Xenopus laevis erythrocyte nuclei have been used as a source of inactive ribosomal RNA genes to study the regulation of gene activity during early embryogenesis. Addition of an oocyte extract to the erythrocyte nuclear transcription assay brings about transcription by RNA polymerase I. This is not seen If an egg extract is used even though the amounts of RNA polymerase I are the same in both preparations. The oocyte-treated nuclei synthesize ribosomal RNA as defined by RNA-DNA hybridizations and sucrose gradient separation techniques. The active component of the oocyte extract has been semipurified to a single peak by Sephadex G100 column chromatography and DEAE cellulose salt elution. Its presence and absence during oogenesis and early embryogenesis parallels the transcription of the ribosomal genes in vivo at these stages. Within the oocyte itself, the active component is located exclusively in the germinal vesicle. The structure of the rlbo6omal genes in treated nuclei has been studied by DNase I digestion. The ribosomal genes in erythrocyte nuclei are Insensitive to DNase I, but treatment by an oocyte extract or egg extract plus the semipurified active component causes the entire gene repeat to become DNase I sensitive. An egg extract alone has no effect. The semipurified active component by itself causes only the 5' end of the transcribed sequence to become DNase 1 sensitive. The component therefore appears to act on the non-transcribed ribosomal genes of the erythrocyte nucleus to alter their chromatin conformation to a more DNase I sensitive one which permits RNA polymerase 1 transcription. The DNase I sensitivity of histone H4, globln, tRNA and 5Sooc RNA genes was also studied. The extracts had no effect on the DNase I sensitivity of any of these genes, but those genes transcribed by RNA polymerase 111 are DNase I sensitive whatever their transcriptional state.

The Mexican axolotl Ambystoma mexicanum is a member of a family of salamanders with large nuclear genomes. The known geographical distribution and evolutionary history of the genus Ambvstoma makes it an ideal model system for the study of the evolution of repetitive DNA sequences in large genome animals. The genome of the axolotl has been examined by various methods, in particular by reassociation kinetic analysis and associated techniques on bulk DNA and by the molecular cloning of several members of the repetitive fraction of the genome. The haploid genome (37 pg) can be resolved into several distinct components. A foldback fraction (4.6%), a moderately repetitive fraction (43.3%), a slow repetitive fraction (20.4%) and a unique fraction (31.7%). Foldback regions appear to form randomly dispersed clusters. Members of the moderately repetitive fraction are predominantly interspersed amongst other members of the same fraction. At moderate fragment length only (57.5%) of the genome is arranged as moderately repetitive DNA interspersed with more slowly repeating sequences. Fragments of axolotl DNA have been cloned by recombinant DNA techniques. Clones containing axolotl DNA were selected. One clone (pAMW113l) has an apparent repetition frequency of 1480 in the axolotl genome and may be part of a larger fragment undergoing reamplification in the genome. A second clone (pAMW1184) is highly repeated with a short repeat length. Some family members appear to be scattered through the genome. Some members are transcribed on lampbrush chromosomes. A third clone (pAMW1199) has several features characteristic of bulk repetitive DNA. It has a complex organisation in the genome and may contain a number of shorter repeats present in the genome at different frequencies. These clones represent different aspects of the organisation of the axolotl genome. Together with the general genome analysis they provide a starting point for comparative evolutionary analyses of the genus Ambvstoma.

Intraspecific genetic, morphological and life history structuring is evident in many taxa. Where such intraspecific structuring exists, study of the nature of the patterns displayed can reveal much about the evolutionary processes that operate during the early stages of divergence. Intraspecific structuring is particularly prevalent amongst fishes that occupy recently glaciated freshwater systems. One such species, the brown trout, Salmo trutta, was the subject of the work presented in this thesis. Genetic and morphological intraspecific structuring of brown trout was examined across a single but large dendritic catchment, the River Foyle, Ireland. Structuring was examined at three spatial scales (large-scale, compared between major sub-catchments; medium-scale, compared between tributaries within sub-catchments; small-scale, compared between streams within tributaries). The two general aims of the study were to look for any structuring in either phenotype or genotype in brown trout across the catchment and, if this was found, to look for landscape or environmental gradients that might be driving such structuring. Using a suite of 21 microsatellite markers that were chose for their ability to resolve population differences in this species elsewhere, this study identified clear and distinct genetic structuring. Brown trout collected from 28 sampling sites, resolved into 21 genetically distinct and discrete populations using a hierarchical approach implemented in STRUCTURE. The structuring was evidence across all three spatial scales. There was strong evidence of isolation by distance and isolation by environment playing a role in shaping the magnitude of the genetic differences between populations. Landscape variables which are shaped by anthropogenic impacts (urbanised area (measured as the number of houses in the catchment), proximity to farmland (measured as the distance to the nearest farm) and concentration of phosphorus in the water) showed the greatest effects in shaping the genetic population structuring (chapter 2). In a parallel study, the morphological structuring of brown trout from across the Foyle catchment was investigated at three spatial scales. Morphology was measured as the shape of brown trout determined by Geometric Morphometric Analysis of fixed position landmarks identified on photographs of trout taken from 22 sampling sites across the catchment. Very clear, statistically significant differences in morphology (fish shape) were evident for all the 21 sampling sites (one sampling site was removed from the analysis due to small sample size) with Canonical Variate Analysis resolving 21 discrete phenotypic groups. Morphological structuring was evident across all spatial scales (large, medium and small). Analysis showed that genetic distance and geographic distance between morphological groups was significantly correlated with morphology of populations, with morphological groups that were most divergent from each other also being most genetically distinct and geographically more distant. The effect of landscape and environmental variables driving morphology of populations was tested. In-stream substrate composition, water pH, stream order, site elevation, river gradient and the number of houses per km2 (representative of urban area) were all found to have a significant effect on morphology of populations. However, once the effect on morphology on these environmental variables were accounted for the residual effect of genetic distance was not significant (chapter 3). To attempt to discriminate between three alternative population genetic hypotheses for the origin of two alternative life history strategies in brown trout; freshwater residency and anadromy, the genetic structuring of brown trout was examined between life history strategy (anadromy or resident), between three sites and across two years (2013/2014) for brown trout collected from the Foyle catchment. There was no evidence of population structuring being attributed to life history strategy (that is no genetic differences between anadromous or resident trout). There was however strong and clear evidence of five genetic populations based on geographical site. Two sympatric populations were identified at each of two locations. However, both populations in each river were composed of both freshwater resident and anadromous brown trout, although the frequency of each life history strategy significantly differed between these rivers. The results of this study support the concept that partial migration in brown trout is most likely driven by a quantitative threshold trait, where the threshold trait value varies both between populations and between individuals within populations (chapter 4). It is critical, for effective management of the relatively high economic value anadromous component of brown trout populations in a catchment, to be able to identify which tributaries are contributing most to their production. A Genetic Stock Identification (GSI) analytical framework was used to determine the tributary of origin for anadromous brown trout captured from a mixed stock within the River Faughan sub-catchment, River Foyle and to look for any evidence of straying. The results showed that three genetic populations from specific parts of the sub-catchment contributed disproportionately to the production of anadromous brown trout. There was also evidence of straying of anadromous trout, particularly to one tributary elsewhere in the catchment (chapter 5). Taken together this body of work has demonstrated strong genetic and morphological structuring amongst brown trout in this large dendritic catchment. Genetic structuring seems to be at its most extreme when driven by factors which could be regarded as anthropogenic. This raises questions about human effects on the process of genetic divergence. Morphological structuring was, if anything even stronger than genetic structuring. Although there was evidence of genetic divergence between populations of differing morphologies, this neutral genetic differentiation was not a significant driver of morphological variation once landscape and environmental variables, such as substrate composition, driving morphological differences were taken into account. This suggests that the environmental drivers of structuring are greater in magnitude than neutral genetic divergence. Examining genetic structuring between two common morphologies of brown trout (anadromous and freshwater resident) in more detail, revealed no genetic differentiation between life history strategies but there was evidence of differences in frequency of life history between populations. Using the genetic structuring of brown trout as a genetic baseline it was possible to determine which tributaries within the River Faughan sub-catchment produce anadromous brown trout. If some discrete populations in a catchment are contributing disproportionately to the anadromous trout population (as they are in the Foyle) there is a strong risk of over exploitation and a need for enhanced attention in the nursery areas for those populations. These results have significant implications for the management of all trout in the Foyle catchment and elsewhere.

Recent studies have indicated the presence of a high degree of cryptic genetic diversity in some clitellate sentinel species. One of these species, the earthworm Lumbricus rubellus, has been recently found to comprise two divergent clades in the UK, and are possibly cryptic species. L. rubellus is commonly used in ecotoxicological assays, where undetected differences in contaminant responses between cryptic lineages may lead to confusing or misleading results. Furthermore, given the key role that earthworm species play in the soil ecosystem, a better understanding of cryptic diversity is necessary to investigate whether divergent lineages play different roles within their ecosystems. In this study, the phylogenomics of the acid-tolerant, cosmopolitan, epigeic species Lumbricus rubellus was investigated, with regard to demography during the glacial stages of the Pleistocene and the recent post-glacial colonization of North Europe using mitochondrial DNA markers, next-generation sequencing and environmental niche modelling tools. The niche suitability of L.rubellus during the last 120.000 years was inferred, allowing hypotheses on survival and recolonisation to be constructed. Phylogenetic, population structure and coalescent-based analyses resulted in the discovery of 11 deep divergent lineages (with levels of divergence up to 18% for mitochondrial markers), which most likely survived in refugia during Pleistocene glaciations. Signatures of expansions point to a possible recolonisation of central Europe during the last Glaciation, survival of one of the clades in a northern cryptic glacial refugium and a consequent recolonisation of northern Europe during the last 10,000 years. Genetic evidence and divergence time ultimately suggest that L. rubellus is a cryptic species complex, which clades diverged as far as ~5MY ago. The entire mitochondrial genome of the species complex is described here for the first time, and a survey of the deep phylogenetic signal over the mitochondrial genomes of eight selected individuals was carried out, supporting and deepening the phylogeny constructed using only two mitochondrial genes. Finally, whole genome analysis of genetic divergence supported the hypothesis of cryptic divergence for the two most divergent lineages selected

The Major Histocompatibility Complex (MHC) is a multi-gene family that includes most vertebrate immune genes. Life history traits have been associated with MHC allelic variation, including offspring survival, reproductive success, kin recognition, inbreeding avoidance, body mass gain, mate choice and parasite resistance. The studies reported in this thesis used laboratory and field investigations to identify differences in MHC genetic variation between truly wild, wild type and domesticated conspecifics and the implications of this for fitness, across the entire life history of a vertebrate, the guppy (Poecilia reticulata). Specifically, the effects of host inbreeding and domestication on parasite susceptibility are assessed in relation to MHC allelic and supertype composition. Laboratory studies showed that inbreeding and domestication lead to increased susceptibility to Gyrodactylus turnbulli, which was also linked to the presence of particular functional groups of MHC. A multi-site field sampling supported this finding; revealing that natural parasite communities reflected host MHC functional groups, as well as the river of origin. Truly wild fish had greater MHC genetic diversity than wild type (wild population maintained in the laboratory for ~ 3 years), which, in turn, were more genetically diverse than ornamental (domesticated) conspecifics. The accidental and deliberate release, into the wild, of domesticated fish is common. The release of infected and uninfected ornamental guppies into a wild type laboratory population increased parasite prevalence and abundance, due to the integration of a more susceptible individual into the social group. Mate preference is often linked to MHC similarity, whereby individuals select mates that are dissimilar or optimally similar at the MHC. The effects of sexual selection, MHC similarity and parasitism on mate choice, were assessed, indicating that a combination of factors are important in a female’s preference. Female guppies spent more time interacting with males with redder colouration and less MHC alleles in common. An experimental F1 generation revealed that offspring with parents sharing more MHC alleles and supertypes were more susceptible to parasitic infection. This research suggests that MHC functionality is at least as important as allelic and supertype diversity, with regards to individual fitness and life history traits.

Microbial communities play important roles in organismal and ecosystem health. High throughput sequencing has revolutionized our understanding of host-associated microbial communities, but the viral component of these communities remains poorly characterized relative to microbes such as bacteria, particularly in non-human hosts. This knowledge gap has implications for global health, as viruses originating in wildlife are responsible for recent disease outbreaks in humans and domestic animals. Although studies have identified factors differentiating viral communities between species, we have little understanding of the variability of viral communities within species. Comparative studies of viral communities are therefore necessary to characterize novel taxa and to evaluate the ecological factors influencing intraspecific viral diversity and distribution. Bats are recognized as “special” reservoirs for viruses because they are associated with diverse viral communities and display deep evolutionary relationships with individual viral taxa. Common vampire bats (Desmodus rotundus) represent a particularly interesting system in which to investigate viral communities, as they are obligate blood feeders that interact ecologically with many different host species, providing opportunities for the acquisition of diverse viruses. The overall objective of this thesis was to advance our understanding of intraspecific wildlife-associated viral communities using an established field network of common vampire bat colonies across Peru. Specifically, I developed a novel method for comparative viral community studies, characterized the viral communities of vampire bats, and examined the ecological correlates of vampire bat viral diversity across Peru. Metagenomic sequencing is a promising technique for comparative studies of viral communities in wildlife, but there is a need to first develop standardized methods that can be applied to samples collected in the field. In Chapter 2 I developed a shotgun metagenomic sequencing approach to characterizing viral communities from non-invasive samples. Specifically, I optimized extraction and sequencing protocols using fecal and oropharyngeal swabs collected from common vampire bats in Peru. Two preliminary sequencing runs were performed, the results of which motivated four pilot studies in which I tested how different storage media, nucleic acid extraction procedures, and enrichment steps affect the viral community detected. Metagenomic sequencing revealed viral contamination of fetal bovine serum, a component of viral transport medium, suggesting that swabs should be stored in RNALater or another non-biological medium. Extraction and qPCR tests were performed on swabs inoculated with known concentrations of virus, which revealed that nucleic acid should be directly extracted from swabs rather than from supernatant or pelleted material. Metagenomic sequencing of paired samples was used to test enrichment by ribosomal RNA depletion and light DNAse treatment, which both reduced host and bacterial nucleic acid in samples and improved virus detection. A bioinformatic pipeline was developed specifically for processing vampire bat shotgun viral metagenomic data. Finally, the optimized protocol was applied to twelve pooled samples from seven localities in Peru, and read subsampling demonstrated that the viral communities detected were consistent at commonly attained depths of sequencing. The protocol developed in this chapter enables minimally biased comparative viral community studies in non-invasive samples collected from wildlife. Having a detailed understanding of viral diversity in key wildlife hosts is an important first step in evaluating the risk of zoonotic disease emergence, but we still lack a holistic view of viral communities in many species including vampire bats. In Chapter 3, I used the metagenomic sequencing protocol developed in Chapter 2 to thoroughly characterize viral communities in the saliva and feces of vampire bats captured across Peru. Viruses were detected from a range of natural host groups including vertebrate-associated taxa that were potentially infecting vampire bats, bacteriophages associated with gut bacteria, and plant- or insect-infecting viruses potentially acquired from the environment. There were broad differences between fecal and saliva viral communities, showing evidence of body habitat compartmentalization. Overall, results established that vampire bat viral communities differ between body habitats and suggested that, for the vertebrate-infecting families analyzed, novel viruses mostly fall within bat-specific clades, without evidence of livestock or humans acting as a major source of viral diversity in vampire bats. Interspecific differences in ecological and life history traits are known to impact viral richness in bats, but the factors structuring viral communities within bat species are less well understood. In Chapter 4, I examined the spatial, demographic and environmental correlates of intraspecific viral diversity in vampire bats. Three measures of viral diversity were calculated at the colony level: richness, a novel measure of taxonomic diversity, and community composition. Generalized linear models were then used to test the effects of broad scale and local ecological variables on saliva and fecal viral diversity. The results showed for the first time that ecological variables can influence intraspecific viral diversity. In summary, the work presented in this thesis advances our understanding of wildlife-associated viral communities in an ecologically important bat host. Future directions in comparative wildlife viral metagenomics, as discussed in Chapter 5, will include exploring the determinants of viral communities across host species, environments and time.

The effects of pupal cold shock on the life cycle and wing morphology of the Speckled Wood butterfly are examined and their genetic assimilation is investigated. Metamorphosis is modelled in terms of changes in stability, and the mediation of cold shock effects by hormones is considered. Current theories of pattern formation are evaluated for the species, and pattern is analysed using manual, photographic and digital methods. The development of wing morphology is modelled, and cold shock effects understood by comparison with normal development. Developmental canalisation is estimated as variability and fluctuating asymmetry. An index is developed that predicts the extent of assimilation. Likely modes of inheritance are suggested, and the possibility of natural cold shock and assimilation in the species is considered. Recent trends in biology indicate that neo-Darwinian concepts cannot adequately account for certain developmental and hereditary phenomena and that a new paradigm is emerging. The two schools are compared with particular reference to Weismann and Waddington, and the phenomenology is re-examined in the light of the new findings.

The okapi (Okapia& johnstoni) is an endangered, evolutionarily distinct giraffid, endemic to the Democratic Republic of Congo (DRC). The okapi is a flagship species for the DRC,a country that contains some of the greatest biodiversity in the world. The okapi is currently under major threat from habitat fragmentation, human encroachment and poaching, yet to date, very little is known about the species in the wild, and no genetic study in the wild or captivity has ever been carried out. This thesis aims to use genetics to aid conservation efforts of okapi,a species that due to its elusive nature, is highly challenging to study using alternative methods.

Rhipicepahlus (Boophilus) microplus is an obligate feeding, hard tick of great economical importance in the cattle industry. Every year billions of dollars of loss is attributed to R.(B) microplus, mainly through loss of cattle due to pathogens transmitted such as Babesia and Anaplasma, but also through damage to hides from blood-feeding. There is conflicting evidence regarding the taxonomic status of R.(B) microplus, however the most recent published research has been in support of the reinstatement of R.(B) australis as a species distinct from R.(B) microplus. The way in which some members of the scientific community have responded to the designation of separate species has implications for vaccine and acaricide research. In this study, we aimed to resolve the taxonomic status of Rhipicephalus (Boophilus) microplus, using morphological and phylogenetic approaches. 1,650 Rhipicephalus (Boophilus) microplus ticks from Australia, Thailand, South Africa, North and Central America and South America were used in this study. 340 specimens consisting of 170 R.(B) annulatus (USA) and 170 R.(B) decoloratus (South Africa) were also used. To maximize the information obtained from morphological observations, three methods were used; a binary scoring system based on previously described features, a standard morphometric method, and the more novel approach of geometric morphometrics. For the phylogenetic analysis three genes were used; the mitochondrial gene COX1 and two functional nuclear genes; Bm86 and βAOR. Morphological scoring is the process of assigning a binary value to any feature as being present or absent, or satisfying a logical comparator. For this study the scoring matrix was based on previously described sets of morphological criteria used for discriminating among species. Each of the populations for which samples were obtained was tested using four two-way analyses, each of which was designed to test whether a sample should be classified as one of two possible species: R.(B) australis versus R.(B) microplus; R.(B) microplus versus R.(B) annulatus; R.(B) microplus versus R.(B) decoloratus; and R.(B) annulatus versus R.(B) decoloratus. The scoring system was highly repeatable for the differentiation of males and females of R. (B) annulatus and R.(B) decoloratus from both of R.(B) microplus and R.(B) decoloratus. However, in the case of R.(B) australis and R.(B) microplus, clear differentiation was not achieved for either male or female ticks. Among females, the Australian population were classified almost evenly as R.(B) australis and R.(B) microplus, with 8 individuals showing a mixture of features and therefore not able to be classified. Ticks from the rest of the regions were mainly classified as R.(B) microplus, which is to be expected as R.(B) australis is reported in Australia. However, only the Mozo isolates were classified as solely R.(B) microplus. The remaining regions included several ticks with mixed features. Six ticks from South Africa, and four of the Juarez isolate were classified as R.(B) australis. Among the males an entirely different pattern emerged. Most male ticks from all geographical locations were classified as either R.(B) australis or showed a mixture of both features, with only a small number scoring as R.(B) microplus. Morphometrics is the linear measurement from one anatomical landmark to another and is a widely-used technique for quantifying phenotypic variation. Twelve features based on previous morphometric work were used. The results obtained from this study varied according to stage and sex. For the larvae, the Fisher Pairwise comparison showed that the Australian ticks tended to have a shorter body length, idiosoma length and narrower scutum width. Among the remaining morphological features, there were no consistent patterns in the different populations and species. A principal components analysis (PCA) was undertaken and in PC1 the strongest feature was scutum length and hypostome length. In PC2 the strongest feature was idiosoma length. The PCA of larval stage ticks didn’t provide conclusive evidence that R.(B) australis is a distinct species from R.(B) microplus, and there was no obvious grouping based on region at all, even when including R.(B) decoloratus and R.(B) annulatus. In relation to the male ticks studied, the Fisher Pairwise comparison and the PCA showed that Australian males (presumed R.(B) australis) were significantly different from the other isolates. As with the larvae, no patterns were seen in the other populations, based on species or region. In PC1 palpal length measures were the strongest features for differentiation and in PC2 the length of the ventral basis capituli had the strongest effect. The adult female samples yielded a mixed result. There was no real trend in the size of Australian ticks observed from the Fisher Pairwise comparison. However, R.(B) decoloratus tended to be smaller for most of the morphological features tested and R.(B) annulatus tended to be larger. This observation was inconsistent with the results from the PCA, in which there was grouping of Australian ticks. Measures of palpal length, width of the basis capituli and the length of the dorsal basis capituli were the strongest for differentiation in PC1. In PC2 the length of the ventral basis capituli was the strongest feature for differentiating populations. Geometric morphometrics is the quantitative representation of shape using coordinates in the form of landmarks, instead of measurements and is intended to give the shape of the feature independent of size. Hence it is useful for eliminating the effect of size distortion occurring with physiological changes. Geometric morphometric analysis did not clearly and consistently enable the differentiation of any of the populations of ticks in this study. Each feature differed among samples in different sets of pairwise relationships. Mitochondrial cytochrome oxidase subunit I gene (COX1) has been presented as a suitable mitochondrial gene to clarify complex groupings that were not resolved when using other mitochondrial genes. COX1 has also been proposed to be the main gene for differentiating between R.(B) microplus and R.(B) australis. The aims of this study were to confirm whether COX1 can be used to resolve complex relationships within the R.(B) microplus clade and to determine whether there is justification for the view that R.(B) australis is a distinct species from R.(B) microplus. Maximum likelihood trees were constructed with a Bootstrap analysis. A relaxed clock Bayesian analysis was then undertaken to estimate topology and divergence timings, using three ticks found in amber covering three genera: Amblyomma, Hyalomma and Ixodes to calibrate the clock. These analyses suggest that R.(B) microplus is a clade, containg five subspecies including R.(B) annulatus, R.(B) australis, and three, regionally based clades of R.(B) microplus: 1. All the South and Central American isolates together with isolates from Cambodia, Thailand, and some of those from Malaysia; 2. Indian and the remaining Malaysian isolates; 3. Most of the Chinese isolates. R.(B) decoloratus shares a common ancestor with R.(B) microplus and R.(B) annulatus however it is clearly divergent, appearing to be more related to R. bursa. All proposed groups of R.(B) microplus also appear to have evolved within the same time scale (within the last 20 million years). Bm86 is the name given to a midgut glycoprotein that is the target antigen of the only commercially available vaccine against ticks. All the prior work on this gene has been conducted using cDNA and suggests a high degree of sequence variation and the presence of different isoforms. The aim was to use genomic DNA to examine the regional variation in the Bm86 sequences and to determine whether Bm86 variation segregated according to the recently proposed taxonomic re-classification of R.(B) microplus and R.(B) australis. / After extensive optimization, it was found that all primer sets, including those previously published and those designed in this project, failed on the extracted genomic DNA from all isolates. High variability in the published cDNA sequences indicated an extremely high mutation rate, which could potentially be linked to variation in the function of the protein and its utility as a vaccine immunogen. Analysis of sequence alignments from publicly available databases did not allow grouping of samples by either geographical location or proposed taxon. These findings are in apparent contradiction to claims by other researchers that regional variation in the efficacy of the vaccine is associated with regional variation in sequence.

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.