Thesis (Ph.D.)--University of Western Australia, 2006.

Thesis (MScAgric)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The exomorphology and size of Leucadendron pollen was examined using a scanning electron and light microscope respectively. Pollen was found to have a consistent triangular shape with three apertures. Pollen grain size however, show difference between species, sections and subsections on Leucadendron. Pollen of all species examined had a woven appearance like the intertwined threads of a fabric with orbicules present on the surface of L. chamelaea, L. elimense subsp. elimense and L. galpinii. Pollen viability was successfully assessed using a solidified agar medium containing 2g agar and 109 sucrose. Pollen germination for all species was found to be above 55% viability. A diallellayout of crosses has demonstrated conclusively that fecundity differs when crossing between species of the genus Leucadendron. Artificial hand pollination was applied successfully on Leucadendron and showed repeatedly that seed set following intraspecific crosses between the male and female inflorescence of the same species gave the same high rate of seed set as found in nature. However, seed numbers declined sharply when crossing between species of the section 'Leucadendron'. Seed set following crosses between species of different sections or sub-sections was the lowest and in most cross combinations there was no seed harvested or no seed germination. The diallellayout was useful in identifying incompatible species and for locating possible incompatibility barriers to interspecific seed development. The morphology of the stigma was examined with a scanning electron microscope. Stigma appearance of all species had a consistent round to oval shape, except for L. rubrum, which had an elongated shape. Stigma surfaces of all species were densely covered with a large number of unicellular papilar cells on the swollen base. The aniline blue staining technique, together with the fluorescent microscope technique was used to follow the growth of the pollen tube following compatible and incompatible cross combinations. Pollen on the stigmas of compatible and incompatible species examined showed signs of germination. Pollen tubes grew between the papilla cells in all directions and only the most vigorous ones reached the upper part of the style. From the upper region of the style, yellow green tubes grew cohesively in the middle of the style towards the ovule. In compatible combinations a not more than 4 tubes reached the ovule region, but was difficult to observe when they entered the micropyle for fertilization. In incompatible species a large number of abnormalities occurred beyond the upper region of the style. / AFRIKAANSE OPSOMMING: Die morfologie en grootte van Leucadendron stuifmeel is deur middel van 'n skandeerelektron - en ligmikroskoop bestudeer. Baie klein verskille in stuifmeel morfologie het voorgekom. Diverse verskille in stuifmeelgrootte het wel voorgekom tussen spesies, groepe en subgroepe van Leucadendron. Stuifmeelvorm was deurgaans driehoekig en die oppervlakte van die stuifmeelkorrel het die voorkoms van geweefde vesels gehad. Klein, bolvormige struktuurtjies was teen verskillende digthede oor die stuifmeeloppervlak van L. chamelaea, L. elimense subsp. elimense en L. galpinii versprei. Stuifmeelkiemkragtigheid is bepaal deur dit op soliede agar medium te ontkiem en was deurgaans bo 55% kiemkragtig. Onderlings dialleliese kruisings van Leucadendron spesies het variasie in saad set getoon. Handbestuiwing is suksesvol uitgevoer en saadset in intraspesie kruisings hoog en soortgelyk aan natuurlike bestuiwing. Saadset en saad ontwikkeling het drasties verswak toe verder vewante spesies as ouers gebruik. As gevolg van hulondeurdringbare saadhuid is neutagtige sade gewoonlik moeiliker ontkiembaar. Die diallel uitleg was ook nuttig om verenigbare en onverenigbare kruisingskombinasies te identifiseer en om onverenigbaarheidskanse op te spoor. 'n Skandeerelektronmiskoop is gebruik om die morfologie van die stigma te bestudeer. Stigmas was deurgaans rond tot ovaalvormig, behalwe die van L. rubrum wat 'n verlengde voorkoms gehad het. Die stigma bestaan uit 'n groot aantal eensellige papilla, wat dig teen mekaar gepak is op 'n geswolle basis. Aniline-blou fluoresserende kleurstof en 'n fluoressensie mikroskoop is gebruik om die pad van die stuifmeelbuis in verenigbare en onverenigbare kruisingskombinasies in Leucadendron te volg. Stuifmeelontkieming het in alle kruisingskombinasies geskied. Stuifmeelbuise het in alle rigtings tussen die papilla gegroei en slegs die mees kiemkragtige stuifmeelbuise het die boonste deel van die styl bereik. In die styl het die buise dig teen mekaar gegroei en was dit moeilik telbaar. 'n Maksimum van vier buise het die vrugbeginsel bereik, maar dit was moeilik om verder te volg nadat hulle die poortjie bereik het. In onverenigbare kruisingskombinasies het stuifmeelbuise abnormale groeipatrone in die boonste gedeelte van die styl getoon.

Flowers from members of the genus Leucadendron have colourful bracts and long vase life that make them highly desirable cut-flowers. Breeding programs based on interspecific hybridization would encounter difficulty if pre- or post-fertilization barriers exist in the distant crosses. Embryo rescue is one of the commonly used approaches to overcome post-fertilization barriers in wide hybridization. In this study, intersectional and intersubsectional hybridization of Leucadendron was attempted. Observation of pollen-pistil interactions revealed that post-zygotic rejection was the main reason for the incompatibility of the crosses, therefore embryo rescue was adopted and a protocol was developed to raise the hybrids. To better understand the genome structure in the genus, karyotypes of selected species were analyzed. Chromosome examination indicated that all (27) Leucadendron species examined were diploid and had a chromosome number of 2n = 26. The chromosomes were small in size and had predominantly median to submedian centromeres. The karyotypes of the species were rather symmetrical and seemed to be primitive according to Stebbins' karyotype classification. DNA based PCR-RFLP and RAMP markers were developed to identify Leucadendron hybrids at an early age. RAMP analysis showed more discrimination in identifying Leucadendron hybrids than did PCR-RFLP. The occurrence of PCR recombination also proved to be a troublesome issue when using the PCR-RFLP method, whereas the clarity of the interpretion of the RAMP method was not influenced by PCR recombination. Interspecific hybridization in a breeding program can provide valuable information on grouping of the species for systematic purposes. Regression analysis between cross success rate and cpDNA character difference revealed that there was a highly significant correlation between them. Patterns of success for intersectional hybridizations in Leucadendron were generally consistent with current taxonomic hypotheses regarding the sectional division of the genus. Success was generally lower for intersectional crosses than for intrasectional crosses.

Thesis (PhD(Agric)--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The bright colouration of involucral leaves in Leucadendron is unfortunately transient in nature. Undesirable colour changes render this cut flower unmarketable, resulting in a considerable loss of profit. A deeper understanding of the mechanism leading to colour change is needed to form the framework on which future manipulation strategies can be built. Yellow Leucadendron possess the ability to degreen and regreen naturally, a phenomenon linked to the controlled degradation of chlorophyll and the lesser degradation of carotenoids, which then impart the yellow colour. This colour change is directly linked to the development of the inflorescence. Involucral leaves degreen towards anthesis and are entirely yellow at full bloom. They begin to regreen again when the last florets on the cone have wilted. Deconing before flowering completely inhibits the colour change. Deconing at full bloom, results in leaves regreening sooner. Therefore the inflorescence appears to be the origin of the cue for colour change. Any factor that expedites the death of the florets, results in sooner regreening of involucral leaves. Ultra-structurally, the degreening and regreening resulted from a transdifferentiation of mature chloroplasts to gerontoplast-like plastids, which upon regreening completely redifferentiated into fully functional chloroplasts. In the red Leucadendron cultivar Safari Sunset, the photosynthetic pigment degradation pattern is identical to that of yellow cultivars. However, colour expression is complexed by the presence of anthocyanins. Anthocyanin concentration was shown to be directly related to the opening of the flower head rather than to the phenological development of the inflorescence. With opening, the previously shaded inner involucral leaf surfaces are exposed to higher levels of irradiance and respond by turning red, presumably for photoprotection. Similar to yellow cultivars, any factor leading to the death of the florets before flowering, not only prevents the degreening of involucral leaves, but also prevents the opening of the flower head and therefore the associated change in anthocyanin levels. The ecological significance of regreening was also investigated. What does a female Leucadendron plant stand to gain by regreening rather than discarding the involucral leaves? Regreened involucral leaves were shown not to play a significant role in providing photosynthates for the developing cone. Although the presence of regreened involucral leaves were shown to provide protection against high irradiance and radiant heating of the cone, they were not essential to ensure survival of the cone. The small floral bracts were shown to be very capable of adaptation. The most plausible reason for regreening is therefore assumed to be based on a cost-benefit relationship. As most Leucadendron are adapted to grow on very nutrient poor soils, the question should maybe be rephrased. Why waste valuable resources? Sclerophyllous leaves, like the involucral leaves, are costly to make and therefore reusing, rather than discarding them does seem a sensible strategy for survival. / AFRIKAANSE OPSOMMING: Leucadendron snyblomme word gekenmerk deur die helder kleure van hul omwindselblare. Die helder kleure is egter slegs vir 'n kort periode aanwesig waarna die snyblomme onbemarkbaar word, met aansienlike verlies aan potensiele inkomste. Die ontwikkeling van manipulasies ten einde die bemarkbare periode van Leucadendron te verleng, berus op die verkryging van 'n dieper insig in die meganisme van kleurverandering. Die kleurveranderinge van geel Leucadendron omwindselblare is te wyte aan 'n unieke vermoë tot die gereguleerde degradasie en heropbou van chlorofiele en karotenoiede onder direkte beheer van die ontwikkelende bloeiwyse. Met die aanvang van blom, lei groter proporsionele degradasie van chlorofiele tot geleidelike vergeling van omwindselblare. Die hele blomhofie verkry uiteindelik met volblom 'n helder geel kleur. Sodra die laaste blommetjies doodgaan, neem chlorofiel- en karotenoiedsintese weer in aanvang en binnekort is die omwindselblare weer net so groen soos voor die aanvang van blom. Die geel verkleuring kan verhoed word deur die keël voor blom uit te breek. Enige faktor wat die dood van die blommetjies versnel, asook die uitbreek van keël tydens volblom, lei tot die vroeëre aanvang van vergroening. Die degradasie van plastiedpigmente hang nou saam met die differensiasie van volwasse chloroplaste tot gerontoplast-agtige plastiede wat op hul beurt weer tydens vergroening tot volkome funksionele chloroplaste herdifferensieer. Soortgelyk aan geel Leucadendron kultivars, vind die veranderinge in plastiedpigmente ook plaas tydens blom van die rooi kultivar, Safari Sunset. Kleurveranderinge in 'Safari Sunset' is egter meer ingewikkeld vanweë die aanwesigheid van variërende konsentrasies antosianiene. Antosianienkonsentrasies en rooi kleur neem toe tydens blom vanwee die blootstelling van die beskutte adaksiale binnekante van omwindselblare aan hoe irradiasie met die oopvou van die blomhofie. Die akkumulasie van antosianiene het moontlik 'n fotobeskermende funksie. Kleurveranderinge in 'Safari Sunset' kan, soos in geel kultivars, voorkom word deur blom te verhoed. Antosianiensintese word voorkom deurdat die blomhofie geslote bly en is nie direk gekoppel aan blom soos wat met plastiedpigmente die geval is nie. Die belang van vergroening is ondersoek na aanleiding van die vraag oor wat dit 'n vroulike Leucadendron baat om omwindselblare te behou na die afloop van blom? Die bydrae van foto-assimilasie deur omwindselblare tot die ontwikkeling van keels is beperk. Alhoewel omwindselblare wel keels teen hoe irradiasie en stralingsverhitting beskerm, is die blomskutblare in staat om aan te pas by hierdie kondisies. Die mees waarskynlike verklaring vir die behoud van die omwindselblare na blom berus moontlik op 'n koste-voordele verwantskap. Alhoewel nie essensieel nie, is die beperkte bydrae van die omwindselblare na die afloop van blom tot die oorlewing en welstand van die keel waarskynlik genoegsaam om hul behoud te regverdig. Verskeie Leucadendron spesies groei in gronde wat baie arm is aan nutriente. Sklerefiele blare, soos die van Leucadendron, is verder duur om te vervaardig. Dit maak dus sin om hulle vir meer as een funksie te herontplooi eerder as om hulpbronne te belê in meer gespesialiseerde en minder durende blombykomstighede. Dus dui die behoud van omwindselblare dalk op 'n strategie wat gemik is op die behoud en besparing van beperkte hulpbronne.

[Truncated abstract] Grevillea and Leucadendron belong to Proteaceae and both have economic importance to the floriculture industry. Grevillea is a highly diverse genus endemic to Australia and very attractive for landscaping. Leucadendron is a South African Proteaceae but is cultivated in Australia and is well known as a cut flower. This thesis focuses on the application of DNA-based molecular markers to these genera. Several groupings within Grevillea were suggested by previous researchers based on morphological characteristics. In this thesis the monophyly of the groupings among 12 Grevillea species from New South Wales was tested using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analyses. To test the robustness of the data, UPGMA using Jaccard similarity, Neighbor Joining using total character difference and Wagner parsimony analyses were undertaken. The relationship trees generated supported monophyly of the groupings. Chloroplast DNA (cpDNA) was used to develop phylogenetic relationships among Leucadendron species. Inheritance and variation of cpDNA were evaluated using PCR-RFLP. The study demonstrated that cpDNA was inherited maternally and a phylogenetic tree of Leucadendron species using parsimony analysis was constructed. ... A fingerprinting study conducted using ISSR, produced a dendrogram showing the relationships among 30 cultivars. From the results, i a fingerprinting key was developed. Three examples of synonymous cultivar pairs were identified. In Leucadendron the male and female flowers develop on separate plants, and sex identification is only possible at time of flowering. ISSR, suppression subtractive hybridisation (SSH), and SSH combined with mirror orientation selection (MOS) were used in attempts of identifying sex-dependent DNA fragments at earlier stages of plant development. Neither of these techniques was able to identify sex-specific markers in Leucadendron. Nevertheless, the results did indicate that cpDNA copy number may differentiate male and female plants. Also, it was demonstrated that the genomes of male and female plants are quite homologous, which increases the difficulty in identifying sex-specific sequences. This thesis highlights the potential of DNA-based markers to determine species relationships in Grevillea and Leucadendron, as well as to identify Leucadendron cultivars. The information produced during the research for this thesis provides a basis for Grevillea and Leucadendron variety development and may be used to assist the design of interspecific crosses, to identify cultivars and the parents of hybrids. In addition, the results offer insights into the likelihood, problems and strategies of finding sex-specific markers for genes controlling sex in Leucadendron. ii

abstract: The Cape Floral Region (CFR) in southwestern South Africa is one of the most diverse in the world, with >9,000 plant species, 70% of which are endemic, in an area of only ~90,000 km2. Many have suggested that the CFR's heterogeneous environment, with respect to landscape gradients, vegetation, rainfall, elevation, and soil fertility, is responsible for the origin and maintenance of this biodiversity. While studies have struggled to link species diversity with these features, no study has attempted to associate patterns of gene flow with environmental data to determine how CFR biodiversity evolves on different scales. Here, a molecular population genetic data is presented for a widespread CFR plant, Leucadendron salignum, across 51 locations with 5-kb of chloroplast (cpDNA) and 6-kb of unlinked nuclear (nuDNA) DNA sequences in a dataset of 305 individuals. In the cpDNA dataset, significant genetic structure was found to vary on temporal and spatial scales, separating Western and Eastern Capes - the latter of which appears to be recently derived from the former - with the highest diversity in the heart of the CFR in a central region. A second study applied a statistical model using vegetation and soil composition and found fine-scale genetic divergence is better explained by this landscape resistance model than a geographic distance model. Finally, a third analysis contrasted cpDNA and nuDNA datasets, and revealed very little geographic structure in the latter, suggesting that seed and pollen dispersal can have different evolutionary genetic histories of gene flow on even small CFR scales. These three studies together caution that different genomic markers need to be considered when modeling the geographic and temporal origin of CFR groups. From a greater perspective, the results here are consistent with the hypothesis that landscape heterogeneity is one driving influence in limiting gene flow across the CFR that can lead to species diversity on fine-scales. Nonetheless, while this pattern may be true of the widespread L. salignum, the extension of this approach is now warranted for other CFR species with varying ranges and dispersal mechanisms to determine how universal these patterns of landscape genetic diversity are. / Dissertation/Thesis / Ph.D. Biology 2013