Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. i 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. Roger L. S. Watkins 2012 iAbstract 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. ii Acknowledgements Firstly, my thanks to my wife Barbara for her forbearance, patience, putting up with: stacks of papers, home fridges packed with samples in transit, and her help with field collections and measurements. A thank you to my son Stephen, who often calmed my panic with his exorcisms of the demonic computer programmes which usually boiled down to my computer na?vet?. Then to A/Prof. Al Rowland the Chief Supervisor of this project, his help, direction, humour and friendship was a constant inspiration. Also thanks to my co-supervisor Prof. Pete Lockhart whose assistance with the molecular trees was much appreciated. A very special thanks to two wonderful people, Trish McLenachan of the Allan Wilson Centre, for her knowledge, patience, friendship and help with the DNA protocols, the molecular sequencing and to Doug Hopcroft of the MMIC for his knowledge and technical expertise of TEM and SEM microscopy. Their friendship was one of the highlights of my time at Massey. The initial period of this thesis was supervised by A/Prof David Fountain whose advocacy helped me negotiate the early academic slalom and his retirement so early in this project was regretted. Another early co-supervisor, Dr Peter Long initiated me into the world of fungi and was sorely missed when he left for China and then retirement, while he was available, his door was always open. I am grateful to Dr. Heather Outred who kindly read this thesis prior to submission and who instilled in me an interest in mycorrhiza at the undergraduate level. She has continued to be a friend throughout my time at Massey. Thanks to A/Prof. Dr. Kathryn Stowell for her assistance in the final months of this project after taking over the chief supervisors role on Al?s retirement. An appreciation also to Massey University, who granted me a Doctoral Scholarship which provided me a three year funding for this thesis. The sample of Solophenyl Flavine 7GFE 500 donated by Mr. Chris McGregor of Huntsman Textile Effects NZ Branch proved to be a worthwhile fluorophore in imaging the hyphal wall of endophytes in samples when using the confocal laser scanning microscope (CLSM). I was fortunate to meet some very interesting people during the location and collection of plants for this thesis and I owe a debt to Margaret Menzies for her hospitality, interest and conveyance to orchid sites in the Matemateonga. Thanks to Margaret and all the others who I have not specifically mentioned, this thesis would be the poorer if it was not for your help and encouragement. iii Table of contents Abstract i Acknowledgements ii Table of contents iii List of figures vi-viii List of tables ix Glossary and list of abbreviations used x-xiii Chapter 1 Introduction and aims of the thesis 1-3 Chapter 2 Methods, materials and sampling sites used in the thesis 4-26 2.1 Sampling 4 2.1.1 The sample sites 5-7 2.2 Wind measurements 8-9 2.3 Pod Harvest 9 2.4 Pod Oscillation 9-10 2.5 Seed Collection 10 2.6 Seed morphology 10-11 2.7 Seed Germination 11-17 2.7.1 Symbiotic germination (series 1) 11-12 2.7.2 Symbiotic germination series 2) 13 2.7.3 Asymbiotic germination. 13-15 2.7.4 Field Germination. 15-17 2.8 Leaf surface and crystal inclusions. 17-18 2.9 Examination of the fungal endophyte 18-19 2.10 Microscopy used in this thesis 19-22 2.10.1 Light Microscopy 19-20 2.10.2 Environmental scanning electron microscopy 20 2.10.3 Scanning electron microscopy (SEM) 20 2.10.4 Confocal laser scanning microscopy (CLSM) 20-22 2.10.5 Transmission Electron Microscopy (TEM). 22 2.11 Molecular analysis, DNA preparation 23-26 2.11.1 Selection of primers (Series 1) 23-25 2.11.2 Sequencing 25 2.11.2.1 Sequencing (Series 2) 25 2.11.3 Tissue disruption 25-26 2.11.4 PCR (Series 2) 26. Chapter 3 Description and distribution of the Corybas alliance (CA) 27-45 3.1 The geographic footprint of the Corybas alliance (CA) 28-29 3.2 Distribution within New Zealand 30-34 3.3 Climatic factors in seed dispersal 34-45 3.3.1 General site climate 34-35 3.3.2 Long distance wind dispersal 36-40 3.3.3 Wind velocity and direction at the plant site 40-41 3.3.4 Results of wind site measurements 41-43 3.4 Discussion 43-45 iv Section A. Ecology Chapter A1 Aspects of seed morphology, anatomy and germination in N. iridescens 46- 72 A1.1 Introduction 46- 47 A1.2 Results 47-49 A1.3 Pod oscillation 49-52 A1.4 Seed mass 52 A1.5 The morphology of N. iridescens seed 52-59 A1.5.1 Volume ratios 54-57 A1.5.2 The micromorphology of N. iridescens seed 57-59 A1.6 Germination trials of N. iridescens Seed 60-70 A1.6.1 Symbiotic trial (Series 1) 62-63 A1.6.2 Symbiotic trial (Series 2) 64 A1.6.3 Asymbiotic germination trial 65-67 A1.6.4 Field germination 67-70 A1.7 Discussion of Section A1 70-72 Chapter A2 Leaf characteristics of N. iridescens 73-101 A2.1 Introduction 73-78 A2.2 Abaxial surface 78-84 A2.3 Adaxial surface 85-86 A2.4 Laminar margin 86-87 A2.5 Epicuticle 87-89 A2.6 Crystal inclusions 89-95 A2.7 Leaf predation 95-99 A2.8 Diatoms 99 A2.9 Discussion 99-101 Section B: The peloton endophyte Chapter B1 The fungal mycorrhiza of N. iridescens 102-121 B1.1 Mycoheterotrophy in New Zealand orchids 102-104 B1.2 Results and Discussion 104-121 Chapter B2 The ultra-structure of the fungal septae 122-131 B2.1 Introduction 122 B2.2 The septal pore 122-126 B2.3 Results and discussion 127-131 Chapter B3 The molecular analysis of the fungal endophyte 132-153 B3.1 Introduction 132-133 B3.2 Results 133 B3.2.1 Series 1 131-135 B3.2.2 Series 2 135-141 vB3.3 Phylogenetic methods 141-150 B3.3.1 Group 1 144 B3.3.2 Group 2 145 B3.3.3 Group 3 147 B3.3.4 The 3A group 147-149 B3.3.5 Group 3b 150 B3.3.6 Group 4 150 B3.4 Discussion 151-153 Section C: Summary of the Thesis 154-161 References 162-193 Appendices 194-229 Appendix 1 1a Botanical description of Nematoceras iridescens 194-195 1b Consent from Te Atiawa tribal authority 196 1c NZ orchid collection data 197 1d Distribution of CA within the N.Z. botanical region areas 198-199 1e Kew Gardens; notification of nomenclature changes to CA 200-202 1f Description of Lepidoptera Scopariade 203-204 Appendix 2 2a Fungal culture media used 205-206 2b Orchid seed culture media 207-209 Appendix 3 Stains used 210-211 Appendix 4 Seed data 212-217 Appendix 5 Culture of N. iridescens root cortex tissue 218- 229 vi List of figures Chapter 2 4 Fig. 2.1 Sampling site locations 5 Fig. 2.2 The site areas from which the samples were found 6 Fig. 2.3 Original Pukeiti collection site 2005 7 Fig. 2.4 Roadworks (2006-2007) have destroyed all plants on the right bank. 7 Fig. 2.5 Illustrating Q1301 anemometer reading positions 8 Fig. 2.6 The measuring points used in collection of wind data Pukeiti 8 Fig. 2.7 The experimental set up used to measure the pod oscillations in a lignified peduncle of N. iridescens. 10 Fig. 2.8 A diagram of seed and symbiont placement 12 Fig. 2.9 A QIAshredder? Mini Spin Column 14 Fig. 2.10 A completed orchid seed envelope. 16 Fig. 2.11 A field marker at Mangamingi 16 Fig. 2.12 Absorbance (dashed line) and emission (solid line) spectra of Solophenyl Flavine (7GFE 500). 21 Fig. 2.13 The resin block section, prior to ultra-microtoming analysis for septa 22 Fig. 2.14 A locus map of specific primers, designed for Basidiomyceteae PCR, 24 Fig. 2.15 Specific primers, designed for Tulasnella spp. 26 Chapter 3 27 Fig. 3.1 A N. iridescens colony, Tarata Tunnel 27 Fig. 3.2 Plants of Singularybas oblongus from the Pukeiti site 27 Fig. 3.3 Frontal view of N. iridescens 28 Fig. 3.4 The footprint? of Corybas alliance distribution 29 Fig. 3.5 NZ ecological regions showing CA areas 31 Fig. 3.6 Differences between ?helmet? and ?spider? orchids 32 Fig. 3.7 Rainfall maps of the Taranaki area 35 Fig. 3.8 Backtracked trajectory paths of air envelopes 37 Fig. 3.9 An Isobaric weather map of the Tasman Sea 37 Fig. 3.10 An holistic air flow diagram of symniotic wind flows 39 Fig. 3.11 Diagrammatic adaptation of alpine valley wind flow directions 40 Fig. 3.12 Vertical average wind speeds 41 Fig. 3.13 Horizontal average wind speeds 42 Fig. 3.14 Wind strengths at Moki 42 Fig. 3.15 Wind strengths at Pukeiti 43 Section A1 Fig. A1.1 Early seed pod formation 48 Fig. A1.2 Fully extended peduncles 48 Fig. A1.3 Peduncle expansion rates of four species of the CA 49 Fig. A1.4 A peduncle, pod and leaf. 50 Fig. A1.5 Oscillations of N. iridescens seed pods, on mature peduncles 51 Fig. A1.6 The average wind strengths at horizontally measured points 51 Fig. A1.7 N. iridescens seed morphology: 54 Fig. A1.8 A seed of N. iridescens with measurement point lines. 55 Fig. A1.9 A N. iridescens seed stained with SafraninO 56 vii Fig. A1.10 Air floatation times of a range of orchid seeds 57 Fig. A1.11 Reticulate testa patterns of N. iridescence 58 Fig. A1.12 A comparison of the seed testa. 59 Fig. A1.13 Comparative measurement of seed embryo the symbiotic trial. 62 Fig. A1.14 Fungal hyphae invading an N. iridescens seed. 62 Fig. A1.15 A predatory mite (Cryptostigmata family spp.) on N. papa seed 65 Fig. A1.16 Imbibed embryos of N. iridescence 67 Fig. A1.17 Field trial seed of N. iridescens at the sample b site 2 68 Fig. A1.18 Fungal hyphae accumulation in field trial seed 68 Fig. A1.19 Seed germination initiated in a field trial seed, after one year. 69 Fig. A1.20 The embryo interior with hyphae of T. calospora 70 Fig. A1.21 An illustration of seed deposit rings. 71 Section A2 Fig. A2.1 Morphological diversity of four leaf forms of N. iridescens. 78 Fig. A2.2 Abaxial stomata of six CA species. 81 Fig. A2.3 Corybas alliance stomata 82 Fig. A2.4 A N. iridescens leaf stained with SafraninO and Alcian blue. 82 Fig. A2.5 A multidimensional, CLSM micrograph, illustrating the topological features (x and y planes) together with a depth (z) field. 83 Fig. A2.6 CLSM images of stomata. 84 Fig. A2.7 A comparison of the adaxial epidermis of six CA species 85 Fig. A2.8 An adaxial N. iridescens leaf section profile 86 Fig. A2.9 The laminar leaf margin of N.iridescens. 87 Fig. A2.10 A waxy cuticle patterning covering the adaxial leaf 88 Fig. A2.11 The wax patternation of the epicuticle of Anzybas rotundifolia 88 Fig. A2.12 A young leaf t/s of N. iridescens with an idioblast containing a developing raphide 89 Fig. A2.13 A N. iridescens root with raphide crystals 90 Fig. A2.14 Scattering of raphide crystals on the abaxial surface 90 Fig. A2.15 ESEMs of crystal inclusion forms found in N. iridescens. 91 Fig. A2.16 Two idioblasts, each containing raphide bundles. 92 Fig. A2.17 ESEM of raphides penetrating root cell walls. 92 Fig. A2.18 EDAX analysis of a raphide crystal. 93 Fig. A2.19 eSEM of raphide crystals showing acute ends and edge serrations. 94 Fig. A2.20 ESEM micrographs of early raphide development 95 Fig. A2.21 A snail traversing a leaf of N.iridescens at Tarata site 96 Fig. A2.22 A heavily predated leaf of N. iridescens. 97 Fig. A2.23 The herbivorous leaf predator found on an N.iridescens leaf: 98 Fig. A2.24 Damage of N. iridescens leaf, the peripheral margin untouched 98 Fig. A2.25 Diatoms found on adaxial leaf surfaces of N. iridescens. 99 Section B: The peloton endophyte Chapter B1: The fungal mycorrhiza of N. iridescens Fig. B1.1 A phylogenic tree, based on the Basidiomycota, of a major orchid mycorrhizal genus, Tulasnella (teleomorph) / Epulorhiza (anamorph) 102 Fig. B1.2 A typical Rhizoctonia form hyphae 105 viii Fig. B1.3 T/s of a young root of N. iridescens 106 Fig. B1.4 Hyphae and trichomes of N. iridescens 107 Fig. B1.5 A N. iridescens root with the degree and location of hyphal penetration 108 Fig. B1. T/s of a new season?s N. iridescens root. 109 Fig. B1.7 A N. iridescens root section incorporating all phases of mycorrhizal distribution. 110 Fig. B1.8 Older N. iridescens root sections 111 Fig. B1.9 An inner cortex cell of N. iridescens containing amyloplasts. 111 Fig. B1.10 Late season N. iridescens root with amyloplasts. 112 Fig. B1.11 An external mature root section 113 Fig. B1.12 Four stages of peloton formation and degradation. 114 Fig. B1.13 T/s of (A) the stem and (B) a leaf section) 115 Fig. B1.14 Large cortex cells showing mega-nuclei. 116 Fig. B1.15 Cortex hyphal forms. 117 Fig. B1.16 A fungal endophyte penetrating cell walls and hyphal morphology. 118 Fig. B1.17 An accumulation of amyloplasts (amyl) in the large mid-cortex 119 Fig. B1.18 CLSM micrograph image of the hyphae within a peloton 120 Fig. B1.19 Late lysis of the peloton hyphae within a giant cortex cell 121 Chapter B2: The ultra-structure of the fungal septae Fig. B2.1 An adaptation of Moore?s illustration,septal pore ultrastructure 124 Fig. B2.2 The fungal SPA?s characteristic of various filamentous fungal genera 124 Fig. B2.3 Ascomycete septa. 125 Fig. B2.4 Ceratobasidium corrigendum septa 125 Fig. B2.5 Tulasnella septa 125 Fig. B2.6 Cerinomyces atacua septa 126 Fig. B2.7 Tremulaccea septa 126 Fig. B2.8 Auricularia spp. septa 126 Fig. B2.9 Tremella globospoa septa 126 Fig. B2.10 A peloton hyphae from a young root of N. iridescens. 127 Fig. B2.11 The hyphal septa from a young root of N. iridescens. 128 Fig. B2.12 The electron dense parallel layers making up the parenthesome 129 Fig. B2.13 An l/s section of a peloton hyphae from an older plant. 130 Fig. B2.14 The parenthesomes are disinteragating 131 Chapter B3: The molecular analysis of the fungal endophyte Fig B3.1 Gel Doc image (BIORAD) of PCR products 137 Fig B3.2 Neighbour-joining tree of dataset 1, the diversity of 131 Tulasnella 143 Fig.B3.3 Group 1 from the phylogenic tree 144 Fig.B3.4 Group 2 from the phylogenic tree in Fig.B3.2 145 Fig.B3 5 Neighbour-Joining tree using dataset 3 (28 taxa, 404bp aligned). 146 Fig.B3.6 Group 3A Neighbour-Joining tree using dataset 3 148 Fig.B3.7 Group 3B Neighbour-Joining tree using dataset 2. 149 Fig.B3.8 Neighbour-Joining tree using dataset 3. 150 ix List of tables Methods, Materials and Sampling Sites used in the thesis Table 2.1 The coding of seed used in the symbiotic germination 12 Table 2.2 A list of the selected library inoculants. 13 Table 2.3 Kauth's six orchid seed germination stages . 15 Table 2.4 Primers used in the first series of PCR amplifications. 24 Description and distribution of the CA Table 3.1. The Acianthinae, showing the New Zealand genera of the CA. 29 Table 3.2 Species distribution of ?helmet and spider? flowering forms 33 Table 3.3 Mean monthly values of 9am relative humidity for the 1971-2000 periods for New Plymouth. 34 Table 3.4 A summary of climate information for selected NZ locations. 35 Section A. Ecology: Chapter A1 Table A1. 1 The average weights of N. iridescens seed. 52 Table A1. 2 Distribution of seed forms found in seed pod samples 53 Table A1.3. Cell measurements, medial section, of the testa cells of three Nematoceras species. 58 Table A1.4. Cell measurements of the medial and long axis of the testa cells of three Nematoceras species. 59 Table A1.5. Trial results, symbiotic germination of N. iridescens seed 63 Table A1.6. Results of light and dark on inoculated N. iridescens seed 64 Table A1.7. Results of the asymbiotic germination trial of N. iridescens. 66 Table A1.8. Record of seed germination in field trials at Mangamingi. 67 Table A2.1. Average stomatal density / mm?, of a selection of Nematoceras and a Singularybas sp. 79 Table A2.1 Stomata size, Nematoceras stomata giving a comparison of polar axis with lateral axis and form. 80 Table A2.3 A comparison of stomata form, size and density from the previous tables. 80 Section B: The peloton endophyte Table B3.1 One of two tables (#1 and #2) showing the results of the investigation to find the identity of the endophyte within the peloton of N. iridescens and other CA species. 134 Table B3.2 A continuation of the earlier table B3.1 135 Table B3.3 Primers used in the second series of PCR amplifications. 136 Table B3.4 PCR Gel results using MagNA Lyser and the ITS mixed ITS-OF primer pair. 136 Table B3.5 PCR Gel results using MagNA Lyser and the ITSTul-4/ ITS 5 primer pair with a lowered annealing temperature of 54oC. 137 Table B3.6 Results obtained from retained DNA samples from the 1st series, using the ITS4-Tul / ITS5 primer pair. 138 Table B3.7 Table of the 2nd series of extractions from root and seed tissue using Basher Bead preparation with ITS4-Tul / ITS5 primer pair. 139 Table B3.8 GenBank accessions used in this study. 140 Table B3.9. Tulasnella NCBI accession numbers from sample sequences gathered in this project 141 xGlossary and Abbreviations used ?? Provisionally identified / diagnosed . Abaxial Ventral aspect of a leaf, the underside Acicular A needle like form, especially in crystals Acotyledonous Without seed cotyledons Adaxial Dorsal aspect of a leaf, top side Aeolian Borne on the wind, (Aeolus, the Greek wind god) Aestivate A period of dormancy / quiescence AFLPs Amplified Fragment Length Polymorphism Agenous Ontogenetic pathway creating stomata without subsidiary stomatal development. An unequal mitosis occurring in protodermal cells, leading to the development of meristemoids. Amphistomatous Stomata found on both sides of the leaf (Generally a higher proportion are found on the Abaxial side of the leaf) Anamorph Describes the fungus when reproducing asexually Anemochory The role of wind in seed dispersal Anomycytic No subsidiary cells surround the guard cells. Anomycytic stomatal complex. Mature guard cells surrounded by epidermal cells that do not deviate morphologically from other epidermal cells in the same plant. Anthesis A period in which a flower is fully open and functional. Apiculus A long sharp pointed tip Apomorphic A derived type, a more contemporary state character ASL Above sea level AWCGS Allan Wilson Centre Genome Services Biseriate Two cell layers in depth. CA Corybas alliance CLSM, Confocal laser scanning microscopy. Connate Joined together, a union between two similar parts cpDNA Chloroplast DNA Crustose. Crust like, having an epidermal surface characteristic. Cyclocytic pattern More than four subsidiary cells surrounding a central pair of guard cells. Cyclocytic stomatal complex. A pattern of subsidiary cells that occur when a greater number of similar subsidiary cells radiate out from the mature guard cells outer circumference. Defensin Small, basic peptides that can inhibit the growth of a broad range of pathogenic fungi but seem nontoxic to either mammalian or plant cells. Dematiaceous Having a dark colour, usually olive, grey, or black. Dermatogen External cuticle of plants in a forming condition. Diacytic Only two subsidiary cells surrounding the stomata. Diacytic stomatal complex Mature guard cells surrounded by a pair of subsidiary cells with their shared wall at right angles to the long axis of the guard cells Disjunct A plant species that is found in two or more widely separated areas with no evidence of that species being represented between the two sites Distichous Two leaves growing oppositely and alternately dNTPs Dinucleotide triphosphates xi Dolipore septum A cross wall found in Basidiomycetes and characterized by special swellings and membranes in association with the septal pore Druse crystals Small open topped clusters of calcium oxalate crystals DSE?s Dark septate endophyte Edaphic When produced or altered by the soil EDAX Energy-dispersive X-ray spectroscopy Endemic Taxa which occur only in a specific area Endophyte In the context of this dissertation the term represents a fungal endophyte that has a significant part of its lifecycle residing internally and asymptomatically in various plant structures and distinguishable from mycorrhiza in not possessing external hyphae or mantels. Endovelamen Inner tangential wall thickening of the external velamen Ensiform Shaped in the form of a double edged sword. Epivelamen External tangential wall thickening of the velamen e-SEM Environmental scanning electron microscope EtOH Ethanol Exo III Exonuclease III FIM Fungal initiating media GA Gibberellic acid Gynostemium A fused or partially fused column of stamens and pistils. Hemimesogenous The meristemoid divides twice to produce a second order meristemoid. Holoepiphytes Epiphytes having a complete life cycle on the host tree. Holomorph Describes the whole shape. Horizontal transmission Transmission of the fungus by sexual or asexual spores. Hyaline Transparent, clear. Hydrophytic Living in /on or by water. Hypostomatous Leaf stomata found on the abaxial surface. Isodiametric Length width and height are of roughly the same dimensions. LM Light microscopy. Ls Life span. Lotus effect Leaf surface cleaning by water droplets running off a hydrophobic waxy cuticle. Lysigenous development A duct in tissue, formed following lysis of cells. Malesia The botanical area of Indo-malaysia and Australia Mesogene cell A second order meristemoid. Mesophytic Neither too wet nor too dry an environment. MGS Massey Genome Service MMN Melin-Norkrans agar MMNL MMN without agar, liquid media Mya Million years ago NCBI National Centre for Biotechnology Information Neoendemic Representing an evolutionarily young taxon that has not had an opportunity to disperse. NGS Next generation sequencing nrDNA Nuclear ribosomal DNA OM Orchid mycorrhiza Paracytic stomatal complex. Mature guard cells surrounded by two flanking subsidiary cells. xii Palaeoendemic Representing relicts of a once broadly dispersed taxon. Parenthosomes Shaped like a parenthesis symbol ?(?. They are bow to dome-shaped double membranes thought to be a modified part of the endoplasmic reticulum (ER), that cover the dolipore and pore channel forming the septal pore cap. PCR Polymerase chain reaction PDA Potato Dextrose Agar, Difco Pedicel The stalk, internode, that arises from the peduncle and terminates at the distal end of the individual flower. Fahn (1997) Peduncle An inflorescence stalk bearing a solitary flower in a one-flowered inflorescence Peloton Intracellular coils of mycorrhizal fungal hyphae found within the root cortex cells in an endosymbiotic association, Burgeff (1936). Perigene cells Cells that arise during stomatal development by the division of protodermal cells around the stomatal meristemoid. Petiole The stalk of a leaf, attaching the blade to the stem Phorophytes Host trees Plasmoptysis The physiological state of mycorrhizal fungi after the cytoplasm has been ejected. See ptyophagy. Plesiomorphic An original type or primitive ancestral state character Pseudovivipary Production of offspring by apomictic or asexual propagules such as plantlets and bulbils. Ptyophagy Fungal cytoplasm is inserted into the transfer cells of roots by the specialized hyphae of the mycorrhizal mutualist. PUA Polyunsaturated aldehyde rDNA Ribosomal DNA. RAPD Random amplified polymorphic DNA Raphides Needle like crystals composed of calcium oxalate Reniform Kidney shaped (renal) RFLP Restriction fragment length polymorphism Rosanoffian crystals Crystals are found within a sheath, bundled within a membrane or specialist cell. Schizogenous development. Development of a duct by division of a common middle lamella that expands to form a lacuna. SAP Shrimp Alkaline Phosphatase SEM Scanning electron microscope SPA Septal pore apparatus Teleomorph Describes the fungus when reproducing sexually. Tetracytic pattern Four, roughly equal sized, subsidiary cells surround the stomatal guard cells. Tolypophagy Intracellular aggregates of coiled fungal hyphae that have been isolated by root or rhizoid cells prior to absorption or excretion. TS Transverse section Uniseriate One cell layer depth. Wewelite Calcium oxalate monohydrate crystal Xeromorphic Morphologically adapted for dry conditions