The biogeography, ecology and endophyte mycorrhiza of the New Zealand Corybas alliance (Orchidaceae) : specifically, Nematoceras iridescens (Irwin et Molloy) Molloy, D.L.Jones & M.A.Clem. (species) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand

Abstract

No research on the fungal endophytes in the green New Zealand terrestrial orchids has been published. Identification of the endophyte resident in Nematoceras iridescens roots was accomplished by comparing hyphal septal ultra-structure and using TEM imaging of the hyphal septa, all of which indicated that the genus Tulasnella was involved. The Tulasnella species was identified using molecular techniques focused on sequencing of the ribosomal RNA locus on the ITS1–5.8S–ITS2 nuclear ribosomal gene. The endophytic fungi, resident in the host plant N. iridescens and the germinating seed of this species, were identified for the first time as strains of Tulasnella calospora. Scanning electron microscopy (SEM), light microscopy (LM) and Confocal Laser Scanning Microscopy (CLSM) was used to investigate spatial distribution of the endophyte hyphae within the plant. The SEM results identified four morphological types of hyphae: initiating, divaricating, intercellular and necrotised, or undergoing lysis. Peloton formation only occurred in specific areas of the root; mainly within the sub-epidermal and mid cortex cells. No hyphal involvement within the stele or the immediately adjacent cortex cells occurred. This thesis, based on meteorological information, proposes that the centre of origin of Nematoceras is likely to be Papua New Guinea, with on-going dispersal being direct or from Australia to New Zealand. Wind vectors and Nematoceras adaptations to seed dispersal, both local and long distance, were investigated and tend to support this hypothesis. Germination of all known orchid seed requires an obligate mycoheterotroph, generally a member of the Basidiomyceteae. For the first time, CLSM has imaged the fungal endophyte within the seed embryo and this was identified by molecular techniques and found to be a unique strain of T. calospora. Three methods of orchid seed germination were trialled: symbiotic, asymbiotic and field envelopes. After a 12-month period, only field envelopes produced germinating seeds of N. iridescens. In all other methods the seed failed to germinate. All Nematoceras spp. are solitary leafed and classified as moist mesophytes. Leaves were found to be hypostomatous. Being single leaved, protection is essential and a number of adaptations to counter herbivoury were found: raphide crystals, wax cuticle, winter maturity with summer–autumn aestivation all provide an antiherbivoury component. The relationship between the genera of Nematoceras plus Singularybas and the associated species, of the far more ancient Hepatophyte order of Metzgeriales, were investigated. The genus Aneura contains peloton like vesicles of various strains of T. calospora. The majority of the Hepatophytes have a parallel geographic-ecological requirement. The T. calospora mycorrhiza of the Metzgeriale genus Aneura was found to have a close association with the Corybas alliance observed.