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.

Reproductive studies in invasive plants are necessary for an understanding of their potential to establish and spread in foreign environments. Elaeagnus umbellata Thunb. (autumn olive) is an invasive woody shrub that flowers early in the spring and is often noted for its abundant fruit set. This study examined the reproductive biology of E. umbellata in Illinois, where it is highly invasive. Hand-pollination experiments were performed to determine the breeding system of E. umbellata, and floral visitors were collected to determine its pollinators. Experiments showed that E. umbellata is a predominantly outcrossing species with a self-incompatible breeding system. However, individual variation was detected in several reproductive characteristics. Pollen tube analyses revealed that a small percentage of individuals allow successful self-pollen tube growth, and self-fruit set resulting from automatic self-pollination (autogamy) was relatively high in a few plants. Automatic self-pollination is possible because the male and female parts of flowers mature sychronously, but the likelihood of autogamy may vary among individuals due to variability in the spatial separation of male and female parts (herkogamy). Variability in the incompatibility system and the level of herkogamy may impact the outcrossing rates and reproductive success of individuals. The majority of floral visitors to E. umbellata were generalist pollinators. Frequently visiting bees included small and large species such as native Andrena spp., Augochlorella aurata, Bombus spp., Ceratina calcarata, Xylocopa virginica, and the introduced Apis mellifera. Bombylius major (large bee fly) and the moth Mythimna unipuncta (armyworm) were also frequent visitors. Most of the above insect taxa are pollinators of E. umbellata based on analysis of pollen on insect bodies. E. umbellata is likely to achieve its abundant fruit set where these common pollinators and other E. umbellata are present. However, in my study sites, many individuals experienced low fruit set on branches that were open to pollinator visitation, suggesting pollen limitation may be common in some years and at certain sites. The discovery of autogamous individuals demonstrates that some E. umbellata individuals may be able to establish and spread even when mates or pollinators are limiting.

The genus Sorbus is an example of a taxonomically complex group (TCG) with diversity derived from hybridisation, polyploidy and apomixis. The focus of this study was to elucidate the evolutionary relationships among nine Sorbus species including endemics of the Devon and north Somerset region of the south west UK, determine main routes of polyploid formation and investigate reproductive sustainability in order to make recommendations for Sorbus conservation. Molecular analysis showed that genetic structure patterns and genotypic diversity support the hypothesis that the study polyploids are a product of rare interspecific hybridisation, of single origins and are maintained through apomictic reproduction. PCoA, Neighbour Joining analysis and parental simulations reveal a reticulated relationship, with diversification the result of hybridisations between sexual diploid Sorbus torminalis and both tetraploid and triploid species. Hybridisation between S. torminalis and tetraploid Sorbus margaretae (subgenus Aria) have likely given rise to the study members of subgenus Tormaria through production of a triploid which has subsequently backcrossed to Sorbus torminalis to form further tetraploids. The discovery of a cryptic hybrid in subgenus Aria also suggests occasional hybridisation events among tetraploids are a possible route for further tetraploid formation These events illustrate key routes of polyploid formation, both illustrating the role of triploids in tetraploid formation via the triploid bridge and the key role in sexual diploids in diversification in Sorbus. Hand pollination experiments showed that self-incompatibility in the triploid species (Sorbus subcuneata) means reliance on congeneric pollen from sympatric tetraploid species for seed production. Reproductive sustainability in this species is severely compromised through spatial isolation from compatible congeners. Our findings are strong support for the development of conservation strategies that aim to safeguard current diversity through actions that increase reproductive sustainability and recruitment opportunities, and promote opportunities for on-going hybridisation for future diversification of Sorbus in this region.

The vast majority of flowering plants rely on pollinators for outcrossed reproduction. Unfortunately, many species of pollinators are in decline. Pollinators face a number of challenges, including shifting land use, climate change and pesticides. In addition, pollinator habitat may be altered or eliminated through urbanization. Fragmented urban landscapes may lengthen the distances among foraging patches, limit resources in a given patch, and reduce the availability of pollinator nesting sites. Here, I examine the effects of urbanization on pollination services by addressing the following questions: (1) Does bee species richness and/or abundance change along an urbanization gradient?; (2) Does the pollen limitation of a focal species vary along the same gradient?; (3) Do plants with a mixed mating system produce more selfed seeds in more urban environments? Using the percentage of impervious land cover in the space immediately surrounding the site as a proxy for site urbanization, 15 study sites were set up across the city of Ottawa to span a range from minimal percent impervious surface (mostly green space) to mostly impervious surface (little green space) at a range of spatial scales. At each site I set up an array of 20 potted Impatiens capensis plants as well as six pan traps in order to collect data on pollen limitation, seed production, and the number of selfed progeny, in addition to information about local pollinator species richness and abundance. Plants in the arrays were randomly assigned to either a hand or open pollination treatment in order to assess the level of pollen limitation. Surprisingly, pollinator species richness and abundance were not correlated with urbanization. Pollen limitation declined with urbanization, however, so did overall seed set, making it difficult to detangle the effects of resource limitation and hand pollination treatments. The number of selfed seeds produced by a plant was also significantly correlated with site urbanization. Further investigation is required to better understand the dynamics of pollination services in urban environments. I recommend that future studies explore how the presence of individual bee species in rural and urban habitats could be influencing pollen limitation and selfing in I. capensis.

The interaction of herbivory and pollination is not well understood. Both topics on their own have been studied thoroughly, yet their interaction has not. Until the 1990s, few studies attempted to explore how herbivory might affect pollination, and vice versa.

To date, 13168 plant species have been naturalized outside their native range. While invasive plants efficiently compete for resources, they can also disrupt vital mutualisms. Pollination is a crucial mutualism required for 87% of flowering plants to reproduce. Invasive species may disrupt patterns of pollinator visitation, pollen transfer dynamics (conspecific [CP] and heterospecific [HP]), and reproductive success. Furthermore, whether invasive species’ effects depend on scale (site vs. patch) is not known. Thus, it is critical to understand the effects of invasive plants at all stages of pollination and how those effects vary with scale. Here, I investigate pollinator visitation, CP and HP deposition, and pollen tube growth between local flowers in sites and plots invaded by Cirsium arvense. For 7 weeks (June-July), pollinator visits were recorded and styles collected from an invaded and non-invaded floral community. Styles were processed for fluorescent microscopy. The number and identity of pollen grains on stigmas and the number of pollen tubes at style bases were recorded. The invaded site received significantly less visits than the non-invaded site, but the effect varied by species. This variation is not explained by local species’ abundance or floral symmetry. Similarly, invaded plots received significantly less visits than non-invaded plots, and no plot-species interaction was found. The invaded site received more CP and HP while invaded plots received less CP and HP. There was no difference in pollen tube growth between sites or plots. My preliminary results suggest that C. arvense affects pollinator visitation and pollen transfer dynamics in local floral communities, but the effects vary depending on species and scale. Conversely, these effects do not seem to lead to differential reproductive success between invaded and non-invaded communities at any scale.

Introduction, Methods Most flowering plants rely on biotic pollination for seed production. However, pollination failure can still occur due to inadequate quantity or quality of pollen delivered (pollen limitation). In fact, 60% of plant populations have been shown to be pollen limited. Pollen limitation can occur due to low pollinator availability or when low quality pollen fails to fertilize the ovules. Co-flowering species share pollinators, and can influence each other's pollination by attracting a larger pollinator pool compared to when flowering alone (pollinator facilitation), resulting in greater pollen loads. On the contrary, a limited pollinator pool can lead to pollinator competition and decrease the amount of pollen received. Lastly, pollinator competition may increase self-pollination or heterospecific pollen transfer, hence increasing pollen quality limitation. Here, we evaluate the contribution of pollen quantity and quality limitation to plant reproductive success across a gradient of co-flowering community diversity. We used four co-flowering Clarkia species in the Sierra Nevada as our study system. Species bloom singly and in combination of 1-3 other Clarkia species. Styles from all species (~60/species) were collected across 25 sites varying in co-flowering diversity. 1600 styles were processed in the lab and the amount of pollen grains and pollen tubes were counted. Results, Discussion Clarkia speciosa received the most pollen grains (477 grains) and Clarkia cylindrica the least (300 grains) on average across all populations. Preliminary data also shows that pollen quantity limitation decreases in communities where all four Clarkia species co-flower compared to when they flower singly or with only one-two other species. This pattern was observed for all Clarkia species. For instance, Clarkia unguiculata alone received 465.68 (± 30.63) pollen grains on average compared to 840.65 (±56.03) when co-flowering with all three other Clarkia species. Clarkia xantiana received 41.50 (± 48.82) pollen grains when flowering alone compared to 407.8 (± 62.19) when co-flowering with all other Clarkia species. We are currently collecting data on pollen tube number to evaluate differences in pollen quality limitation. Our preliminary data suggests pollinator facilitation can be a key mechanism reducing pollen quantity limitation, promoting species co-existence, and increasing plant reproductive success in this system. However, it is still not known if higher pollen loads lead to higher fertilization (work in progress). Clarkia species can self-pollinate (low quality pollen) or receive pollen from a different Clarkia which will not lead to fertilization and thus pollen quality could still affect reproductive success and mediate co-existence in these communities.

Compounding evidence suggests a current or impending sixth mass extinction event and pollinator crisis. While several factors contribute to pollinator declines, the most notable driver is habitat loss and degradation. Agricultural grasslands provide crucial habitat for wild and domesticated fauna, regulate water and nutrient cycles, store carbon, and maintain soil stabilization. However, conventional tall fescue pastures, which dominate the southeastern United States, limit pollinator habitat, reduce ecosystem services, and diminish cattle productivity if infected with toxic endophytes. Establishing wildflowers (WFs) and native warm season grasses (NWSGs) into tall fescue pastures has the potential to boost both pollinator ecosystem services and cattle productivity. This study monitored the differences in honey bee colony health, productivity, and pollen foraging behavior between diversified and conventional grazing pastures in south west Virginia. Chapter 1 sought to evaluate the health and productivity of hives within NWSGW+ diversified and conventional grazing systems. Chapter 2 sought to 1) determine whether honey bees used sown wildflower species in diversified pastures as significant sources of pollen, 2) compare species composition and nutritive value of pollen collected from hives within diversified and conventional pasture systems, and 3) evaluate temporal trends in pollen collection. Floral surveys revealed diversified pastures had almost 4x greater mean bloom density than conventional pastures, with over half of all blooms recorded in diversified pastures belonging to unsown species. Results from this study suggest that colonies in diversified pasture systems may have a slight advantage in population resource acquisition, population growth, and winter survival following the first year of establishment, though further research is needed. Pollen DNA metabarcoding revealed that honey bees in both diversified and conventional pasture systems have similar diets, and that sown species were foraged upon primarily in the fall. Samples collected from diversified pasture systems yielded greater pollen weight, species richness, and protein content. In conjunction with previous research, these results indicate that diversified pastures could ultimately provide a more complex nutritional support system for pollinators in grazing agroecosystems. However, individual pasture renovation may not be large enough to yield statistically significant differences in honey bee colony success. / Master of Science / Many studies have suggested that we are currently experiencing or entering a sixth mass extinction event and pollinator crisis. While several factors contribute to pollinator declines, the most notable driver is habitat loss and degradation. Agricultural grasslands provide crucial habitat, regulate water and nutrient cycles, store carbon, and maintain soil stabilization. However, conventional tall fescue pastures, which dominate the southeastern United States, limit pollinator habitat, reduce ecosystem services, and can diminish cattle productivity. Establishing wildflowers (WFs) and native warm season grasses (NWSGs) into tall fescue pastures has the potential to boost both pollinator and cattle health and productivity. This study monitored the differences in honey bee colony health, productivity, and pollen foraging behavior between pastures diversified with WFs and NWSGs and conventional grazing pastures in south west Virginia. Chapter 1 evaluated the health and productivity of hives within diversified and conventional grazing systems. Chapter 2 sought to 1) determine whether honey bees used sown wildflower species in diversified pastures as significant sources of pollen, 2) compare species types and value of pollen collected from hives within diversified and conventional pasture systems, and 3) evaluate temporal trends in pollen collection. Floral surveys revealed diversified pastures had almost 4x greater mean bloom density than conventional pastures, with over half of all blooms recorded in diversified pastures belonging to species we didn't purposefully plant. Results from this study suggest that honey bee colonies in diversified pasture systems may have a slight advantage in resource acquisition, population growth, and winter survival following the first year of establishment, though further research is needed. A Pollen DNA analysis revealed that honey bees in both diversified and conventional pasture systems have similar diets, and that they collected pollen and nectar from the flowers we planted primarily in the fall. Honey bee colonies in diversified pastures collected more protein-rich pollen from a wider variety of flowers. These results indicate that diversified pastures could ultimately provide a more complex nutritional support system for pollinators in grazing agroecosystems. However, individual pasture renovation may not be large enough to yield large differences in honey bee colony success.

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