The reproductive ecology of two terrestrial orchids, Caladenia Rigida and Caladenia tentaculata.

The reproductive outcome of plants is often determined by a multitude of interacting factors operating at both the plant level and the population level. For many plants, fruit production and the subsequent release of seeds are paramount for the persistence of the species. Understanding the processes that influence variation within and among populations is therefore crucial for the successful long-term management of threatened plants. While abiotic factors such as resource availability and environmental conditions can influence seed production directly through their effects on plant growth, biological interactions such as those between plants and pollinators or herbivores can be equally important. The relative intensity and direction of such interactions are often determined by the nature of the plants themselves, or by characteristics of the plant population or the habitat in which it occurs. This thesis examines the processes that influence spatio-temporal variation in the reproductive success of two terrestrial orchids, Caladenia rigida and Caladenia tentaculata. The study was carried out over three years (2005 – 2007), in several populations located in the Mount Lofty region of South Australia. A detailed investigation of the pollination strategy employed by C. rigida revealed that this species is a generalist, being pollinated by a suite of food-seeking insects, possibly attracted by the presence of small amounts of nectar. Successful pollination and seed release for C. rigida was highly variable across space and time. Furthermore, both measures were consistently higher than for the sexually deceptive species, C. tentaculata, leading to the suggestion that the highly specialised pollination syndrome of the latter species may place it at a reproductive disadvantage. Pollination success of C. rigida was influenced by the height of flowers, but not by the local density of conspecifics. Small populations of C. rigida did not produce capsules when environmental conditions were stressful, suggesting that resource availability may indirectly restrict reproductive success by limiting the availability of pollinators. Poor seed quality in some populations may also be attributed to reduced population size. Both orchid species were subject to intense levels of vertebrate florivory and capsule predation, leading to significant reductions in seed output. A herbivore exclusion experiment was carried out to help elucidate the size and type of herbivores, and video surveillance identified birds as a predominant florivore in some populations. The intensity of florivory varied within and among populations, as well as among years, in response to several factors including flower height, the local density of conspecifics, concealment amongst neighbourhood vegetation and proximity to the habitat edge. Spatio-temporal variation in seed release was thus the net outcome of processes acting on both mutualistic and antagonistic interactions. This work provides valuable baseline data of factors that influence the reproductive ecology and, hence, population dynamics of Caladenia species. Implications for the conservation and management of threatened populations are discussed, with respect to both short-term and long-term goals. The thesis is presented as a series of five manuscripts. Two of these have been published, and the remaining three have been prepared for submission as publications.Thesis (Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 201

Developmental Activation of the Rb–E2F Pathway and Establishment of Cell Cycle-regulated Cyclin-dependent Kinase Activity during Embryonic Stem Cell Differentiation

To understand cell cycle control mechanisms in early development and how they change during differentiation, we used embryonic stem cells to model embryonic events. Our results demonstrate that as pluripotent cells differentiate, the length of G(1) phase increases substantially. At the molecular level, this is associated with a significant change in the size of active cyclin-dependent kinase (Cdk) complexes, the establishment of cell cycle-regulated Cdk2 activity and the activation of a functional Rb–E2F pathway. The switch from constitutive to cell cycle-dependent Cdk2 activity coincides with temporal changes in cyclin A2 and E1 protein levels during the cell cycle. Transcriptional mechanisms underpin the down-regulation of cyclin levels and the establishment of their periodicity during differentiation. As pluripotent cells differentiate and pRb/p107 kinase activities become cell cycle dependent, the E2F–pRb pathway is activated and imposes cell cycle-regulated transcriptional control on E2F target genes, such as cyclin E1. These results suggest the existence of a feedback loop where Cdk2 controls its own activity through regulation of cyclin E1 transcription. Changes in rates of cell division, cell cycle structure and the establishment of cell cycle-regulated Cdk2 activity can therefore be explained by activation of the E2F–pRb pathway

Grazing orchids: impact of florivory on two species of Caladenia (Orchidaceae)

Herbivory is considered a major threat in many of the orchid-species recovery plans in Australia. Kangaroos and rabbits are the most commonly implicated herbivores; however, no studies have attempted to confirm their role. Regular monitoring of several populations of Caladenia rigida R.S.Rogers and C. tentaculata Schldl. during 3 years in the Mount Lofty Ranges, South Australia, revealed that up to 94% of flowers and 36% of seed capsules were browsed, whereas leaf herbivory was less prevalent. Furthermore, patterns of herbivory varied markedly among sites and across years. In two seasons, predation of C. rigida flowers inside a kangaroo- and rabbit-proof exclosure was equal to or higher than outside the exclosure. Florivory within populations was influenced by proximity to the habitat edge, although the direction of this response differed among sites. Various types of mesh cages were erected around plants to elucidate the size and type of herbivores. Plants protected from florivores were almost three times more likely to produce seed than were exposed plants; however, some cage types reduced pollination. Video surveillance confirmed the role of the white-winged chough, Corcorax melanorhamphos, as a florivore. The present study is the first one to identify a herbivore unequivocally, quantify the intensity and extent of floral herbivory across a range of populations, and assess the potential cost of florivory to the direct reproductive output of orchids.Renate Faast and José M. Facell

Investigating DNA barcoding options for the identification of Caladenia (Orchidaceae) species

© CSIRO 2009The application of molecular techniques for defining evolutionary units in Caladenia has largely focussed on addressing relationships at the subgeneric and deeper levels. However, in light of the morphological complexity present in this diverse genus, molecular markers offer additional characters for the refinement of taxonomy at the species level. In the present study, we explored the utility of marker systems with demonstrated application for defining fine-scale units, both in terms of phylogenetic information and in the context of DNA barcoding. We also provide a working example of the use of molecular techniques for identifying the source plants of pollinia collected from passively sampled pollinators and for identifying sterile plants.Lachlan Farrington, Phyllis MacGillivray, Renate Faast and Andrew Austi

The lectin dolichos biflorus agglutinin recognizes glycan epitopes on the surface of murine embryonic stem cells: A new tool for characterizing pluripotent cells and early differentiation

Copyright © 2007 by AlphaMed Press.Cell surface markers are key tools that are frequently used to characterize and separate mixed cell populations. Existing cell surface markers used to define murine embryonic stem cells (mESCs) such as stage-specific embryonic antigen 1 (SSEA1), Forssman antigen (FA), alkaline phosphatase (AP), and CD9 are limiting, however, because they do not unambiguously define the pluripotent state and are not reliable indicators of differentiation commitment. To identify glycan cell surface markers that would circumvent this problem, we used a panel of 18 lectins to identify epitopes specifically elevated on the surface of mESCs, which, during differentiation, decrease with kinetics that precede currently used markers such as CD9, SSEA1, FA, and AP. The anticipated outcome of this analysis was to identify glycans that have utility as reliable mESC markers and high-resolution readouts for early differentiation commitment. Here, we show that the lectin Dolichos biflorus agglutinin (DBA) recognizes -N-acetylgalactosamine (GalNAc) cell surface epitopes on mESCs (CD9high SSEA1high APhigh DBAhigh). These glycan epitopes decline markedly in cells undergoing the first definable step of differentiation, the transition from mESCs to primitive ectoderm (CD9high SSEA1high APhigh DBAlow). Loss of GalNAc epitopes is, therefore, the earliest cell surface change that can be assigned to differentiating cells, and the only cell surface marker known to be tightly associated with the pluripotent state. The lectin DBA is, therefore, a useful tool to characterize mESC cultures by nondestructive approaches, an indicator of differentiation commitment, and a predictor of developmental potency.Rodney Nash, Lori Neves, Renate Faast, Michael Pierce and Stephen Dalto

Bees and white spiders: unravelling the pollination syndrome of Caladenia rigida (Orchidaceae)

© CSIRO 2009Orchids of the genus Caladenia have been shown to utilise two quite different pollination strategies, namely species-specific sexual deception of thynnine wasps and a more generalist strategy attracting a larger spectrum of foraging insects. While baiting techniques have enabled the identification of numerous pollinators of sexually deceptive Caladenia, insects that pollinate food-advertising species have received little attention. The present study employed a multidisciplinary approach to better evaluate the pollination syndrome of the white spider orchid, Caladenia rigida R.S.Rogers, a species previously reported to utilise both food and sexual deception. This included the observation and capture of potential pollinators of C. rigida through direct observation, pantraps, Malaise traps and pollinator-baiting experiments, and the use of molecular techniques to identify orchid pollinia isolated from carrier insects. We describe a suite of generalist insects visiting and bearing pollinia from C. rigida. In addition, samples collected from the labellum and column of C. rigida contained sugars at levels comparable to those of a known nectar-producing orchid, Microtis parviflora R.Br. Potential osmophores in the clubs and calli stained positively with neutral red and although this character is often associated with sexual deception, we found no evidence for this secondary pollination syndrome in C. rigida. The present study is the first one to provide a detailed description of the pollinators and pollination syndrome of a non-sexually deceptive species within the genus Caladenia and the first report to provide evidence of nectar production by a species within this genus.Renate Faast, Lachlan Farrington, José M. Facelli and Andrew D. Austi