Cotton is a high-value commercial crop in Australia. Although cotton is largely self-pollinating, previous researchers have reported that honeybees, Apis mellifera, can assist in cross-pollination and contribute to improved yield. Until recently, use of bees in cotton had, however, been greatly limited by excessive use of pesticides to control arthropod pests. With the widespread use of transgenic (Bt) cotton varieties and the associated reduction in pesticide use, I decided to investigate the role and importance of honeybees in Bt cotton, under Australian conditions. I conducted two major field trials at Narrabri, in the centre of one of Australia’s major cotton-growing areas, in the 2005-6 and 2006-7 seasons. In the first trial, I particularly assessed methods of manipulating honeybee colonies by feeding pollen supplements of pollen/soybean patties, and by restricting pollen influx by the fitting of 30% efficient pollen traps. I aimed to test whether either of these strategies increased honeybee flight activity and, thus, increased foraging on cotton flowers. My results showed that although supplementary feeding increased bee flight activity and brood production, it did not increase pollen collection on cotton. Pollen traps initially reduced flight activity. They also reduced the amount of pollen stored in colonies, slowed down brood rearing activity, and honey production. However, they did not contribute to increased pollen collection in cotton. In the second trial, I spent more time investigating honeybee behaviour in cotton as well as assessing the effect of providing flowering cotton plants with access to honeybees for different time periods (e.g. 25 d, 15 d, 0 d). In this year, I used double the hive stocking rate of (16 colonies / ha) than in the previous year, because in 2005-6 I observed few bees in cotton flowers. I also conducted a preliminary investigation to assess whether there was any gene flow over a 16 m distance from Bt cotton to conventional cotton, in the presence of a relatively high honeybee population. Both of my field experiments showed that honeybees significantly increased cotton yield via increased boll set, mean weight of bolls, number of seeds / boll, and weight of lint / boll. It was obvious that cotton flowers, and particularly cotton pollen, were not attractive to honeybees, and this was also reflected in the low proportion (5.3% w/w) of pollen from cotton collected in the pollen traps. However, flower visitation rate was generally above the 0.5% level regarded as optimal for cross-pollination in cotton, and this was reflected in increased yield parameters. I recorded a gene flow of 1.7 % from Bollgard®II cotton to conventional cotton, over a distance of 16 m. This is much higher than had previously been reported for Australia, and may have been a result of high honeybee numbers in the vicinity, associated with my managed hives. In an attempt to attract more honeybees to cotton flowers, I conducted an investigation where I applied synthetic Queen Mandibular Pheromone (QMP) (Fruit Boost®) at two rates, 50 QEQ and 500 QEQ / ha, and for two applications, 2 d apart. Neither rate of QMP increased the level of bee visitation to flowers, either on the day of application or the subsequent day. There was also no increase in boll set or yield in plants treated with QMP. My observations of honeybee behaviour in cotton brought some interesting findings. First, honeybees totally ignored extra floral nectaries. Second, most flower-visiting honeybees collected nectar, but the overwhelming majority of them (84%) collected floral nectar from outside flowers: this meant these bees did not contribute to pollination. Those nectar gatherers which entered flowers did contribute to pollination. However, they were observed to exhibit rejection of cotton pollen by scraping pollen grains from their body and discarding them, prior to returning to their hives. Pollen gatherers collected only small, loose pellets from cotton. SEM studies showed that cotton pollen grains were the largest of all pollen commonly collected by bees in my investigations, and that they also had large spines. It is likely that these characteristics make cotton pollen unattractive to honeybees. Another possible reason for the unattractiveness of cotton flowers was the presence of pollen beetles, Carpophilus aterrimus, in them. I conducted a series of studies to determine the role of pollen beetles in pollination of cotton. I found that they did not contribute to pollination at low levels; at high populations they damaged flowers (with ≥ 10 beetles / flower, no flowers set bolls); and that honeybees, when given the choice, avoid flowers with pollen beetles. Because the insecticide fipronil was commonly used in Australian cotton at flowering time, and because I had some experience of its toxic effects against honeybees in my field investigations, I conducted a series of laboratory and potted plant bioassays, using young worker bees. The studies confirmed its highly toxic nature. I recorded an acute dermal LD50 of 1.9 ng / bee, and an acute oral LC50 of 0.62 ppm. Fipronil’s residual toxicity also remained high for an extended period in both laboratory and potted plant trials. For example, when applied to cotton leaves in weather-exposed potted cotton plants, it took 25 d and 20 d for full and half recommended rates of fipronil, respectively, to become non- toxic to honeybees. I had previously investigated whether a shorter period of exposure of cotton plants to honeybees would contribute adequately to increased yield, and concluded that a 10 d window within a 25 d flowering period would contribute 55% of the increase in total weight of bolls contributable to honeybee pollination, but only 36% of the increase in weight of lint. Given the highly residual activity of fipronil I recorded, the only opportunity for an insecticide-free period during flowering would be at its commencement. I concluded that, while there is evidence that honeybees can contribute to increased cotton yield in Bt cotton in Australia, this is unlikely with the continued use of fipronil at flowering. / Doctor of Philosophy (PhD)

Thesis (Ph. D.)--University of Wisconsin, 1965. / Includes bibliographical references.

Thesis (Ph.D.) -- University of Western Sydney, Hawkesbury, 1999. / Thesis submitted for the degree of Doctor of Philosophy. Includes bibliographical references (leaves 210-230).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 96-112).

Thesis (M. Sc.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 130-149). Also available via the World Wide Web.

Thesis (M.S.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 44-52). Also available on the World Wide Web.

For over twenty years, flower-visitation networks have been used to assess the effects of pollinator decline, linked to habitat loss, climate change and invasive species, on entire communities. However, most rely on flower visit frequency as a proxy for pollination; very few sample pollen from flower visitor's bodies or from stigmas and so do not include a quantitative measure of pollination success. Here, I add pollinator effectiveness (as single visit pollen deposition) into a traditional flower visitation network, creating a pollinator importance network that better evaluates the flower visitor community from the plant's perspective. Given recent interest in pollination in urban areas, I use an urban garden habitat, and compare visitation, pollen transport and pollinator importance networks, giving several novel conclusions. Firstly, although there are similarities in the structure of my networks, interactions were most specialised in the pollinator importance network, with pollen transport proving to be a better proxy for pollinator importance than visitation alone. Secondly, the specialisation of individual plants and the role of individual flower visitors varied between the networks, suggesting that community-level patterns in simple visitation networks can mask important individual differences. Thirdly, the correlation between flower visit frequency and pollinator importance largely depends on bees, and may not hold in plant-pollinator communities that are not bee-dominated. Fourthly, heterospecific pollen deposition was relatively low, despite the unusually diverse plant community of a garden. Finally, bees (particularly Bombus and non-eusocial halictids) carried the largest pollen loads and were the most effective at depositing pollen on to the stigma during a single visit in this garden habitat. The implications of this thesis highlight the strengths and limitations of each network for future studies, and raise important questions for the future of urban pollination studies.

Bibliography: leaves 256-323.

Orchids provide a model system for addressing evolutionary and ecological questions both because of their species diversity, and because the packaging of their pollen into pollinia facilitates the estimation of male and female pollination success. This thesis focuses on the ecology and evolution of pollination systems in the African orchid genus Eulophia, with an emphasis on deceptive pollination, mechanisms promoting cross-pollination, and pollinatordriven speciation. Pollination in the deceptive species E. zeyheriana is shown to depend on flower colour and proximity to the rewarding model species, Wahlenbergia cuspidata (Campanulacae). This study demonstrates the functional importance of colour matching between model and mimic in a floral Batesian mimicry system, as well as the importance of facilitation by the rewarding model [chapter 2]. The pollinaria of the vast majority of Eulophia and Acrolophia species undergo reconfiguration following removal by pollinators, similar to the phenomena first described by Darwin in some European orchids and which he hypothesised to be adaptations to limit pollinator mediated self-pollination. In chapter 3, a less common mechanism – anther cap retention – is described for E. foliosa. Observations of reconfiguration times were compared to the respective visit times by pollinators in a number of orchids (including Eulophia and Acrolophia) and asclepiads. In 18 of 19 species, pollinarium reconfiguration times exceed the average visit times, providing empirical support for Darwin’s cross-pollination hypothesis [chapter 4]. All of the 25 species of Eulophia examined are deceptive, but two of the three species in the small, closely related Cape genus Acrolophia examined in chapter 5 are rewarding. This translates into very high levels of pollen transfer efficiency in the rewarding A. cochlearis relative to the deceptive A. capensis and species of Eulophia. In addition, A. cochlearis exhibits high rates of pollinator-mediated self-pollination, as quantified using a novel method based on levels of inbreeding depression during embryo development. In chapter 6 the evolutionary divergence of long- and short-spurred forms of E. parviflora in response to different pollinators is investigated. This shows that divergence has occurred in floral morphology, scent chemistry and flowering phenology and that this can be attributed to adaptations to the respective bee and beetle pollinators of each form. This thesis also includes case histories of bee pollination in an additional five Eulophia species, and beetle-pollination in two other species of Eulophia with dense inflorescences and slow pollinarium reconfiguration [chapter 7]. In addition, four taxa were found to undergo auto-pollination [chapter 8]. The main conclusions of this thesis are that pollination of food-deceptive species can be enhanced by spatial proximity to, and floral colour matching with, sympatric rewarding species; that selection strongly favours traits that promote cross-pollination; that pollinatorshifts can drive speciation; and that floral adaptations for bee-, beetle-, and auto-pollination are found in South African representatives of Eulophia. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

The morphology, anatomy and pollination biology of Mystacidium Lindl., a small, epiphytic genus of orchids, was investigated within a phylogenetic context. Morphological and anatomical studies were carried out in order to obtain characters for a cladistic analysis of the genus using Cyrtorchis arcuata (Lindl.) Schltr. as an outgroup. The phylogenetic analysis indicated that the genus may not be monophyletic. Two species of the closely related genus Diaphananthe Schltr., D. caffra (H.Bol.) Linder and D. millarii (H.Bol.) Linder, appear to be nested within Mystacidium. Mystacidium species grow in habitats varying from mistbelt forest to dry savanna. Analysis of stable isotope composition (Ȣ¹³C values) of leaves and roots showed that all Mystacidium species, as well as D. caffra and the outgroup C. arcuata, employ CAM photosynthesis. The Ȣ¹³C values were significantly negatively correlated with mean annual rainfall at the collection sites. Breeding system experiments revealed that Mystacidium is dependent on pollinators for fruit set, and that self-pollination results in substantially reduced seed set due to either inbreeding depression or partial self-incompatibility. Field observations revealed that M. venosum Harv. ex Rolfe and M. capense (L.f.) Schltr. are hawkmoth-pollinated, and that M. gracile Harv. and M. pusillum Harv. are pollinated by settling moths. The spurs of the flowers contain dilute, sucrose-dominant nectar. Mystacidium venosum and M. capense showed evidence of nectar reabsorption. Nocturnal emission of scent occurred in all species except M. aliceae H. Bolus and M. brayboniae Summerh., which are unscented, and was composed largely of a combination of monoterpenes and benzoids. Despite substantial variation in spur length (1 - 4.7 cm) among species, no evidence for directional selection on spur length was found in M. venosum, M. capense or M. gracile. Hand pollinations significantly increased fruit set in M. capense in two consecutive seasons at different sites, indicating pollen limitation. Although pollen removal was greater than pollen receipt in M. venosum, M. capense and M. gracile, suggesting transport loss or insufficient visitation, a remarkably high percentage of removed pqllen reached stigmas (35 - 50%). Experiments on M. venosum revealed that flower longevity is reduced by pollination, and that pollinia removed from flowers remained viable for up to 20 days under field conditions. The phylogeny indicated that long-spurred, hawkmoth-pollinated species are basal within the genus, and that shorter-spurred species pollinated by noctuid moths are derived. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2002.