Petal spots are aggregations of pigmented cells in distinct regions of the petal and are known to function in pollinator attraction across multiple systems. They occur within many plant lineages, but petal spots of the South African daisy species Gorteria diffusa are unusually complex. These elaborate structures are richly pigmented, deeply textured, and include three distinct cell types. G. diffusa petal spots function in attracting male bee-fly pollinators, in one of only two known cases of sexual deception outside of the Orchidaceae. G. diffusa is comprised of geographically discrete floral morphotypes, defined by extreme intraspecific variation in capitulum phenotype. Between-morphotype variation in the position and complexity of petal spots is associated with differential pollinator behavioural responses. As such, this system has much potential for understanding the molecular development of an ecologically relevant trait within a powerful intraspecific comparative framework. The sexually deceptive Spring morphotype was the focus for this project, which had two main aims: to investigate Spring population genetic structure and to characterise the genes regulating anthocyanin pigmentation within petal spots. For the latter, two additional morphotypes were studied to improve the robustness of conclusions based on comparisons between spotted and plain petal tissues.
The anthocyanin cyanidin 3-glucoside was found to pigment G. diffusa ray floret petals. In petal spots there was a high proportion of malonated anthocyanin, that was absent from other petal regions. In the first comprehensive characterisation of a G. diffusa petal spot developmental pathway, a small family of subgroup 6 R2R3 MYB transcription factors (GdMYB8 proteins) were identified as potential petal spot anthocyanin regulators. The genes encoding these proteins were found to be upregulated within petal spots and induced ectopic anthocyanin production when stably transformed into Nicotiana tabacum. Potential downstream targets of GdMYB8 proteins within the anthocyanin synthesis pathway were identified. The expression patterns of genes encoding these enzymes, and the ability of GdMYB8 proteins to bind to promoter regions of the anthocyanin synthesis genes (in yeast), imply that GdMYB8 proteins are likely to regulate G. diffusa petal spot anthocyanin production through co-regulation of several anthocyanin synthesis enzymes. Extending our developmental approach into one which addresses the evolution of petal spot regulators across morphotypes, requires fundamental understanding of the genetic nature of morphotypes and in-depth characterisation of their floral phenotypes. Toward this aim, a genotyping by sequencing analysis of genetic structure within the Spring morphotype was conducted, along with floral phenotypic measurements of the individuals sequenced. Limited floral trait variation was detected within the Spring morphotype, but there was no grouping of phenotypes by locality. The genetic analysis indicated strong isolation by distance patterns, hypothesised to be due to limited seed dispersal. These findings suggest that limited dispersal may be a key component contributing toward the evolution of G. diffusa floral morphotypes, pending further investigation. Ultimately, this research enhances our understanding of the genetics underlying G. diffusa petal spot development. It also demonstrates that isolation by distance is a major determinant of gene flow within a subset of G. diffusa, providing a first insight into the mechanisms that may facilitate the evolution of extreme intraspecific variation.