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Despite over a century of research, malaria parasites (Plasmodium) still remain a major cause of mortality and morbidity worldwide. In recent years, the application of theoretical principles from ecology and evolutionary biology to the study of these parasites has started to provide insight into variety of fundamental subjects from the evolution of virulence to the facultative strategies (i.e. phenotypic plasticity) that parasites use to maximize their transmission. It is now becoming increasingly clear that to understand and predict population level patterns of virulence and transmission, the processes that occur at the between-host level must be studied in light of the interactions that happen within hosts (between parasites and between parasites and hosts). In this thesis I combine concepts from evolutionary biology and ecology with tools from molecular and cellular biology and evolutionary genetics, which allow me to study rodent malaria parasites at both evolutionary and ecological timescales. The work I present in this thesis has the following four components: 1. Phylogenetics (chapter 2): I applied recently developed phylogenetic methods to a large DNA sequence dataset that I generated, to provide a better understanding of the phylogeny of rodent malaria parasites and investigate how selection has shaped their genomes. I show that all rodent malaria subspecies can be considered species, provide the first time line for the evolution of this group of parasites and demonstrate that most loci are under purifying selection. 2. Hybridization and reproductive isolation (chapter 3): I show that hybridization between two rodent malaria parasites (P. berghei and P. yoelii) can occur, but only occurs at high levels when one of two proteins (P230 or P48/45) is absent from the surface of female gametes, which indicates that these proteins are involved in gamete recognition. I find that P230, P48/45 and P47 (a possible interaction partner) are evolving under positive selection, a feature often observed in gamete recognition proteins of other taxa. Finally, I show that the fertilization success of P. berghei is reduced in the presence of P. yoelii, but not vice-versa, which indicates asymmetric reproductive interference. 3. Sex allocation (chapter 4): I carry the first test of sex allocation’s assumption that immunity impacts on the fertility of Plasmodium male gametocytes/gametes more than on the fertility of females. I show that while the fertility of both males and females is equally affected, males are affected during gametogenesis and females are mostly affected through gamete dysfunction (i.e. gametes can mate but zygotes fail to develop), which is in agreement with the assumptions of theory. In collaboration, I incorporate these effects into sex allocation theory and predict that malaria parasites can minimize the effects of factors that kill gametocytes/gametes by adjusting their sex ratios. On the other hand sex ratio adjustment cannot compensate for gamete dysfunction or zygote death. These results have applied implications for transmission-blocking vaccines. 4. Infection dynamics of mixed-species infections (chapter 5): I develop a series of experiments to test how a focal parasite species (P. yoelii) is affected by competition with heterospecifics (P. chabaudi) and how the interaction between the two species is mediated by immunity and resource availability. I show that P. chabaudi can boost P. yoelii above its single species level (i.e. facilitation) and that this is mediated by resource availability. On the other hand, P. yoelii’s performance can also be hindered in mice that were exposed to a P. chabaudi infection. My results also reveal that host mortality is exacerbated in mixed-species infections of naïve mice, which may be due to an inability of the host to achieve the right balance between the production and the destruction of red blood cells, when dealing with a mixed-species infection. The work I present here tackles fundamental questions concerning the transmission biology and the within-host interactions of malaria parasites The results presented demonstrate the importance of interactions between hosts and parasites and between different parasite species (at the molecular and the whole organism levels) for determining the outcome of transmission, virulence and within-host parasite performance.

This study evaluated comparative genomic hybridisation (CGH) as a tool to detect candidate regions of the genome associated with chemoresistance. Using a variation on conventional CGH, DNA from three cell lines that were resistant to thymidylate synthase (TS) inhibitors (tomudex [TDX] or 5-fluorouracil [5-FU]) and their sensitive parent cells were evaluated. In MCF-7 and H630, cells that were resistant to TDX, a specific TS inhibitor with no other known cytotoxic potential, only a single region of change (18p gain) was apparent. The third cell line H630R10, which was resistant to 5-FU, had changes in several genomic regions following the acquisition of resistance, including 18p. Gain in the chromosomal region containing the TS gene (18pll.32) was detected by CGH in all three resistant cell lines. However, additional novel regions of interest were identified in the cells that were resistant to 5-FU, a cytotoxic agent known to have several other modes of cytotoxicity besides TS inhibition. These results suggested that CGH is of potential use in the detection of regions of the genome involved in chemoresistance. Having shown the potential of CGH as a tool for assessing chemoresistance at the genomic level, steps toward clinical application of this technique were evaluated. A prerequisite for study in archival pathology samples was successful DNA extraction and universal amplification of tumour DNA from paraffin-embedded tumour sections for CGH analysis. Degenerate oligonucleotide primed - polymerase chain reaction (DOP-PCR) was performed on minute quantities (50ngs) of fresh cell line DNA (H630R10) and tumour DNA (osteosarcoma), as well as paraffin-embedded DNA from the same case. The results of these DOP-PCR CGH reactions were compared with conventional CGH using l|0.g quantities of fresh DNA from both H630R10 cell line and osteosarcoma. The CGH profiles of the conventional CGH and DOP-PCR CGH did not show a high level of concordance, only 55% of the gains and 83.3% of losses detected by conventional CGH were detected by DOP-PCR CGH The use of universal amplification by DOP-PCR in paraffin-embedded sections was not taken forward into clinical evaluation. A study of colorectal cancer (CRC) was initiated which involved the microdissection of 29 Dukes' C CRC tumours from fresh frozen material for CGH analysis. This conventional CGH analysis of CRC tumours involved assessing each tumour twice by reversal of fluorochromes. Only genomic regions that were detected as changed in both forward and reverse profiles were accepted. This approach detected several regions of genome as changed across the 29 tumours. In all, 108 gains (a mean number of 3.7 aberrations per tumour, range 1-12) and 85 losses (a mean number of 2.9 aberrations per tumour, range 0-11) were detected in the 29 tumours. CGH analyses identified certain chromosomal regions as more likely to be changed than others. The most frequent aberrations detected across the 29 tumours was a loss of chromosomal arm 18q, seen in 31% of the tumours assessed. Gain was also common at some sites throughout the genome, for example, gain of chromosomal arms, 13q and 20q was seen in 27.6% of cases. Mann-Whitney U tests investigating the association between specific chromosomal aberrations such as gain of 20q or loss of 18q and known markers of CRC tumourigenesis (p53, p27, p21, Rb, cyclin Dl, PCNA, P-catenin, e-cadherin, c-erbB-2, bcl2, EGFR and c-erbB-2) assessed by immunohistochemistry (IHC) in 29 tumours found no association. Testing of the total number of genomic aberrations detected (loss + gain = genetic grade) rather than the frequency of aberration at specific chromosomal loci also found no association with the CRC tumour markers. Finally, the association between the chromosomal aberrations detected by CGH was investigated in relation to patient survival. This thesis has demonstrated the value of a global approach to the study of chemoresistance and tumourigenesis through the application of powerful technology such as CGH.

Two of the most rapid drivers of evolution are hybridisation and polyploidisation. Hybridisation allows the rapid introduction of novel genetic material, potentially much faster than mutations, but this process is impeded by reproductive barriers between species. Differencesinploidylevelcanformsuchabarrier. Hybridisationaswellaspolyploidyareknown to occur frequently in the plant kingdom, including the genus Betula, which is investigated in this thesis. Three species of the Betula genus that exist in the United Kingdom are studied here: B. nana (dwarf birch), B. pendula (silver birch), and B. pubescens (downy birch). They differ in ploidy: B. nana and B. pendula are diploid and B. pubescens is a tetraploid. HybridisationandgeneflowbetweenthesethreespecieswasanalysedbyusingaRAD-seq dataset derived from 196 wild individuals. It was found that introgression acts unidirectionally from the diploid into the tetraploid species and that there is a cline of introgression between the north and south of the UK. This result suggests a range shift of the species from different distributions in the past. Gene flow from B. nana to B. pubescens could be a neutral or even maladaptive consequence of their past species distributions. Alternatively, it could be an adaptive process: alleles from B. nana could be helping B. pubescens to adapt to harsher, more northerly populations. To gain a preliminary understanding of the possible effects of introgression, the loci in close linkage to RAD tags introgressed from B. nana into B. pubescens were investigated and their putative function inferred by comparing their homologs in related species. To enhance the analyses, a draft whole genome sequence assembly of a B. nana individual was improved with long read data generated by PacBio sequencing, as well as the addition of RNA-seq data. This produced a more contiguous and complete reference sequence, enabling a closer look at more genes in linkage to the RAD tags.

Three subspecies of house mice of different geographic origins have reached New Zealand; M. m. domesticus (10 haplotypes), M. m. musculus (1 haplotype) both from Europe, and M. m. castaneus (3 haplotypes) from Asia. Identifying the sources of the multiple historical introductions of the house mouse is a complex issue, particularly during the peak colonisation period of 1830-1880. The early European settlers came with many bags, crates, plants, seed and livestock, which provided ample travel opportunities for stowaway rats and mice. With the assistance of volunteers, I have collected mice from various locations throughout the New Zealand region, to confirm the previously recorded haplotypes and to look for evidence of hybridisation between the colonising subspecies. Morphological characteristics traditionally used for identification of subspecies were compared with genetic characters, to establish whether the use of morphology is still a viable method of identifying subspecific distinctions between mice in New Zealand. While no M. m. musculus haplotypes were found among these samples, some mice still had the coat colouration typical of M. m. musculus. Data from mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) markers revealed some surprising results. I have found six new M. m. domesticus haplotypes, and three new M. m. castaneus haplotypes. The data have also revealed extensive hybridisation, particularly between M. m. domesticus and M. m. castaneus. The finding of the new haplotypes supports the previous assumption that there were multiple introductions of mice into New Zealand, but the finding of M. m. castaneus in inland towns and cities does not support the associated assumption that European mice were initially the only mice present in New Zealand. Rather, the wide distribution of M. m. castaneus suggests that this subspecies probably arrived during the early nineteenth century. The house mouse has long been recognised as an ideal organism for hybridisation studies, and the finding of a hybrid zone within New Zealand would provide an excellent opportunity to extend our knowledge of hybridisation and gene transfer. Previous studies found M. m. domesticus in Napier, and a M. m. musculus/M. m. castaneus hybrids in Wellington. A systematic sampling programme was undertaken between Wellington and Napier following State Highway 2 (SH2), in an attempt to locate a hybrid zone. Analysis of mtDNA and nDNA showed that, M. m. castaneus was found as far North as Dannevirke, and M. m. domesticus as far South as Featherston. There was also extensive evidence of integration of M. m. domesticus nDNA markers into mice with M. m. castaneus mtDNA. There was no clear support for a hybrid zone, but this does not mean that one has not existed in the past and that it has since been overrun by movement of mice with humans and their goods. Identification of subspecies by morphological characteristics, while used with confidence in other countries, is of little use in New Zealand. Extensive hybridisation/and or introgression between the subspecies has long since blurred any morphological distinctions the colonising mice may have had. I propose that these physical characters should be used in support of genetic analysis of the subspecies. The official classification of the New Zealand house mouse currently stands as M. m. musculus, however, data collected does not support this and it is proposed that the official classification should be changed to M. m. domesticus.