The leishmaniases are neglected tropical diseases with an urgent need for effective drugs. Better understanding of the metabolism of the causative parasites will hopefully lead to development of new compounds targeted at critical points of the parasite’s biochemical pathways. In my work I focused on the pentose phosphate pathway of Leishmania, specifically on transketolase, sugar utilisation, and comparison between insect and mammalian infective stages of the parasites. The pentose phosphate pathway (PPP) is the major cellular source of NADPH, an agent critical for oxidative stress defence. The PPP uses glucose, reduces the NADP+ cofactor and produces various sugar phosphates by mutual interconversions. One of the enzymes involved in this latter part is transketolase (TKT). A Leishmania mexicana cell line deleted in transketolase (Δtkt) was assessed regarding viability, sensitivity to a range of drugs, changes in metabolism, and infectivity. The Δtkt cell line had no obvious growth defect in the promastigote stage, but it was more sensitive to an oxidative stress inducing agent and most of the drugs tested. Most importantly, the Δtkt cells were not infective to mice, establishing TKT as a new potential drug target. Metabolomic analyses revealed multiple changes as a consequence of TKT deletion. Levels of the PPP intermediates upstream of TKT increased substantially, and were diverted into additional reactions. The perturbation triggered further changes in metabolism, resembling the ‘stringent metabolic response’ of amastigotes. The Δtkt cells consumed less glucose and glycolytic intermediates were decreased indicating a decrease in flux, and metabolic end products were diminished in production. The decrease in glycolysis was possibly caused by inhibition of fructose-1,6-bisphosphate aldolase by accumulation of the PPP intermediates 6-phosphogluconate and ribose 5-phosphate. The TCA cycle was fuelled by alternative carbon sources, most likely amino acids, instead of glucose. It remains unclear why deletion of TKT is lethal for amastigotes, increased sensitivity to oxidative stress or drop in mannogen levels may contribute, but no definite conclusions can be made. TKT localisation indicated interesting trends too. The WT enzyme is present in the cytosol and glycosomes, whereas a mutant version, truncated by ten amino acids, but retaining a C-terminal targeting sequence, localised solely to glycosomes. Surprisingly, cells expressing purely cytosolic or glycosomal TKT did not have different phenotypes regarding growth, oxidative stress sensitivity or any detected changes in metabolism. Hence, control of the subcellular localisation remains unclear as well as its function. However, these data are in agreement with the presumed semipermeable nature of the glycosome. Further, L. mexicana promastigote cultures were grown in media with different combinations of labelled glucose and ribose and their incorporation into metabolism was followed. Glucose was the preferred carbon source, but when not available, it could be fully replaced with ribose. I also compared metabolic profiles from splenic amastigotes, axenic amastigotes and promastigotes of L. donovani. Metabolomic analysis revealed a substantial drop in amino acids and other indications coherent with a stringent metabolic response in amastigotes. Despite some notable differences, axenic and splenic amastigotes demonstrated fairly similar results both regarding the total metabolic profile and specific metabolites of interest.

The Earth’s climate is changing at an unprecedented rate due to increasing use of fossil fuels and widespread deforestation. This means that the concentration of atmospheric carbon dioxide is increasing, elevating mean global temperatures. In addition, the oceans act as a huge carbon sink and are absorbing more carbon dioxide than they have in the last 650, 000 years causing the oceans to become more acidic. At the beginning of this study the guidelines stated that the control pCO2 concentration for laboratory studies should be 380 µatm (Meehl et al., 2007), concordant with the then current atmospheric pCO2. Four years later, the most recent literature reports that current concentrations of atmospheric pCO2 have now risen to 400 µatm (IPCC, 2013). This demonstrates the unprecedented speed at which our climate is changing and highlights the urgency for research into the potential implications that this change may have on marine systems. In many coastal marine systems calcifying organisms construct biogenic formations that can underpin the ecosystem and form biodiversity hotspots. Calcifying algae and cold water corals are two such organisms. These habitats are economically, politically and socially important. However our knowledge of how these keystone marine organisms may respond in the future is still ambiguous. In general marine calcifiers are likely to be negatively affected, although within some taxa there is considerable variability in their response during climate change studies (e.g. coralline algae and corals). Furthermore, natural variability will overlay the environmental changes associated with anthropogenic global climate change, and as such is likely to significantly influence the response of marine biota to the projected environmental changes. Currently, there are few studies that consider global climate change in the context of natural variability and/ or run long enough to assess acclimatisation potential. Thus, this research aims to provide a better understanding of the impact global climate change may have on key marine calcifiers in the context of natural variability and acclimatisation. This was achieved through a number of laboratory- and field-based studies, utilising well established and adapted techniques. This research focused primarily on red coralline algae. Projected changes in seawater carbonate chemistry mean that marine organisms that utilise dissolved inorganic carbon species as a substrate for multiple physiological processes (i.e. photosynthesis and calcification) are likely to be amongst those most greatly impacted by environmental change. Chapter 3 is the longest laboratory study (24 months), to date, investigating the effects of global climate change on a marine calcifier. Results suggest that seasonal variability in environmental conditions will greatly impact the response of coralline algae to elevated temperature and pCO2. In addition, while calcification may be maintained or increase in response to elevated temperature and/ or pCO2 (chapter 3 and 5) and despite evidence of acclimatisation potential, overall growth was significantly hampered by elevated temperature in the long term. This supports the hypothesis that dissolution may in fact be the primary threat to marine calcifiers, as opposed to impaired calcification. Irradiance is key in coralline algal photosynthesis yet the role of light availability on mediating coralline algal responses to multiple stressors remains scant. The present study examined net photosynthesis and photosynthetic characteristics in the free-living coralline algae, Lithothamnion glaciale in response to sub-diel changes in irradiance in algae exposed to elevated temperature and pCO2 (chapter 4). Observations suggest that light availability will mediate the response of coralline algae to global climate change in the future, as optimal light for photosynthesis increases with increasing temperature and pCO2 (chapter 3, 4 and 5). Cold-water corals make up some of the most heterogeneous, biologically diverse, three-dimensional ecosystems known in the deep sea. However, due to the difficulty in accessing these habitats, to date there is little information about how these organisms may respond to global climate change. The present study provides evidence of intraspecific variability in the response of cold water corals to global climate change that may be dependent upon their prior environmental experience (chapter 7). In situ acclimatisation to variable and low pH may provide cold water corals with the physiological flexibility to acclimatise and adapt to global climate change in the future. Evidence of intraspecific differences in physiology and morphology were also observed in situ in the brown partially calcifying alga, Padina gymnospora between algae located on the reef crest and more environmentally variable reef flat (chapter 6).

Understanding physiological processes is key to answering the questions of why organisms behave in the way they do, and how they interact with each other, and their environment. Despite technological innovations in recent decades, assessment of physiological state in free-living animals still generally requires subjects to be trapped and handled, so tissues or blood can be sampled, or so measurement devices can be attached or implanted. Such methods limit research to species and individuals that can be caught, potentially restricting the generalisability of findings, and introducing bias. Additionally, natural behaviours are interrupted, and subsequent physiology, behaviour or performance may be altered as a result of the stress of capture, the burden of attached apparatus, or the effects of surgery. Consequently, alternative techniques such as inferring physiological state from traits that do not require invasive sampling would be a valuable development. Body temperature is a particularly promising candidate trait, linked with an array of physiological functions, and having previously been used as a proxy for metabolic activity, stress state and immune challenge. With the advent of low cost, highly portable thermal imaging cameras, physiological ecologists are now presented with unprecedented opportunities to measure body surface temperature non-invasively, and at high frequencies from free-living animals. In this thesis, I investigated relationships between body surface temperatures, measured using thermal imaging from free-living blue tits or captive zebra finches, with physiological measures or situations relevant to the assessment of physiological state. I developed reliable thermal imaging techniques to take non-invasive measurements of body surface temperatures in a variety of contexts, allowing characterisation of physiological responses in real time. My studies of captive birds revealed that activity levels influence body surface temperatures measured from free moving animals, and so should be accounted for in experimental designs. I also successfully acquired body surface temperatures from overwintering blue tits visiting food-baited traps, and from breeding blue tits entering and leaving their nest. Using this data, I showed that body surface temperature exhibits a characteristic response to acute stress, which differs with stressor type. While the mechanisms require explanation, much potentially useful information appears to be stored within body surface temperature dynamics during acute stress. Additionally, I established links between body surface temperature and longer term physiological processes in free-living blue tits. I observed near identical correlations between body surface temperature and body condition across differing seasons and life history stages. Also, I found evidence suggesting both that repeated acute stressors (predation risk and human disturbance) had a chronic effect on body condition breeding blue tits, and that surface temperature in those birds was linked to body condition. If confirmed, these results would be particularly interesting in a conservation physiology context, as it may prove possible to detect a signal of persistent physiological effect(s) relating to human disturbance, non-invasively. Furthermore, my discovery of a further correlation between baseline plasma glucocorticoids and body surface temperature in overwintering birds implies links with the hypothalamic-pituitary-adrenal axis. All of these results combined suggest that body surface temperatures measured using thermal imaging are highly likely to prove useful in determining aspects of physiological state non-invasively from free-living animals. While further investigation and validations are necessary, this work has laid the foundations for an exciting new methodology that could help solve many questions that remain unanswerable using current techniques.

Animal colouration is associated with a multitude of ecologically adaptive traits known to drive biological diversification, from predator avoidance to physiological regulation. As such, it is an ideal system in which to study the evolutionary patterns and processes that generate and maintain biological diversity. Within the terrestrial vertebrates, amphibians display some of the greatest complexity and variation in terms of colour patterning, with the salamander genus Salamandra particularly renowned for its colour diversity. Typically, Salamandra species present bright, highly variable yellow-black patterns consisting of spots and/or stripes, which are thought to hold an aposematic (warning) function related to their toxic secretions. In addition to this, individual species and populations have evolved melanic, fully yellow and fully brown colourations, with gradations seen in-between. Importantly, there are also indications of parallel colour pattern evolution, making Salamandra an attractive system for studying the repeated evolution of adaptive phenotypes. However, the genus currently lacks phylogenetic resolution, and the molecular mechanisms underlying amphibian colouration are poorly understood. In this thesis, I aim to fill both of these knowledge gaps through the use of next-generation sequencing (NGS) techniques, which offer both unpredicted opportunities to resolve systematically challenging relationships and allow us to study the genetic basis of ecologically adaptive phenotypes in wild non-model organism. In Chapter 2 we reconstruct the controversial interspecies phylogeny of Salamandra using three largely independent phylogenomic data sets. First, using restriction site associated DNA sequencing (RAD-Seq), I genotyped representatives of all six currently recognised Salamandra species (and two outgroup species from its sister genus Lyciasalamandra). This was combined with nuclear protein-coding sequences derived from RNA-Seq and full mitochondrial genomes. Analyses of concatenated RNA-Seq and RAD-Seq data retrieved well supported, fully congruent topologies that placed: (1) S. infraimmaculata as sister to all other species in the genus; (2) S. algira sister to S. salamandra; (3) these two species sister to a clade containing S. atra, S. corsica and S. lanzai; and (4) the Alpine species S. atra and S. lanzai as sister taxa. The phylogeny inferred from mitochondrial genomes differed from this in its placement of S. corsica, as did species tree analyses of RNA-Seq and RAD-Seq data. However, the general congruence among topologies recovered from the RNA-Seq and RAD-Seq data sets gives us confidence in our methodologies and results. In Chapter 3, I perform more in-depth phylogenomic analyses, using RAD-Seq to genotype 231 salamanders from across the taxonomic and geographic breadth of Salamandra. Both Bayesian and maximum likelihood based analyses of concatenated RAD-loci (comprising 187,080–294,300 nt of sequence data) returned well-supported, largely congruent topologies that supported the monophyly of all six currently recognised species. However, the placement of S. corsica was again unclear, and data filtering parameters were found to have a great impact on downstream analyses. Further, I identified undescribed diversity within the North African species (S. algira) and find that 43% of S. salamandra subspecies do not meet a criterion of monophyly. Following this, I use the phylogenetic hypothesis generated to assess the parallel evolution of reproductive (parity) mode and two colour phenotypes (melanism and stripe formation) through ancestral state reconstruction analyses. I find that pueriparity (giving birth to fully metamorphosed juveniles) has independently arisen in at least four lineages, melanism in at least five, and a striped phenotype in least two, all from a common yellow-black spotted larviparous (larvae depositing) ancestor. Finally, in Chapter 4, I leverage and highly colour-variable lineage of the European fire salamander (S. salamandra bernardezi) to identify genetic associations with colour, test for selection on colouration, and test the relationship between colour phenotype and toxicity (the functional basis of aposematism). I show that, within a geographically restricted region, colour phenotypes form a gradient of variation, from fully yellow to fully brown, through a yellow-black striped pattern. Population genetic analyses suggest a sympatric evolutionary origin for this colour variation, and I found no association between a salamanders colour pattern and the metabolomic profile of its toxic secretions, which calls into question the adaptive significance attributed to these striking colourations. Following this, I identified significantly differentially expressed genes between skin colours using transcriptomic (RNA-Seq) analyses and genomic loci associated to representative colour phenotypes (yellow, brown and striped) using RAD-Seq approaches. I also found signals of selection on genomic loci between representative colour phenotypes, several of which overlap with genomic analyses. Overall, my results provide greater phylogenetic resolution for the genus Salamandra than ever before, revealing the need for taxonomic revisions and confirming the convergent (or parallel) evolution of both reproductive and colour phenotypes. My data also represents a significant contribution to our understanding of the genetic basis of amphibian colouration, providing a valuable resource for future comparative research on vertebrate colour evolution.

A number of species of wood decay fungi important for the damage they cause to timber and timber products in Korea were cultured. Trametes versicolor, which is one of the most important, was also cultured from a collection made in the UK and likewise the ascomycete Daldinia concentica was obtained for comparative studies. In the initial testing of the effectiveness of the two wood preservatives, ammoniacal copper quaternary (ACQ) and copper azole (CuAz) preserve injected blocks of Japanese Red Pine and Yellow Poplar were inoculated with T versicolor, Pleurotus ostreatus and D. concentrica. Weight loss(%) of the wood blocks showed that Japanese Red Pine possessed greater natural resistance to decay by the white rot basidiomycete fungus T versicolor, than to the white rot ascomycete D. concentrica. The results for Yellow Poplar were the opposite. It was also found that both preservatives had an inhibitory effect on all three test fungi regardless of tree species. Furthermore ACQ was the most effective preservative in relation to T versicolor, which is the most damaging wood decay fungus in Korea. It has also been found that the absorption of the preservatives by the two different wood types differed with Yellow Poplar exhibiting a slightly greater absorption than the Japanese Red Pine, which might be a result of differences in the anatomical structure of the woods. Fungal biomass was also determined using chitin and ergosterol assays. The results regarding levels of decay caused by T versicolor, P ostreatus and D. concentrica are in close agreement with the weight loss determinations. The assays also confirmed the effectiveness of the copper based preservatives. The application of Scanning Electron Microscopy (SEM) has allowed observations on the damage caused by the test fungi to the untreated blocks of the two wood species and the reduction in damage on blocks treated with the preservatives. Linked studies using the SEM and Atomic Force Microscopy have demonstrated differences in the micromorphology of the hyphal tips of the test fungi.

Redshank breeding biology is examined and reviewed in relation to other sandpiper studies. "Primarily, general aspects are described. The mean clutch size was 3.82 eggs. Fourth eggs laid were significantly smaller and more likely to hatch last. Nest hatching success varied annually from 30-741, with tidal flooding the main cause of nest failure. In successful nests, hatch success was less variable. Chick size at hatch was positively correlated with egg size. Pre-fledging mortality is discussed in relation to overall fledging success. Surviving adults generally remained mate faithful. Experienced birds were found to nest earlier in the season and to produce larger eggs. In all years, female age correlated positively with egg volume. Inexperienced birds laid smaller eggs later in the season. Older birds were more successful at hatching eggs. Experienced breeders were more likely to return to their former breeding grounds than were younger birds, particularly when they were successful the previous year. Birds unsuccessful in the previous year were more likely to divorce. Divorced females were less site faithful than males and were more likely to disperse. Natal philopatry is discussed in relation to pre and postfledging mortality. Philopatry is non sex biased and is estimated at being very high. Chick growth and development is considered and an age determining formula devised for ageing chicks from their weight and bill length. Growth rates were constant between and within years. Annual adult survival (0.75), life expectancy (3.48 years), and study area population are calculated. An estimated 175 pairs breed in the restricted area (168 pairs/km ) and approximately 500 pairs on the Nature Reserve. A model of population dynamics estimates pre-fledging mortality at between 66-74%. Timing of breeding in other waders and a comparison with the related Greenshank are reviewed in the Appendices.

A study of the properties of tree-ring density and ring-width chronologies from five sites in the British Isles, two in Sweden and two sub-fossil sites in Northern Ireland is described. The technique of x-ray densitometry is used to measure density. It is shown that it is possible to use x-ray densitometry on well preserved sub-fossil pine. Chronologies have been constructed for parameters of earlywood and latewood widths, ring-width, maximum and minimum densities for all sites. The statistical properties of chronologies are related to the latitude and altitude of the sites. Sub-fossil chronologies behave differently to any of the living tree chronologies. Response functions on monthly temperature and precipitation data are calculated for the five tree-ring parameters for the living tree chronologies. A principal component analysis involving 25 ring-width chronologies from northwestern Europe is used to examine the spatial relationship between British and European ring-width chronologies. The continuous pattern of density variation across the annual ring is measured for trees from two scottish sites, at Glen Derry and Glen Affric from 1900 to 1979. A method of constructing and comparing annual density profiles by fitting cubic spline functions to the density data is described. This has enabled the effects of growing season climate on density to be examined. The importance of temperature in governing tree-ring density is demonstrated. The use of image analysis techniques to measure the continuous variation in cell dimensions across the annual ring is described. Variations in ring density are explained in terms of changes in wall thickness and lumen diameter. A comprehensive literature review on the physiological mechanisms controlling the response of tree-ring width and density in P. sylvestris to climate is described. The physiological causes-for the climate-growth response in earlywood and latewood widths and densities are summarised seperately. It has been possible to explain some of the results of the response function analysis and the density profile study in terms of physiological processes.

This study develops a fractographic method to diagnose hammerstone- and carnivore induced fracture. This is important because interpretations of hominin entry into the carnivore guild and evolution of meat-eating are based on rare tool and tooth marks in Oldowan (2.5-1.8mya) fossil assemblages. Consequently, estimating hominin and carnivore involvement is difficult, and questions remain about Oldowan hominin’s position in the carnivore guild and socioecology. One aspect of bone damage, fracture surfaces, is ubiquitous, but largely unstudied. The fractographic (study of fracture surfaces) method is based on fracture principals, particularly how differences in static- and impact-loading affect material response and fracture features resulting from loading extremes. The method is applied to analysis of fracture features in a) the Amboseli Hyaena Den assemblage, b) an experimental hammerstone-broken assemblage, c) a Plio-Pleistocene assemblage previously interpreted as a carnivore accumulation, FLK-NN2 (Olduvai Gorge), and d) the zooarchaeological assemblage from FLK-Zinj, (Olduvai Gorge). This is the first zooarchaeological/taphonomic study to demonstrate that a) static and impact fracture differ fundamentally in applied load size and material responses to loading extremes, b) impact-forces are significantly greater than the maximum carnivore bite-force, c) cones, incipient flakes, radiating cracks, and lateral stress features are characteristic of impact fracture, and e) Oldowan hominins at FLK-Zinj were responsible for breakage of 54% of the limb assemblage (a 37% – 40% increase over estimates based on percussion marks). The socioecological implications of the habitual transport of food from death and/or kill sites to secondary locations are explored by examining reasons why social carnivores transport food. Aspects of modern carnivore behaviour suggest general mammalian constraints that may have predicated food transport by early Homo. Early Homo food transport behaviour was structured by anti-predator defense strategies associated with a) foraging in an open habitat rich with competing predators, b) the lack of masticatory and digestive apparatus to quickly consume animal tissue, and c) the presence of altricial young in the hominin group.

O conceito de homologia similaridade entre partes com ancestralidade comum é um dos mais importantes conceitos dentro das análises evolutivas. Desde a sua origem, muitas definições que diferem na definição e na aplicação foram propostas. A presente dissertação tem como objetivo reconstruir a história da homologia para esclarecer teoricamente os debates modernos. Após esta historiografia, foi feita uma análise filomemética dos principais autores que discutiram a teoria e a prática do conceito. E, por último, foi feita uma discussão teórica do papel da homologia dentro da sistemática filogenética, abordando as principais discussões atuais. Os resultados demonstram que é possível esclarecer algumas discussões através da análise histórica do conceito sob a perspectiva da teoria da evolução. A análise filomemética reconstruiu os grupos de autores de acordo com suas escolas e propostas sobre a homologia. A análise teórica do conceito na metodologia filogenética evidencia que o conceito de homologia é equivalente ao conceito de sinapomorfia e uma nova visão sobre a relação de ausências, homoplasias e homologias é proposta. A discussão sobre as nomenclaturas e definição do conceito de homologia demonstra que as terminologias homologia primária e secundária são as melhores propostas para representar a relação da origem e legitimação de homólogos. Uma crítica às análises filomeméticas conceituais é feita. / The concept of homology similarity between parts due to common ancestrality is one of the most important concept in evolutionary analysis. Since it origin, many definitions which differ on definition and application were proposed. This dissertation has as the main goal reconstruct the history of homology to clarify theoretically the modern debates. After this historiography, was made a phylomemetic analysis of main authors that discussed the theory and pratics of homology concept. Lastly, was made a discussion on the importance of the role of homology in phylogenetic systematics, approaching actual discussions. The results support that is possible to clarify some discussions through historical analysis of this concept with an evolutionary background. The phylomemetic analysis reconstruct groups of author accordingly to their methodological schools and propositions. The historical analysis of the concept in phylogenetic analysis evidence that homology concept is equivalent to synapomorphy and a new look into the relation of absence, homoplasy and homology is proposed. The discussion on nomenclatures and definition of the concept of homology support that primary and secondary homology are the best proposition to represent the relation of origin and legitimation of homologs. A criticism to phylomemetic analysis is pointed.

The banteng (Bos javanicus lowi) is an endangered wild bovid that is endemic to the island of Borneo. Within their last stronghold, the Malaysian state of Sabah, their population is believed to be less than 500 individuals, which are threatened with extinction by habitat loss and hunting. The banteng is highly elusive and rarely seen, and their preference for dense and remote tropical forest habitat makes them a highly challenging species to study. No extensive quantitative surveys have been undertaken in Sabah, and there is little information available to underpin their conservation and management. This thesis provides the first baseline data on the Bornean banteng in Sabah using ecological and molecular techniques. In Chapter 2, I created the first extensive natural history account of the banteng, which will help further the knowledge of this species. This compilation helped identify gaps in the knowledge, which were then addressed by this thesis. In Chapter 3, I test non-invasive survey techniques and individual identification, and estimate the population size in two forest reserves. In Chapter 4, I demonstrate that logged forests undergo dramatic changes in structure and ambient temperature, and that banteng mitigate these changes by altering their behaviour to avoid thermal-stress. Chapter 5 presents new information of the population genetic structure of banteng in four forest reserves in Sabah. Using mitochondrial markers I show that the ancestral lineage of the Bornean banteng reinforces the suggestion that they should be recognised as a separate subspecies to the Burmese and Javan banteng. I also show that the banteng experienced a population expansion following their colonisation of Borneo, and that the present genetic diversity indicates the population may be managed as two geographically-distinct units. Chapter 6 summarises the main findings of this thesis and the implications for the conservation of the Bornean banteng in Sabah.