This thesis investigates speciation in Neotropical orchids, with a focus on the subtribe Oncidiinae, which has undergone a dramatic radiation in the Neotropics. A near-complete species-level phylogeny of the Tolumnia clade (a subset of the Oncidiinae), comprising 47 of the 51 currently recognised species, based on multiple plastid (trnH-psbA, trnL-F, rps16) and nuclear DNA regions (ITS and ETS) provides a well resolved and robust framework from which to address questions about the evolutionary history of this group. It also permits the clarification of the taxonomic status of certain anomalous taxa, including the genus Amparoa which is found to be embedded within Rhynchostele. Significant levels of rate heterogeneity are observed between the lineages of this phylogenetic tree, and potential causes, both physiological and ecological, of these are examined, although no single causal factor is identified.

Historically, Ophrys morphological taxonomy and species delimitation have been controversial, with treatments inferring from 16 species plus 34 subspecies to as many as 252 species forming 32 complexes. Highly variable, yet possibly convergent, morphologies and paucity of sequence variation have hindered production of a robust phylogenetic framework. This project generated phylogenies and ordinations from nuclear and plastid DNA sequencing, amplified fragment length polymorphism (AFLP) and morphometric data, with the ultimate aims of producing a robust framework as a basis for more rigorous species delimitation and conservation recommendations for the genus. The project also elucidates genetic and morphological relationships within Ophrys section Ophrys, and within the 0. fuciflora and 0. sphegodes aggregates, and assesses the congruence between these contrasting datasets.

Members of the Orchidales produce dust seeds so lacking in nutrient and carbon reserves that they are dependent upon appropriate fungal partners to supply the essential additional carbon and nutrients required for successful establishment in nature. These plants are thus initially mycoheterotrophic before they normally go on to develop green leaves and become autotrophic as adults. However, at least 210 species of orchid remain fully mycoheterotrophic into adulthood. The metabolic pathways involved in the nitrogen and carbon fluxes from fungus-to -plant in mycoheteratrophic are still poorly understood, but result in characteristic enrichment in 13C and 13N. In this thesis in vitro studies on agar are employed to investigate the effects of different N sources, with and without sugar or cellulose carbon sources on the heterotrophic growth of mycorrhizal orchid seedlings and metabolite pools in the plants and their fungal partners. It is hypothesised that glutamine, a central metabolite in assimilation of mineral by basidiomycete fungi, passes from fungus-to -plant as in traditional models of N assimilation by ectomycorrhizal plants. A range of nitrogen and carbon sources including isotopically labelled 14C and 13C-glucose together with 15N labelled N sources including nitrate, ammonium, ammonium nitrate, glutamine, glycine and arginine were supplied to orchid mycorrhizal fungi in pure culture and to mycorrhizal seedlings of Goodyera repens or Dactylorhiza fuchsii. In three compartment Petri dishes in which the seedlings were grown with their fungal partners colonising the two other compartments, simultaneous additions of DC and 15N tracers provided compelling evidence of fungal assimilatory pathways providing co-transport of C and N into heterotrophic seedlings of G. repens. Nitrate was a better N source than ammonium or amino acids for the plants, and metabolomic analysis showed strong up-regulation of relative abundance of glutamine in the fastest growing plants. The findings presented in this thesis provides strong circumstantial evidence that glutamine serves as a major combined N and C source transported from fungus-to-plant in heterotrophic orchid seedlings.

No description available.

The genus Restrepia is well known to orchid enthusiasts but its micromorphology has not been described, and its pollination and breeding systems have not been investigated. The aim of this investigation was, therefore, to add to existing knowledge so that the resultant data could be used to facilitate ex situ conservation initiatives. A detailed electron microscopy study (SEM) of the floral organs was performed. This confirmed the structure of the dorsal sepal and lateral petal osmophores, their secretory nature together with that of the synsepal and the labellum. It was postulated how, by manipulating different labellar surface textures, the flower might use these ‘tactile guides’ to steer the insect (fly) through the flower. The cirrhi were postulated to help by destabilising the pollinator in flight, trapping it and bringing about pollination. The papillate structure of the calli was established and their optical properties investigated. Media comparison investigations established that Western medium supported the highest germination rates and, with the addition of banana supplement, the highest rates for seedling growth and development. This represented the first protocol for axenic germination of Restrepia in the literature (Millner et al., 2008) and provided a tested methodology for investigating breeding systems and producing Restrepia plant material for both scientific and horticultural purposes. Self-pollinations were found to produce fewer embryos compared to cross-pollinations. The operation of self-incompatibility (SI) was confirmed by the study of pollen tube growth which further confirmed the time interval between pollination and fertilisation. A time line from pollination/fertilisation to flowering was established. The type of SI in operation was best explained by gametophytic incompatibility. This demonstrated that it was possible to raise Restrepia hybrids and species from seed, by performing intraspecific crosses so helping to preserve them for posterity and relieve pressure on wild populations. Narrow endemic Restrepia species face combined threats from habitat loss, habitat degradation and problems of viable seed production due to the effects of SI and inbreeding depression (ID). Recently developed online resources, such as GeoCAT, were used to perform a Red List assessment in order to identify the degree of threat individual species faced, both globally and nationally. All species were classified as facing substantial levels of threat; although this was lessened for populations in protected habitats. Conservation is needed for cultivated collections as well as these wild populations by keeping alive existing knowledge and expertise in growing these species.