early development. In contrast, the spatial and temporal expression of AmqIgTIR1 suggests it might encode a receptor that is specifically involved in the detection of metamorphic cues in larvae. A real-time quantitative PCR (qPCR) study performed on a pool of adult sponge cDNAs indicates that the expression levels of AmqIgTIR1, AmqIgTIR2, AmqMyD88 and AmqTollip are significantly affected by a nine-hour incubation in 50 µg/ml of lipopolysaccharide (LPS), and to a lesser extent by 105 colony forming units (cfu)/ml of live Vibrio harveyi. The gene expression of AmqIgTIR1 and AmqIgTIR2 suggests that they may encode proteins with antagonistic immunological functions. While AmqPellino and AmqNF-ĸB do not appear to be affected by LPS and Vibrio exposure, it is possible that these genes do not participate in the early immune response of poriferans. Together, my data indicate that the sponge genes surveyed might encode proteins that perform developmental, sensory and immunological functions, suggesting their roles could have also been multifaceted in the last common ancestor to all living metazoans. As is observed in other invertebrates, poriferans display an ontogenic shift in allorecognition; genetically different individuals can fuse during early development but, in most instances, not as adults. However, there is a limited understanding of the cellular organisation of sponge chimeras and the onset of poriferan allorecognition response. By following the fates of fluorescently tagged cells derived from genetically distinct Amphimedon larvae that are fused together at metamorphosis, I establish that there is a rapid ontogenic shift in the sponge allogeneic response about two weeks after the initiation of metamorphosis. Moreover, the molecular basis of the poriferan allorecognition system is possibly involved in creating differential cell affinities, which underlie the construction of the sponge body plan. Compatible with this scenario is the observation that cells from postlarvae that are allowed to develop for two weeks before contact do not fuse, and form a distinct boundary between genotypes. The molecules responsible for sponge cell reaggregation, the aggregation factors (AFs), have been proposed to drive the allorecognition response in poriferans. Notably, the Amphimedon genome encodes six putative AFs, of which five occur in a cluster. These findings indicate that the polymorphic variation observed in other poriferan AFs is probably the result of allelic variations of multiple genes belonging to the same family.

It has become well known that an evolutionary tree is inadequate to represent fully the history of life. Two possible ways of dealing with this are the rooted subtree prune and regraft distance between a pair of trees, which measures how different they are, and the slightly more biologically sound hybridisation number of a set of trees that attempts to determine the minimum number of hybrid events that must have occurred for a given set of evolutionary trees. When characterised via agreement forests both problems are, although NP hard, fixed parameter tractable---meaning the problem can be converted to a similar problem with a smaller input size. This thesis investigates ways of improving existing algorithms for calculating the minimum rooted subtree prune and regraft distance and hybridisation number for a pair or, in the latter case, set of trees. In both cases a technique is used that allows the problem to be rekernelised during the run of the program. Another, less effective method, is also looked at which finds the rooted subtree prune and regraft distance or hybridisation number solely on what cannot be contained within any agreement forest. Additionally the characterisation of the minimum rooted subtree prune and regraft distance via maximum agreement forests is extended to non-binary trees and the hybridisation number of a set of phylogenetic trees is extended to unrooted trees.

The pea weevil (Bruchus pisorum L.) is the most significant pest of field pea (Pisum sativum L.) in Australia. The only available means for controlling pea weevil at the present time is with chemical pesticides. The aim of this study was to introgress natural pea weevil resistance, derived from the wild pea species, Pisum fulvum Sibth. & Sm. into cultivated field pea and devise strategies for screening for the resistance with breeding applications. Traditional breeding methods were used to transfer pea weevil resistance from P. fulvum accession ‘ATC113’ to cultivated field pea, cv. ‘Pennant’. Progeny derived from this population were examined for inheritance of pod and seed resistance. Seed resistance in F2 plants segregated in a ratio of 1:37:26 (resistant: mixed response: susceptible), indicating a trigenic mode of inheritance (1:63), with at least three major recessive genes controlling pea weevil resistance. Seed resistance was conserved over consecutive generations (F2 to F5) and was successfully transferred to populations crossed with a second adapted field pea variety‘Helena’. Pod resistance presented as a quantitative trait in the F2 population, but this resistance was not retained in subsequent generations. Amplified fragment length polymorphisms (AFLPs) were sought in the parents and in resistant and susceptible F3 plants. Restricted maximum likelihood (REML) analysis was used to identify 13 AFLP markers with a statistically significant association with pea weevil resistance and 23 with pea weevil susceptibility. Principal coordinate analysis (PCO) showed that the AFLP marker loci formed clusters in the PCO space, which could indicate the three proposed gene locations. Eight AFLP markers were cloned, sequenced and converted to sequence characterised amplified regions (SCAR). Two SCAR markers, SC47359 and SC47435 were polymorphic between the resistant and susceptible parents. Both markers co-segregated with the resistant lines and with 30-36% of susceptible lines. Plants which did not possess either band were highly susceptible. The other PCR products were either monomorphic between the resistant and susceptible parents or produced more than one band product. A range of phenotypic traits was measured in the F2 population derived from the hybridisation between P. fulvum and P. sativum and associations with pea weevil resistance were made. In the F2 population, pea weevil resistance was not correlated with any of the negative traits originating from the wild parent, such as increased basal branching, dark seed coat or small seed size, neither was resistance correlated with flower colour, flowering time or seeds per pod. Pea weevil resistance should therefore be transferable with minimal linkage drag. A convenient morphological marker, such as flower or seed colour was not identified in this study based on these results. Using principal component analysis (PCA) as a visual tool, resistant and semi-resistant plants in the F3 and ‘backcross’ introgression populations were identified with improved trait performance compared with the wild parent

Public sector reforms of recent decades in Europe have promoted managerialism and aimed at introducing private sector thinking and practices. However, with regard to public sector executives' self-understanding, managerial role identities have not replaced bureaucratic ones; rather, components from both paradigms have combined. In this article, we introduce a bi-dimensional approach (attitudes and practices) that allows for different combinations and forms of hybridity. Empirically, we explore the role identities of public sector executives across Europe, building on survey data from over 7,000 top public officials in 19 countries (COCOPS survey). We identify country-level profiles, as well as patterns across countries, and find that administrative traditions can account for these profiles and patterns only to a limited extent. Rather, they have to be complemented by factors such as stability of the institutional environment (indicating lower shares of hybrid combinations) or extent of reform pressures (indicating higher shares of hybrid combinations).