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.

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.

The peptide hormone, ghrelin, exerts its physiological effects through a G-protein-coupled receptor called the ghrelin-R. The ghrelin-R displays a high degree of constitutive activity, signalling through the inositol phosphate pathway in the absence of bound agonist. TMs III and VI have been reported to be central to the activation of Family A GPCRs, with interactions between the two helices stabilising the ground state. During activation conformational rearrangements result in these interactions being broken, with new contacts forming and stabilising the active state. Investigation of the ghrelin-R constitutive activity gives an insight into the mechanisms involved in receptor activation. In this study the role of specific individual residues in the ghrelin-R has been investigated and the effect of disrupting or introducing intramolecular interactions was addressed. Site-directed mutagenesis and functional assays revealed that ghrelin-R constitutive activity can be increased and decreased with mutation of residues within the TM domains, specifically TMs III, VI and VII. The extracellular loops have been found to be involved in ligand binding and activation in a number of Family A GPCRs. The residues within ECL2 of the ghrelin-R were systematically mutated to alanine to determine their role. In particular, one residue, Asn196, was identified as being critical in ghrelin-R function and may be forming stabilising interactions which maintain ghrelin-R constitutive activity. The data presented in this thesis provide an insight into the structure and function of the ghrelin-R and the underlying molecular mechanisms of ghrelin-R constitutive activity.

Circadian rhythms are biochemical, physiological, or behavioural over 24 hours. The avian circadian system is complex, involving numerous oscillators in the brain. I characterised two hypothalamic input mechanism (melatonin receptors and light) and one output mechanism (vasotocin) in the zebra finch. Melatonin receptors were cloned and expression levels investigated in the brain and in peripheral tissues. Receptors were found in all tissues, with some pronounced rhythmic mRNA expression. Tissue-specific differences in temporal distribution, peak expression and amplitude suggests melatonin have varied roles in different tissues and different receptors control/influence these roles. Effect of light in the hypothalamus was investigated by exposing light into the dark phase of an LD cycle and studying the difference in C-FOS expression. C-FOS was found in hypothalamic nuclei associated with photic transduction. C-FOS-IR cells were also found in the two known avian hypothalamic oscillators, the LHN and SCN. Arginine-Vasotocin is a neuropeptide involved in numerous bodily and nervous tissue functions, secreted within the hypothalamus and pituitary gland. Immunofluorescent experiments showed marked differences in expression, as different zeitgeber times and between species. This study has improved our understanding of avian circadian systems, providing new insights into the hypothalamic oscillator of a complex circadian organisation.

Phenotypic differences within a species significantly contribute to the variation we see among plants and animals. Plasticity as a concept helps us to understand some of this variation. Phenotypic plasticity plays a significant role in multiple ecological and evolutionary processes. Because plasticity can be driven by the environment it is more likely to produce beneficial alternative phenotypes than rare and often deleterious genetic mutations. Furthermore, differences in phenotypes that arise in response to the environment can affect multiple individuals from the same population (or entire populations) simultaneously and are therefore of greater evolutionary significance. This allows similar, beneficial alternative phenotypes to increase quickly within a single generation and allow new environments to produce and select for new phenotypes instantly. The direction of the present thesis is to increase our understanding of how phenotypic plasticity, coupled with contrasting environmental conditions, can produce alternative phenotypes within a population. Plasticity provides a source of variation for natural selection to act upon, and may lead to genetic isolation as a by-product. For example, there are multiple cases of polymorphic populations of fish, where groups belonging to multiple isolated gene pools, have arisen in sympatry. Here it is shown that although plasticity is important in sympatric speciation events, plasticity alone is not responsible for the frequency in which sympatric polymorphic populations occur. The most frequently observed differences among sympatric polymorphic populations are morphological differences associated with parts of the anatomy used in the detection, handling and capture of prey. Moreover, it is shown here that there are physiological effects associated with foraging on alternative prey that may significantly contribute towards ecological speciation. It is also shown in this study that anthropogenic abiotic factors can disrupt developmental processes during early ontogeny, significantly influencing morphology, and therefore having ecological consequences. Phenotypic structuring in postglacial fish is most frequently based around a divergence towards either pelagic or littoral benthic foraging specialisms. Divergences that deviate from this pattern are of greater scientific interest as they increase our understanding of how evolutionary processes and selection pressures work. Here we describe a rare divergence not based around the typical pelagic/littoral benthic foraging specialisms. Finally, in this study, the effectiveness of local level conservation policy shows that species of fish which are highly variable in their life history strategies are harder to effectively manage and often poorly represented at a local level.

The endangered population of Amur tigers (Panthera tigris altaica) in the Russian Far East (RFE) faces an increasing risk of extinction due to infection with canine distemper virus (CDV). Short-lasting CDV infections are unlikely to be maintained in small populations of species with limited connectivity like tigers, where viruses fade out as susceptible hosts are depleted. Multi-host pathogens can persist in more abundant host species that can act as reservoirs of infection for threatened populations. This study combines assessments of host demography, serology and viral phylogeny to establish the relative contribution of domestic dogs and small bodied wild mesocarnivores to the maintenance of CDV, and as sources of infection for tigers. No antibodies were detected among tigers sampled prior to 2000 (n=19), but were measured in 35.7% of tigers in subsequent years (n=56), with at least five discrete transmission events occurring in one well-monitored population. Viral sequences from three tigers and one Far Eastern leopard (P. pardus orientalis) aligned within the Arctic-like clade of CDV, and shared recent common ancestry with viruses from 22 other wild carnivores from the region. Extensive spatial mixing of wild carnivore lineages suggested long chains of transmission consistent with a maintenance population. The exposure of tigers following 2000 coincides with increases in sable (Martes zibellina) numbers and hunting pressure, which could lead to greater pathogen prevalence and potential for spill over from a wild reservoir. The ratio of humans to dogs in rural areas in the RFE are among the lowest in the world (1.73), but the overall number of dogs has been stable during the period of increased CDV exposure in tigers. The only CDV sequence obtained from dogs shared high identity with Asia-4 clade viruses from dogs in Thailand, and was distantly related to wildlife sequences from the RFE. Serum antibodies were detected in dogs in all 26 communities where households were surveyed, but seroprevalence was higher in remote, less densely populated areas, suggesting possible transmission from wildlife. Although the maintenance of CDV in Russian dogs remains unconfirmed, the strong support for a wildlife reservoir limits options for managing the impact of CDV on tiger populations. The high turnover of large and often inaccessible populations of mesocarnivores combines with limitations in vaccine safety, efficacy and delivery, to render the control of CDV in a wildlife reservoir untenable. Managing the impact of CDV on Amur tigers must therefore focus on restoring the size and integrity of remaining tiger populations to withstand future outbreaks. The safety and efficacy of vaccine products for tigers should also be investigated, for use in low coverage vaccination strategies that could enhance the long-term persistence of tiger populations.