Application of microsatellite markers for population genetics of three New Zealand Corybas taxa (Orchidaceae) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Manawatū, New Zealand

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Many New Zealand Corybas orchid species are endangered due to habitat destruction caused by changes in land use. Conservation efforts are needed to secure survival of those endangered species. Unfortunately, there are very few studies on these orchids and several aspects of their ecology, biology, and reproductive biology remain unknown. It is difficult to develop efficient conservation plans for those species without this information. This study describes the first genetic investigation in Corybas species and provides insight to their reproductive methods. Genetic diversity assessment was conducted in population(s) of three Corybas taxa, and the results were used to infer their genetic structure and reproductive methods. Pollination experiments and pollinator observations were carried out in one species, Corybas carsei, to understand the pollination strategies in this endangered species. Twelve previously developed microsatellite markers were used to genotype 37 Corybas “Remutaka” individuals collected from one population; these detected a medium level of genetic variation within population. No correlation between genetic and geographic distances were found. The genetic assessment results indicated that self-pollination, cross-pollination, and asexual reproduction likely occur in this population. As the readily available Corybas microsatellite markers were not transferable to C. carsei, novel microsatellite markers were developed using next-generation sequencing. Eleven markers were used to genotype 29 C. carsei individuals sampled from the single remaining C. carsei population. With the finding of only monomorphic loci, no, or possibly extremely low, genetic variation was detected within the population and, hence, reproductive methods employed by the C. carsei population could not be inferred. Conversely, 11 microsatellite markers used to genotype 67 Corybas macranthus individuals from three populations detected low to medium genetic variation within populations, and a low degree of genetic differentiation between populations. The results of within-population genetic assessment indicated that cross-pollination, self-pollination, and clonal propagation occurred. Pollination experiments in C. carsei showed that this orchid is self-compatible and autonomous self-pollination may occur. Its putative pollinator, flies, approaching the flower was captured on camera. No pollination events were observed and the species of IV those flies were not identifiable. Overall, the findings contribute to our understanding of New Zealand Corybas orchids and offer future directions for their conservation.