Ecological responses to a changing climate have been well documented in a broad range of species, predominantly in terms of range movements and phenological changes. When faced with a changing environment, species survival will depend on the ability to: 1) evade changes in climate, via dispersal; 2) evolve, via natural selection; and/or 3) plastically change their phenotype, without underlying genetic changes. The potential for an organism to evade, evolve or plastically respond to a changing environment can be predicted by inferring relationships with current climatic conditions. Altitudinal gradients have been proposed as being particularly suitable for environmental change studies due to the rapid variation in climate even over short geographical distances. Species that inhabit altitudinal gradients experience a range of climatic conditions across their range and are thus subject to varying selection pressures. Changes in temperature are predicted to particularly influence ectotherms due to the direct effect on physiological processes. The common frog (Rana temporaria) occurs from zero to over a thousand metres along altitudinal gradients in Scotland, offering the opportunity to assess the influence of temperature on organism responses. The overall aim of this thesis was to assess population-level relationships with climate, in order to make predictions regarding susceptibility to a changing climate, focussing on R. temporaria in Scotland. In Chapter 2, I inferred colonisation patterns within Europe following the last glacial maximum by combining new and previously compiled mitochondrial cytochrome b DNA sequences. I found that the mitochondrial DNA sequences from my Scottish samples were identical to, or clustered with, the common haplotype previously identified from Western Europe. This clade showed very low mitochondrial genetic variation, consistent with a leptokurtic model of range expansion, where low numbers of long-distance dispersers cause multiple founder events. Second, I assessed fine-scale genetic variation in relation to current temperature gradients using microsatellites. No population structure was found within or between altitudinal gradients at any scale (3-50km; average FST= 0.02), despite a mean annual temperature difference of 4.5°C between low- and high-altitude sites. Levels of genetic diversity and heterozygosity were considerable but did not vary by site, altitude or temperature. In Chapter 3, common temperature treatments were used to assess phenotypic differentiation and phenotypic plasticity variation in relation to altitude in terms of larval fitness traits. Local adaptation to altitude was assessed using QST-FST analyses and adaptive phenotypic divergence was then related to environmental parameters using Mantel tests, to look for drivers of selection. I found that R. temporaria showed evidence of local adaptation in all larval fitness traits measured. However, only variation in larval period and growth rate was consistent with adaptation to altitude. Moreover, this was only evident in the three mountains with the highest high-altitude sites (at least 900m). Adaptive divergence in traits that were locally adapted to altitude was correlated with spring temperature, suggesting that temperature acts as a strong environmental selection pressure influencing local adaptation even in the face of high gene flow. In Chapter 4, the physiological and behavioural responses that facilitate survival in high-altitude environments were evaluated, in terms of routine metabolic rate and freeze tolerance in tadpoles, and breeding temperature in adults. I found that routine metabolic rate was lower for individuals sampled from high- than low-altitude sites but only from the three mountains with the highest high-altitude sites (at least 900m). Glucose accumulation during freezing was not significantly different based on altitude. However, individuals from low-altitude survived freezing significantly better than those from high-altitude, across all mountains. Breeding did not occur below 5˚C at any site and there was no significant difference in breeding temperature between high- and low-altitude sites, leading to high-altitude individuals spawning 30 days later than those at low-altitude. My results suggest that tadpoles are adapted physiologically to surviving at high-altitude via reduced routine metabolic rate, but only at the highest breeding sites. Finally, in Chapter 5, I assessed the spatial variation in species presence and composition of parasitic water moulds in the genus Saprolegnia found on R. temporaria eggs. Thirteen samples isolated from four sites were identified as members of the Saprolegniaceae. Four putative species of Saprolegnia were isolated overall, multiple Saprolegnia water moulds were isolated from within sites, and species composition varied between sites. Acidity was significantly lower at sites where 4 Saprolegniaceae were present, but genetic distance between samples was not correlated with environmental or geographic distance. These findings question the previous focus on S. ferax as the primary agent of Saprolegnia infection in amphibians and suggest that future studies of virulence need to consider the synergistic effect of multiple Saprolegnia species. In conclusion, R. temporaria show the potential for evasion, evolution and plastic responses to a changing climate and my results suggest that the outlook is positive for survival of the common frog in Scotland.

To induce pluripotency in differentiated cells, it is necessary to remodel the epigenetic marks on the regulatory regions of pluripotency genes to enable their expression. The induction of Nanog expression is crucial for establishing pluripotency. However, the epigenetic mechanisms associated with the reprogramming of Nanog expression are not fully understood. In mammalian chromatin, epigenetic control of gene expression includes DNA methylation and histone modifications. In undifferentiated cells, regulatory regions of the pluripotency genes Nanog and Oct4 are demethylated and enriched with activating histone marks while being relatively depleted of repressive marks. One route to investigate mechanisms of cellular reprogramming is through treatment of cells with oocyte extracts from amphibians, such as Xenopus and axolotl. Previously, our lab demonstrated that a major difference between these extracts is that the axolotl oocyte can reprogram expression of the mammalian Nanog, while Xenopus oocyte cannot. In this study, I used extracts from oocytes of axolotl (AOE) and Xenopus (XOE), and focused on the mechanisms underlying the reversal of epigenetic marks in regulatory regions of the mouse Nanog gene during its reactivation. I demonstrated that AOE remodels the mouse somatic chromatin by increasing the level of 5hmC on both mouse Nanog enhancer and promoter sequences as well as adding the activating histone marks H3K27ac and H3K4me1 specifically to the Nanog enhancer. XOE was unable to induce these modifications. The expression of Nanog ortholog axNanog and histone variant H2A.Z in axolotl oocytes, but not in Xenopus oocyte, is likely to be one of the reasons for the differences. Indeed, I demonstrated the binding of axNanog and H2A.Z on the mouse Nanog gene in response to AOE, but not XOE treatment. Furthermore, my experiments have elucidated the sequence of chromatin remodelling events during oocyte extract reprogramming that begins with H2A.Z deposition at the Nanog enhancer which allows axNanog binding, followed by epigenetic alterations such as 5hmC, H3K27ac. Taken together, this study refines our understanding of the step-wise events necessary for remodelling of somatic cell chromatin and underlies the difference in the reprogramming capacity of different Amphibian oocytes.