A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, South Africa in fulfilment of the requirements for the degree of Master of Science, 2019 / The study of flower colour is a particularly valuable approach to investigating fundamental evolutionary questions such as the maintenance of variation within species, the role of natural selection and genetic drift in maintaining polymorphisms, and how such polymorphisms contribute to biodiversity. Flower colour is a phenotype that is easily measured and it provides a strong indicator of the outcomes of selection pressures. It is both driven and maintained by either non- pollinator mediated selection agents, pollinator selection agents, or a combination of both. The overall aim of this study was to investigate the maintenance of flower colour using Rhodohypoxis baurii (Baker) Nel. var. confecta Hilliard and Burtt as a study system. This involved understanding the genetic mechanisms that regulate flower colour and examining the environmental variables that drive this trait variation across populations. Molecular and ecological methods were used in combination to understand flower colour by investigating the genes responsible, as well as the environmental selection pressures acting on this phenotype. Rhodoypoxis baurii var. confecta is a Drakensberg near-endemic species that has naturally occurring populations of magenta, pink, and white flower colour morphs. These flower colour morphs are discrete variants that co-exist in single populations and are thus considered to be true polymorphisms. Three populations of R. baurii var. confecta that occur in Sentinel Peak in the Royal Natal National Park, South Africa were studied. Non-pollinator mediated selection agents were investigated as potential drivers of flower variation in R. baurii var. confecta. The frequency of unpigmented (white) and pigmented (pink/magenta) flower colour morphs was measured over the flowering season. No pollinator visitation was observed at populations of R. baurii var. confecta and therefore pollinator-mediated selection was excluded. The extent to which non pollinator selection agents drive this intra-population variation was investigated by measuring the change in soil moisture, temperature, and precipitation over the flowering season. One population shifted from a greater proportion of unpigmented morphs at the beginning of the flowering season to a relatively greater proportion of pigmented morphs at the end of the season. In this population, a positive correlation was found between the proportion of pigmented morphs and soil moisture. This suggests that the accumulation of water in the soil promotes the production of pigmented flowers. The anthocyanin biosynthetic pathway (ABP) is responsible for the production of pigment in R. baurii var. confecta. The ABP is pleiotropic and in addition to pigment production is responsible for traits related to plant physiology and environmental stress response. Consequently, the ABP is cited as being well conserved among angiosperm species. The presence of expression in four ABP genes (CHI, F3H, F3’H, and F3’5’H) was tested for and compared between unpigmented and pigmented R. baurii var. confecta flowers. As there is no sequence data for Rhodohypoxis or any Hypoxidaceae species, primers for this molecular work were designed using closely related monocotyledon ABP gene sequences. Sequencing results showed that the amplified PCR products were not the targeted ABP genes. These results suggested that the designed primers were non-specific. The similarity of the four ABP genes within angiosperm species was investigated. All available complete sequences of the four regions of interest were aligned and sequence similarity was quantified. The results from this alignment indicate that the four investigated ABP gene sequences are polymorphic and are likely not well conserved within angiosperm species as a whole and, to some extent, within monocotyledon and dicotyledon species respectively. This suggests that the basic structure of the ABP is well conserved amongst angiosperm species however, the gene sequences that make up the pathway are polymorphic and less well conserved. The study highlights the importance of non-pollinator mediated selection on the presence and maintenance of flower colour polymorphisms. In addition, it provides some insight on the ABP and its conservation amongst angiosperm species. It also contributes to understanding how flower colour polymorphisms are maintained in natural populations both on a molecular and ecological level and is the foundational work in understanding the polymorphisms of R. baurii var. confecta. / TL (2020)